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Philip Morris

Date: 16 Mar 1998 (est.)
Length: 447 pages
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nRnFr biomonitoring data and animal studies, support the scientific position that environmental tobacco smoke cannot be classified as a known human carcinogen. 0002150.01 3/18/98 4:54pm -4-
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DRAFC detoxification process predominates. This suggests that though TSNAs may be animal carcinogens (consistent with the metabolic profile reported here), in humans, the metabolic profile of TSNAs is not consistent with carcinogenic activity. • The on-going research program sponsored by Philip Morris which is evaluating the biological activity of an ETS surrogate (room-aged sidestream smoke (RASS)). We have provided experimental details of an on-going lifetime exposure study. This study meets Office of European Community Development (OECD) testing guidelines. Our laboratory, which is an approved Good Laboratory Practices (GLP) facility, has completed a one-year interim sacrifice. We are progressing and anticipate study completion in less than two years. We have thoroughly described the RASS test atmosphere to an extent not previously reported. The biological effects seen to date are indicative of mild irritation that is both reversible and non-progressive. • Philip Morris' 1997 submission to the California Environmental Protection Agency (Cal/EPA) addressing the alleged association between reported exposure to ETS and human cancer. We believe that the underlying science, including ETS exposure as determined by personal monitoring, distributional analysis of exposure information, human epidemiological and 0002150.01 3/18198 4:54pm -3-
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DRAFT Executive Summary Philip Morris U.S.A. submits the following materials to the National Toxicology Program (NTP) for consideration in NTP's review of environmental tobacco smoke (ETS) for listing in the Ninth Report on Carcinogens. Included in this submission are the following items: • A discussion of the recent report from the International Agency on Research on Cancer (IARC) on its multi-center epidemiologic study of ETS exposure and lung cancer risk in Europe (IARC Biennial Report 1996/1997). The IARC study, the largest, and according to IARC, the "most comprehensive" epidemiological study specifically designed to assess the potential lung cancer risk of spousal, workplace and childhood ETS exposure, reported no statistically significant risk estimates for spousal, workplace, spousal plus workplace or childhood exposures; • An additional analysis of the United States Environmental Protection Agency's (U.S. EPA) 1992 Risk Assessment on ETS, which addresses, using Monte Carlo simulations, the uncertainty associated with two critical factors that influence the attributable deaths estimated by EPA. Our analysis indicates that proper consideration of just these two factors reduces the EPA's attributable risk number by two-thirds. Further consideration of factors such as diet, socio-economic status, family history of lung disease, etc., as well as data from 0002150.01 3/18198 4:54pm ' 1"
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DRAFT epidemiologic and exposure studies in the workplace, social settings, etc., would be expected to further reduce this estimate. • Our analysis of methodologies utilizing cotinine as a measure of either smoking status or ETS exposure. The technological measurement short-comings and the metabolic complexities of these issues leads us to describe what we believe to be a more systematic and rigorous approach to this question. Given the current situation, we believe that the use of single point measurements using cotinine alone cannot be used as a quantitative measure of ETS exposure; • An analysis of the smoke component, benzo(a)pyrene (B(a)P), that has been purported to be causally associated with lung cancer. This analysis suggests the following: (i) that the levels of B(a)P that would be expected in environments where ETS is present are within the background levels found in urban air and (ii) that sources other than ETS account for more than 90 percent (> 90%) of the total body burden of B(a)P. • An analysis of the smoke component, tobacco specific nitrosamines (TSNAs), that have been purported to be causally associated with lung cancer. A comparative analysis of TSNA metabolism in lung and liver from the A/J mouse, Fischer 344 rat and human strongly indicates that activation predominates in the mouse and rat whereas in the human tissues a 0002150.01 3118198 4:54pm -2-
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Tricker, A.R., N-Nitroso Compounds and Man: Sources of Exposure, Endogenous Formation and Occurrence in Body Fluids, European Journal of Cancer Prevention, 6: 226-268 (1997) van Rooij, J.G.M., Veeger, M.M.S., Bodelier-Bade, M.M., Scheepers, P.T.J., and Jongeneelen, F.J., Smoking and Dietary Intake of Polycyclic Aromatic Hydrocarbons as Sources of Interindividual Variability in the Baseline Excretion of 1-Hydroxypyrene in Urine, International Archives of Occupational and Environmental Health, 66: 55-65 (1994) von Meyerinck, L., Scherer, G., Adlkofer, F., Wenzel-Hartung, R., Brune, H., and Thomas C., Exposure of Rats and Hamsters to Sidestream Smoke from Cigarettes in a Subchronic Inhalation Study, Experimental Pathology, 37: 186-189 (1989) Witschi, H.P., Oreffo, V.LC., and Pinkerton, K.E., Six-Month Exposure of Strain A/J Mice to Cigarette Sidestream Smoke: Cell Kinetics and Lung Tumor Data, Fundamental and Applied Toxicology, 26: 32-40 (1995) 0159528.01 7
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Coggins, C.R.E., The OSHA Review of Animal Inhalation Studies with Environmental Tobacco Smoke, Inhalation Toxicology, 8: 819-830 (1996) Cress, R.D., Holly, E.A., Aston, D.A., Ahn, D. K., and Kristiansen, J. J.,Characteristics of Women Nonsmokers Exposed to Passive Smoke, Preventive Medicine, 23: 40-47 (1994) Domino, E.F., Estimating Exposure to Environmental Tobacco Smoke - Letter to the Editor, Journal of the American Medical Association, 276(8): 603 (1996) Einhaus, M., Holz, 0., Miessner, R., Krause, T., Wamcke, K., Held, L, Scherer, G„ Tricker, A.R., Adlkofer, F., and Rudiger, H.W., Determination of DNA Single-Strand Breaks in Lymphocytes of Smokers and Nonsmokers Exposed to Environmental Tobacco Smoke Using the Nick Translation Assay, The Clinical Investigator, 72: 930-936 (1994) Finch, G.L., Nikula, K.J., Belinsky, S.A., Barr, E.B., Stoner, G.D., and Lechner, J.F., Failure of Cigarette Smoke to Induce or Promote Lung Cancer in the A/J Mouse, Cancer Letters, 99: 161-167 (1996) Friedman, G.D., Petitti, D.B., and Bawol, R.D., Prevalence and Correlates of Passive Smoking, American Journal ofPublic Health, 73: 401-405 (1983) Givens, G.H:, Smith, D.D., and Tweedie, R.L., Publication Bias in Meta-Analysis: A Bayesian Data-Augmentation Approach to Account for Issues Exemplified in the Passive Smoking Debate, Statistical Science, 12(4): 221-250 (1997) Gorgels, W.J.M.J., van Poppet, G., Jarvis, M.J., Stenhuis, W., and Kok, F.J., Passive Smoking and Sister-Chromatid Exchanges in Lymphocytes, Mutation Research, 279: 233-238 (1992) Gori, G.B., and Mantel, N., Mainstream and Environmental Tobacco Smoke, Regulatory Toxicology and Pharmacology, 14: 88-105 (1991) Haley, N.J., Adams, .LD., Axelrad, C.M., and Hoffinann, D., Sidestream Smoke Uptake by Syrian Golden Hamsters in an Inhalation Bioassay, Proceedings oflndoor Air '87, 2: 68-73 (1987) Heavner, D.L., Morgan, W.T., and Ogden, M.W., Determination of Volatile Organic Compounds and Respirable Suspended Particulate Matter in New Jersey and Pennsylvania Homes and Workplaces, Environment International, 22(2): 159-183 (1996) Hoepfner, L, Dettbam, G., Scherer, G., Grimmer, G., and Adikofer, F., Hydroxy-Phenanthrenes in the Urine of Non-Smokers and Smokers, Toxicology Letters, 35: 67-71 (1987) 2 0159528.01 I
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Lee, P.N., An Assessment of the Epidemiological Evidence Relating Lung Cancer Risk in Never Smokers to Environmental Tobacco Smoke Exposure, In: Environmental Tobacco Smoke, H. Kasuga (ed.), Springer-Verlag, United States: 28-84 (1993) Lee, P.N., ETS and Lung Cancer: A Summary of the Epidemiological Evidence, Submission to OSHA, (1995) Lee, P.N., Lung Cancer and ETS: Is the Epidemiologic Evidence Conclusive?, In: Proceedings of the Section on Statistics and the Environment, American Statistical Association, 65-73 (1994) LeVois, M., and Switzer, P., Differential Exposure Misclassification in Case-Control Studies of Environmental Tobacco Smoke and Lung Cancer,.7ournal of Clinical Epidemiology, 51(1): 37-54 (1998) LeVois, M.E., and Layard, M.W., Comment Re: "Inconsistency between Workplace and Spousal Studies of Environmental Tobacco Smoke and Lung Cancer", Regulatory Toxicology and Pharmacology, 25: 194-195 (1997) LeVois, M.E., and Layard, M.W., Inconsistency between Workplace and Spousal Studies of Environmental Tobacco Smoke and Lung Cancer, Regulatory Toxicology and Pharmacology, 19: 309-316 (1994) Mahanama, K.R.R., and Daisey, J.M., Volatile N-Nitrosamines in Environmental Tobacco Smoke: Sampling, Analysis, Emission Factors, and Indoor Air Exposures, Environmental Science and Technology, 30: 1477-1484 (1996) Martin, F., Hoepfner, I., Scherer, G., Adlkofer, F., Dettbam, G., and Grimmer, G., Urinary Excretion of Hydroxy-Phenanthrenes After Intake of Polycyclic Aromatic Hydrocarbons, Environment International, 15: 41-47 (1989) Matanoski, G., Kanchanaraksa, S., Lantry, D., and Chang, Y., Characteristics of Nonsmoking Women in NHANES I and NHANES I Epidemiologic Follow-up Study with Exposure to Spouses Who Smoke, American Journal ofEpidemiology, 142(2): 149-157 (1995) McAughey, J., Black, A., Strong, J., and Dickens, C., Comparison of Particulate Dose From Exposure to Environmental Tobacco Smoke (ETS) and Mainstream Cigarette Smoke Using Radiotracers, Annals of Occupational Hygiene, 41 (Supplement 1): 724-727 (1997) Miller, S.I., Branoff, S., and Nazaroff, W.W., The Contribution of Environmental Tobacco Smoke to the Exposure of Californians' for Sixteen Toxic Air Contaminants, In: Abstract K2. 01, Society for Risk Analysis, p. 140 (1996) 0159528.01 4
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Fu39erstr. 3, 51149 Ko1n ILSI Sponsored by the International MONOGRAPHS Life Sciences Institute U. Mohr Editor-in-Chief Toxic and Carcinogenic ~ffects of S olid Particles in the Respiratory Tract D.L. Dungworth J.L. Mauderly G. Oberdorster Editors ri_S1 ILSI Press Washington, D.C. /39Cfi R I r i . v ~
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Table 1. Concentration otsmoke componcnLs in FSS and RASS. Pararnctcr fSS RASS Hmpty roond aging Wool curtain Wool carpet Wooden book shelf Painted wallpaper All materials together TPM (µSIL) 10.8 7.1 4.1 6.0 6.4 6.7 3.9 Nicotine (NF/L) 2.49 0.77 0.39 0.81 0.61 0.39 0.23 Ammonia (pf/L) 3.95 3.73 0.46 2.07 1.32 1.49 0.24 I IyJrogcn cyanide (pg/L) 0.18 0.15 (1.11 0.09 0.12 0.10 0.08 Formaldehyde (ppm) (1.51 0.49 0.21 0.25 0.35 0.23 0.11 AccialJchyJc (ppm) 0.75 0.70 0.68 (1.71 Q68 (1.72 0.68 Acrolcin (ppm) 11.11 11.08 O./N) t).08 0.09 0.10 0.09 9Lti8ti9690Z
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Influence of Aging and Surface Cootact on Cigarette Sidestream Smoke 641 were reproducibly generated. No notable trends for the concentrations of all components determined were observed (see Figure 2 for TPM). Summary Large differences in the composition of FSS and RASS were found. The concentrations of RASS components vary considerably depending on the material present. W ith the setups used, significantly different ETS models were reproducibly generated. Acknowledgment. This work was sponsored by Philip Motris, USA. References Eatough D7, Hansen LD, Lewis EA (1990) The chemical characterization of environmental tobacco smoke. Environ Technol 11:1071 Guerin NII2, Jenkins RA, Tomkins BA (1992) The chemistry of environmental tobacco smoke: composition and•measurement. Lewis Publishers, Inc., Chelsea, MI
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Hoffinann, D., Subproject 6: Sidestream Smoke Carcinogenesis Progress Report, In: Application for Continuation Grant, Experimental Tobacco Carcinogenesis, Public Health Service Department of Health and Human Services, pp. 71-81 (1988) Holz, 0., Krause, T., Scherer, G., Schnvdt-Preuss, U., and Rudiger, H.W.,'ZP-Postlabelling Analysis of DNA Adducts in Monocytes of Smokers and Passive Smokers, International Archives of Occupational and Environmental Health, 62: 299-303 (1990) Holz, 0., Meissner, R., Einhaus, M., Koops, F., Wamcke, K., Scherer, G., Adlkofer, F., Baumgartner, E., Rudiger, H.W., Detection of DNA Single-Strand Breaks in Lymphocytes of Smokers, International Archives of Occupational and Environmental Health, 65: 83-88 (1993) Husgafvel-Pursiainen, K., Sister-Chromatid Exchange and Cell Proliferation in Cultured Lymphocytes of Passively and Actively Smoking Restaurant Personnel, Mutation Research, 190: 211-215 (1987) Idle, J.R., Titrating Exposure to Tobacco Smoke Using Cotinine -- A Minefield of Misunderstandings, Journal of Clinical Epidemiology, 43(4): 313-317 (1990) Jenkins, R.A., Palausky, A., Counts, R.W., Bayne, C.K., Dindal, A.B., and Guerin, M.R., Exposure to Environmental Tobacco Smoke in Sixteen Cities in the United States as Determined by Personal Breathing Zone Air Sampling,.7ournal ofExposureAnalysis and Environmental Epidemiology, 6(4): 473-502 (1996) Klus, H., Begutter, H., Scherer, G., Tricker, A.R., and Adlkofer, F., Tobacco-Specific and Volatile N-Nitrosamines in Environmental Tobacco Smoke of Offices, Indoor Environment, 1: 348-350 (1992) Lee, C.K., Brown, B.G., Reed B.A., Rahn, C.A., Coggins, C.R.E., Doolittle, D.J., and Hayes, A. W., Fourteen-Day Inhalation Study in Rats, Using Aged and Diluted Sidestream Smoke from a Reference Cigarette, II. DNA Adducts and Alveolar Macrophage Cytogenetics, Fundamental and Applied Toxicology, 19: 141-146 (1992) Lee, C.K., Brown, B.G., Reed E.A., Coggins, C.R.E., Doolittle, D.J., and Hayes, A.W., Ninety-Day Inhalation Study in Rats, Using Aged and Diluted Sidestream Smoke from a Reference Cigarette: DNA Adducts and Alveolar Macrophage Cytogenetics, Fundamental and Applied Toxicology, 20: 393-401 (1993) Lee, C.K., Fulp, C., Bombick, B., and Doolittle, D.J., Inhibition of Mutagenicity of N-Nitrosamines by Tobacco Smoke and Its Constituents, Mutation Research, 367: 83-92 (1996) 0159528.01 3
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Scherer, G., Westphal, K., and Adlkofer, F., Urinary Mutagenicity, Hydroxyphenanthrene, and Thioether Excretion after Exposure to Environmental Tobacco Smoke, In: Indoor Air Quality, H. Kasuga (ed.), Springer-Verlag, Berlin, pp. 138-146 (1990) Scherer, G., Westphal, K., Biber, A., Hoepfner, H., and Adlkofer, F., Urinary Mutagenicity after Controlled Exposure to Environmental Tobacco Smoke (ETS), Toxicology Letters, 35: 135-140 (1987) Sonnenfeld, G., and Wilson, D.M., The Effect of Smoke Age and Dilution on the Cytotoxicity of Sidestream (Passive) Smoke, Toxicology Letters, 35: 89-94 (1987) Sorsa, M., Husgafvel-Pursiainen, K., Jarventaus, H., Koskimies, K., Salo, H., and Vainio, H., Cytogenetic Effects of Tobacco Smoke Exposure Among Involuntary Smokers, Mutation Research, 222: 111-116 (1989) Stehlik, G., Richter, 0., and Altmann, H., Concentration of Dimethylnitrosamine in the Air of Smoke-Filled Rooms, Ecotoxicology and Environmental Safety, 6: 495-500 (1982) Sterling, T.D., Glicksman, A., Perry, H., Sterling, D.A., Rosenbaum, W.L., and Weinkam, J.J., An Alternative Explanation for the Apparent Elevated Relative Mortality and Morbidity Risks Associated with Exposure to Environmental Tobacco Smoke, Journal of Clinical Epidemiology, 49(7): 803-808 (1996) Strong, J.C., Black, A., McAughey, J.J., and Knight, D.A., Dosimetry of Environmental Tobacco Smoke and 'Attached' Thoron Progeny, Proceedings ofIndoor Air '93, 1: 541-546 (1993) Teredesai, A., and Pruhs, D., Histopathological Findings in the Rat and Hamster Respiratory Tract in a 90-Day Inhalation Study Using Fresh Sidestream Smoke of the Standard Reference Cigarette 2R1, In: Toxic and Carcinogenic Effects of Solid Particles in the Respiratory Tract, Dungworth, D.L., Mauderly, J.L., and Oberdorster, G., (eds.), International Life Sciences Institute Press, Washington, D.C., pp. 629-635 (1994) Thompson, D.H., and Warburton, D.M., Dietary and Mental Health Differences between Never- Smokers Living in Smoking and Non-Smoking Households, Journal ofSmoking-Related Disease, 4(3): 203-211 (1993) Thornton, A., Lee, P., and Fry, J., Differences Between Smokers, Ex-Smokers, Passive Smokers and Non-Smokers, Journal of Clinical Epidemiology, 47(10): 1143-1162 (1994) Tricker, A.R., Klus, H., Begutter, H., Scherer, G., and Adlkofer, F., Tobacco-Specific and Volatile N-Nitrosamines in Environmental Tobacco Smoke, Proceeding ofIndoor Air '93, 3: 47-52 (1993) 0159528.01 6
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DRAFT carcinogenesis study with the determination of a no-effect level. Also, doses have not been selected based primarily on the concept of maximum tolerated dose (MTD), but rather are linked to certain multiples of realistic human environmental concentrations. In the following, critical parameters of the study concept will be discussed. Test Atmosphere Definitions Environmental tobacco smoke (ETS) is a complex mixture mainly composed of aged sidestream smoke (S S) as well as of small amounts of exhaled mainstream smoke (MS) (First, 1985; Baker and Proctor, 1990). Since "real" ETS cannot be generated for long-term laboratory research, RASS will be used in the long-term study as a surrogate for ETS. It will be obtained by diluting and aging SS generated from the standard reference cigarette 1 R4F in a controlled, noninert environment. International standards applying to the generation of MS will be adapted to produce SS. Generally, the same chemical compounds are found in SS, RASS, and ETS, but there are quantitative differences in the concentrations and phase distribution (Guerin et al., 1992; Voncken et al., 1994). DEE is a complex combustion aerosol like SS. The composition of DEE depends on N the fuel and lubricants used as well as on the engine and its mode of use. In the long-term study, ~ ~ - W -, m ,_- N 0002427.01 3/18/98 3:03pm -3-
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References Adlkofer, F., Scherer, G., Heller, W.-D., Sunkeler, X.; and Heintz, T., The Significance of Urinary Hydroxyproline Excretion in Smokers and Passive Smokers, In: Indoor Air Quality, H. Kasuga (ed.), Springer-Verlag, Berlin, pp. 213-218 (1990) Adlkofer, F., Scherer, G., Wenzel-Hartung, R., Brune, H., and Thomas, C., Exposure of Hamsters and Rats to Sidestream Smoke of Cigarettes: Preliminary Results of a 90-Day-Inhalation Study, In: Indoor and Ambient Air Quality, R. Perry and P. W. Kirk (eds.) Selper Limited, pp. 252-258 (1988) Armitage, A.K., Ashford, J.R., Gorrod, J.W., and Sullivan, F.M., Environmental Tobacco Smoke -- Is It Really a Carcinogen?, Medical Science Research, 25: 3-7 (1997) ASHRAE, Table 7 Total-Body Emission of Some Gaseous Pollutants by Humans, 1995 ASHRAE Applications Handbook, p. 41.6 (1995) Aviado, Domingo M., Suspected Pulmonary Carcinogens in Environmental Tobacco Smoke, Environmental Technology Letters, 9: 539-544 (1988) Black, A., McAughey, J.J., Knight, D.A., Dickens, C.J., and Strong, J.C., Estimation of ETS Retention in Volunteers from Measurements of Exhaled Smoke Composition, Proceedings ofIndoor Air '93, 3: 41-46 (1993) Bombick, D.W., Ayres, P.H., Nelson, P.R., Coggins, C.R.E., France, D., Fulp, C., Lee, C., and Doolittle, D.J., Assessment of the Biological Activity of Mainstream or Environmental Tobacco Smoke (ETS) using a Cellular Smoke Exposure Technique, In: Abstracts of the Twenty-Second Annual Scientific Meeting of the Environmental Mutagen Society, no. 26 (1991) Buratti, M., Fustinoni, S., Pellegrino, 0., Xaiz, C., Colosio, C., and Colombi, A., Evaluation of Urinary T,T-Muconic Acid as a Biomarker of Benzene Exposure Related to ETS, Proceedings of Healthy Buildings '95, pp. 605-609 (1995) Cerioli, A.S., DNA and Hemoglobin Adducts in People Exposed to Environmental Tobacco Smoke (E.T.S.). A Literature Survey, Proceedings of Healthy Buildings '95, pp. 633-638 (1995) Coggins, C.R.E., Ayres, P.H., Mosberg, A.T., Ogden, M.W., Sagartz, J.W., and Hayes, A.W., Fourteen-Day Inhalation Study in Rats, Using Aged and Diluted Sidestream Smoke from a Reference Cigarette, I. Inhalation Toxicology and Histopathology, Fundamental and Applied Toxicology, 19: 133-140 (1992) Coggins, C.R.E., Ayres, P.J., Mosberg, A.T., Sagartz, J. W., and Hayes, A.W., Subchronic Inhalation Study in Rats Using Aged and Diluted Sidestream Smoke from a Reference Cigarette, Inhalation Toxicology, 5: 77-96 (1993) 0159528.01 1
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DRAFT According to the principle of maximum tolerated dose (MTD), the highest test substance dose in a carcinogenicity study should be "sufficiently high to elicit signs of minimal toxicity without substantially altering the normal life span due to effects other than tumors. Signs of toxicity are those that may be indicated by alterations in certain serum enzyme levels or slight depression of body weight gain (less than 10 %)" (OECD Guideline 451, 1986). Setting the upper limit of doses to the MTD level should prevent toxicity from substantially interfering with tumorigenicity. On the other hand, dosing as high as this limit should prevent false negative carcinogenicity studies. Historically, the MTD has been frequently determined in pilot subchronic/90-day studies, and histopathological lesions other than those that may be related to carcinogenesis were also considered (Morrow et al., 1996). However, in only approximately 20 % of the NTP inhalation studies, reduced body weight was used as the rationale for selecting the highest dose. In one study, a multiple of the human therapeutic dose was applied. In the long-term studies completed by the NTP, only 60 % had reduced body weight at the highest dose, and the magnitude of these body weight effects did not correlate with the carcinogenic response of the test substances. This is in line with recent trends within the NTP in the interpretation of the MTD concept, i.e., from the "maximum tolerated dose" to the "minimally toxic dose," which would also consider slight body weight effects in the long-term study as minimally toxic. A special workshop dealt with establishing a rationale 4h - ~, ~ for aerosol exposure concentrations in long-term inhalation studies (Lewis et al., 1989). Emphasis ~::- 0002427.01 3/18/98 3:03pm -8-
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DRAFT (Voncken et al., 1994). In general, particles and particle-associated components decreased, while most vapor phase components remained unchanged. In order to provide reasonable aging of the SS and to optimize the reproducibility of the setup, a noninert empty room with defined characteristics will be used in the long-term study. In most field studies as well as in all published experimental inhalation studies, the particle concentration has been used as the key analyte to determine the concentration of ETS and ETS surrogates. In order to be able to compare our analytical and biological results with published data, TPM concentration will be used as the key analyte in the long-term study. In addition, the particle concentration has been the key analyte in all DEE inhalation studies as well as the basis for DEE risk assessment. DEE will be generated using standard fuel and lubricant and a passenger car engine frequently in use in Europe. The engine will be driven using a standardized protocol to simulate city driving behavior. Fresh DEE will be used as the test atmosphere to keep comparability with previously published DEE long-term inhalation studies (Karagianes et al., 1979; Iwai et al., 1986; Ishinishi et al., 1986; Vallyathan et al., 1986; Mauderly et al., 1987; Brightwell et al., 1989; Heinrich et al., o00z4z7.ot 3/18/98 3:03 pm -6-
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DRAFT 1986, 1992, and 1995; Nikula et al.,1995). Fresh DEE may be less environmentally relevant but its use in long-term inhalation studies is consistent with published literature. A comparison of DEE composition in published inhalation studies revealed some differences. The most important difference may be the proportion of elemental carbon in the particulate phase. In the most recent published long-term DEE inhalation studies, elemental carbon was reported to be 60 % (Heinrich et al., 1995) and 92 % (Nikula et al., 1995) of TPM. It is well known that the proportion of elemental carbon in DEE particulates fluctuates (Hering et al., 1990; Watts, 1995). Major factors contributing to this fluctuation include, apart from emission control devices, the engine type, duty cycle, fuel, and lubricant consumption. Elemental carbon generally accounts for about 40 to 60 % of DEE particulate matter mass (Klingenberg et al., 1991; Zaebst et al., 1991). As for SS, DEE concentrations in experimental studies are commonly based on particulate matter mass as collected on glass fiber filters. Sometimes, soot seems to be synonymously used. Dose Levels P 1 - 0002427.01 3/l8/98 3:03pm -7-
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DRAFT Concept of the Long-Term Inhalation Study The study will correlate mechanistic data gained from intermediate biomarker assays with tumor development, as well as compare these data for the two test materials. The study is designed to investigate the test materials at toxicologically relevant concentrations that bear a more realistic relationship to human exposure. This study will also contribute to the discussion on the biological plausibility of the tumorigenic risk purportedly attributable to ETS and DEE exposure. Mechanistic investigations on the interaction between the test model and the test materials will be an integral part of the study in order to provide a comprehensive interpretation of effects. The integration of mechanistic endpoints in long-term bioassays and the use of these data in risk assessment has been recommended by the NTP Board of Scientific Counselors (1992) and the International Agency for Research on Cancer (IARC) Working Group (1992), as well as in the Guidelines for Carcinogen Risk Assessment recently proposed by the U.S. Environmental Protection Agency (EPA) (1996). The study has been designed to generally comply with regulatory requirements and recommendations (OECD guideline 451, 1986; NTP, 1991). There are two major exceptions from these guidelines in the present study design. The use of only two instead of three dose levels is motivated by the comparative nature of the study design; it is not the intention to perform a classical aoo2a27.o1 3118198 3:03pm -2-
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DRAFT Introduction Philip Morris is funding a long-term inhalation study on room-aged sidestream smoke (RASS) and diesel engine exhaust (DEE), currently underway at INBIFO (Institut fiir biologische Forschung, Ko1n, Germany), with the objective of comparing classic and mechanistic endpoints considered to be relevant in experimental carcinogenesis. Appended to this chapter are the following manuscripts prepared and submitted or accepted for publication based on several completed pilot studies: • Haussmann, H: J., Anskeit, E., Becker, D., Kuhl, P., Stinn, W., Teredesai, A., Voncken, P., Walk, R.-A., Comparison of fresh and room-aged cigarette sidestream smoke in a subchronic inhalation study on rats, Toxicol. Sci. 41: 100-116 (1998). [Tab A] • Haussmann, H.-J., Anskeit, E., Gerstenberg, B., Gocke, W., Kuhl, P., Schepers, G., Stabbert, R., Stinn, W., Teredesai, A., Terpstra, P., Tewes, F., Twelve-month inhalation study on room-aged sidestream smoke in rats (Submitted). [Tab B] • Voncken, P., Stinn, W., Haussmann, H.-J., Anskeit, E., Influence of aging and surface contact on the composition of cigarette sidestream smoke -- Models for environmental tobacco smoke, In: Dungworth, D.L., Mauderly, J.L., Oberdorster, G. (Eds.): Toxic and carcinogenic effects ofsolid particles in the respiratory tract, Washington: ILSI Press, ILSI Monographs, pp. 637-641 (1994). [Tab C] 0002427.01 3/18/98 3:03 pm - 1 -
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DRAFT clearance as well as associated inflammation, cell proliferation, and fibrotic changes can also be found in humans, such as coal workers, occupationally exposed to particles Oberdorster, 1995). Deposition of soot particles in the lungs, carbon-loaded alveolar macrophages, and influx of PMNL and lymphocytes were also observed in our subchronic DEE pilot inhalation study. The magnitude of these biomarker responses suggests that the dosing regimen selected would be sufficient to elicit lung tumors in a long-term study. The tumorigenic response to DEE in long-term inhalation studies on rats varies from 4 (spontaneous tumor prevalence) to 54 % lung tumor prevalence at approximately 7 µg particles/1 (Karagianes et al., 1979; Brightwell et al., 1986; Mauderly et al., 1986; Heinrich et al., 1992). This large variance has been mainly attributed to differences in the exposure regimen (Heinrich et al. 1986), but major differences in test atmosphere generation are also probable. For weekly particle doses, a threshold of approximately 120 hours x,ug/1 has been suggested for lung tumorigenicity (Nikula et al., 1995). The lung soot burden is generally considered to be the major determining factor for particle-associated tumor responses. No relevant difference in the lung particle burden or associated effects were found when changing the daily exposure pattern or rate of delivery (Henderson et al., 1992). Thus, the relatively short daily exposure duration applicable to head-only exposure can be compensated by increasing the DEE particle concentrations over those usually used in whole-body exposure studies for longer daily durations. 0002427.01 . 3/18/98 3:03 pm - 12 -
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Nilsson, R., Is Environmental Tobacco Smoke a Risk Factor for Lung Cancer?, In: What Risk?, R. Bate (ed.), Butterworth Heinemann, Oxford: 96-150 (1997) Ozkaynak, H., Xue, J., Spengler, J., Wallace, L., Pellizzari, E., and Jenkins, P., Personal Exposure to Airborne Particles and Metals: Results from the Particle Team Study in Riverside, California, Journal ofExposure Analysis and Environmental Epidemiology, 6(1): 57-78 (1996) Reasor, M.J., and Will, J.A., Assessing Exposure to Environmental Tobacco Smoke: Is It Valid to Extrapolate from Active Smoking?, Journal of Smoking-Related Disease, 2(1): 111-127 (1991) Redhead, C. Stephen, and Rowberg, Richard E., Environmental Tobacco Smoke and Lung Cancer Risk, Congressional Research Service, November 14, 1995 Richter, E., Branner, B., Kutzer, C., Donhaerl, A.M.E., Scherer, G., Tricker, A.R., and Heller, W.-D., Comparison of Biomarkers for Exposure to Environmental Tobacco Smoke: Cotinine and Haemoglobin Adducts from Aromatic Amines and Tobacco-Specific Nitrosamines in Pregnant Smoking and Nonsmoking Women, Proceedings of Healthy Buildings '95, pp. 599-604 (1995) Roberfroid, M.B., Correlating Exposure to Environmental Tobacco Smoke Exposure with Increased Incidence of Lung Cancer in Non Smokers: Is Cotinine a Valid Marker?, The Cancer Journal, 7(3): 108-114 (1994) Rodgman, A., Enviromnental Tobacco Smoke, Regulatory Toxicology and Pharmacology, 16: 223- 244 (1992) Roe, F.J.C., Laboratory Studies in the Prediction of Lung Cancer Risk, Indoor and Built Environment, 5: 196-204 (1996) Romano, G., Mancini, R., et al., Immunohistochemical Analysis of 4-Aminobiphenyl-DNA Adducts in Oral Mucosal Cells of Smokers and Nonsmokers, Anticancer Research, 17: 2827-2830 (1997) Ruppert, T., Scherer, G., Tricker, A.R., and Adlkofer, F., trans, trans-Muconic Acid as a Biomarker of Non-Occupational Environmental Exposure to Benzene, International Archives of Occupational and Environmental Health, 69: 247-251, (1997) Scherer, G., Daube, H., and Adlkofer, F., DNA Adducts as Biomarkers for Exposure to Genotoxic Substances in Tobacco Smoke, Journal of Toxicology and Environmental Health, 40: 460 (1993) Scherer, G., and Richter, E., Biomonitoring Exposure to Environmental Tobacco Smoke (ETS): A Critical Reappraisal, Human and Experimental Toxicology, 16: 449-459 (1997) Scherer, G., Conze, C., Tricker, A.R., and Adlkofer, F., Uptake of Tobacco Smoke Constituents on Exposure to Environmental Tobacco Smoke (ETS), Clinical Investigator, 70: 352-367 (1992) 0159528.01 5
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DRAFT Taking together the foregoing discussion on the applicability of the MTD concept to the present test atmospheres, the dose levels chosen seem to be compatible with the minimally toxic dose concept. Another aspect for setting dose levels is the intention to correlate the responses of selected biomarkers to the lung tumor response. This requires the use of doses which have been shown to be positive in previously published long-term inhalation studies. This rationale fully applies to DEE. It cannot be applied to RASS, since only few data are available on the long-term responsiveness of mice (Witschi et a1.,1995; Witschi and Pinkerton, 1996; Witschi et al., 1997) but none on rats. Maximum mean ETS concentrations in terms of smoke-related, respirable suspended particles (RSP) are reported to be approximately 0.1 F.cg/1 in residences, offices, transportation vehicles, or other places where smoking occurs (EPA, 1992; Guerin et al., 1992). 600-1000 Ag/m3 seems to be the upper limit for the most extreme ETS concentrations reported for all types of occupied spaces. In the present study, the highest RASS particle concentration of 10 µg/1 will exceed extreme human exposure concentrations by a factor of 10 and typical concentrations by a factor higher than 100. For the low dose group of this study, a TPM concentration of 3Ag/1 will be used. As for the planned high RASS concentration, the high DEE concentration in the present study will exceed extreme and normal mean environmental DEE concentrations by factors of 10 and 100, na ' _- respectively (Woskie et a1.,1988; Lehmann, 1991; Pott,1991; Elbers and Muratyan, 1991; Kuhn and 'a" , 4. ~ . Bireft, 1992; projected ambient concentrations by McClellan, 1986; Brightwell et al., 1989). 10 ra 0002427.01 3/18/98 3:03 pm -13- I
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DRAFT different kinetics, the ratio of particle/nicotine mass concentrations changes with time and has thus been considered as a determinant to characterize ETS and fresh SS (Eatough et al., 1990; Nelson et al., 1992; Guerin et al., 1992; Sterling et al., 1996). Average concentration ratios of 4 up to 100 were determined in field studies and 2 to 4 in fresh SS.' This ratio may be distorted at lower ETS concentrations by unaccountable portions of nonsmoke-related particles (dust). In order to approach "reaP"ETS conditions as far as possible, RASS with a mean age of at least 30 min will be used in the long-term study. A mean air residence time of 30 min (corresponding to 2 air changes per hour) can be found in less ventilated rooms. The RASS concentrations (3 and 10 ,ug/1) required for the long-term study may be obtained either by aging SS at the high TPM concentration and subsequent dilution to the lower concentrations or by aging SS separately at each of the RASS concentrations required. As the intention of the long-term study is to investigate a single test atmosphere at two different concentrations, the former approach is preferred. In our previous investigations, the chemical composition of RASS was found to change with the amount and type of surface materials in the aging room (e.g., with paper or wool) N) ~ . O ; ~ ... 1. The ratio indicative of fresh SS was also found in previous subchronic and chronic inhalation .is U] ~ :: _ studies on rats (Adlkofer et al., 1988; von Meyerinck et al., 1989; Coggins et al., 1992; ... m ~ Coggins et al., 1993; Rajini and Witschi, 1994; Witschi et al., 1995). 0002427.01 3/I8/98 3:03pm -5-
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DRAFT was given to substances which are relatively insoluble and of low systemic and respiratory tract toxicity. This workshop concluded that the highest dose should only minimally affect pulmonary clearance. This is in line with the concept of a functionally defined MTD (MFTD) for highest exposure concentrations in long-term bioassays (Muhle et al., 1990a), which should permit the extrapolation of observed biological effects to realistic concentrations to which humans are exposed. The MFTD concept considers biokinetic and mechanistic aspects (NTP Board of Scientific Counselors, 1992). However, more specific recommendations with regard to the type or degree of respiratory tract responses that constitute evidence of an appropriate minimally toxic response or an MFTD were not made (Lewis et al., 1989; Morrow et al., 1996). Especially, the relevance of particle overload in the lungs and the resulting inflammatory response and cell proliferation with regard to risk assessment remains open (Oberdorster, 1995). Altered serum enzyme levels or hematological changes as signs of toxicity were not found in a subchronic SS inhalation study at a TPM concentration of 4,ug/1(Adlkofer et al., 1988). At concentrations of up to 10 ~cg/l, no clinical signs of toxicity were found using "aged and diluted SS" (Coggins et al., 1993). The rationale for the selection of the SS concentration of 4,ug TPM/l in one fully published long-term SS inhalation study was not made quite clear (Witschi et al., 1995). At this concentration, there was no significant effect on body weight. In a previous (pilot?) study performed 0002427.01 3/18198 3:a3 pm - 9 -
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Influence of Aging and Surface Contact on the Composition of Cigarette Sidestream Smoke. Models for Environmental Tobacco Smoke P. VoNcx>,+,' W. STL,\,,' H.-7. Hnuss.wkN*N,' ANn E. A.sxErr' 'ITBIFO Institut fur biologische Forschun_¢. Fuggerstr. 3, D-51 l49 KBIn, Germany 'CRC Contract Research Center, Tollaan l0le, B-1932 Zaventem, Belgium Introduction Environmental tobacco smoke (ETS) is a complex mixture derived from sidestream smoke of burning cigarettes and mainstream smoke exhaled by smokers. Its composition is highly variable in indoor environments as it undergoes chemical/physical changes due to dilution and aging influenced by contact with materials. Due to the discussed health impact of ETS on nonsmok- ers, numerous studies have been performed (Eatough et al. 1990, Guerin et al. 1992), for example, to chemically characterize ETS or to determine its irritative potential. Since it is practically impossible to reproducibly generate "real" ETS, only models of ETS can be used for analytical and biological testing. However, a major problem in developing ETS models is the lack of generally accepted standards for reproducible generation. To obtain information about the influence of aging and surface contact on the composition of cigarette sidestream smoke, fresh sidestream smoke (FSS) was room-aged under vari- ous conditions. because FSS is not considered to bean adequate model for ETS. Method Smoke Generation Diluted FSS, age approximately I second, was continuously generated via a 30-port automatic smoking machine using 2R1 University of Kentucky stan- dard reference cigarettes. Room-aged sidestream smoke (RASS) was gener- ated by continuously passing diluted FSS at a rate of 20 m'/hour through a 30- m' empty experimental room (epoxy-coated walls and ceiling, PVC floor, door, window pane, fluorescent lights, heat exchangers, ceiling fan). The mean age of RASS was 1.5 hours.
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DRAFT standard fuel and lubricant will be used, and international standards for diesel engine operation and the dilution of DEE will be applied (EPA, 1990). Both ETS and DEE are reported in the environment at similar concentrations. The most significant difference in the chemical composition of the two combustion aerosols is the insoluble carbon core of DEE particles, whereas ETS particles are almost completely soluble (Zaebst et al., 1991). Test Atmosphere Generation RASS will be generated from the SS of the standard reference cigarette 1 R4F, a lower yield filter cigarette supplied by the University of Kentucky (Tobacco and Health Research Institute, 1990). The 1R4F is in line with current consumer preference. Dilution and aging of SS involve physicochemical changes (e.g., particle/gas phase distribution and particle size distribution), particle losses due to deposition and adsorption on surfaces, and chemical reactions (Benner et al., 1989; Eatough et al., 1989 and 1990; Baker and Proctor, 1990). In comparison to other SS constituents, nicotine has been shown to interact most readily with surfaces (Neurath et al., 1991; Voncken et al., 1994). To quantify ETS concentrations in field studies, particle and nicotine concentrations have most frequently been determined. Due to oo0z4n.m 3/1&!9s 3:03 pm -4-
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DRAFT The survival of DEE-exposed rats in the study by Heinrich et al. (1995) was not affected, as was the case for female rats in the study by Nikula et al. (1995). However, the male rats in the latter study showed reduced survival in the high dose group. Slight changes in serum clinical chemical and hematological data were reported following DEE inhalation (Ishinishi et a_l., 1986; Lewis et al., 1986). The relevance of these changes is unclear. These parameters have not been regularly investigated in long-term DEE inhalation studies. DEE inhalation at the high doses mentioned above reproducibly resulted in an impairment of the macrophage-associated particle clearance from the lungs, which is connected to a particle overload phenomenon (Proceedings of Symposium on Particle Lung Interactions: Overload Related Phenomena, 1990; HEI, 1995). According to a current hypothesis, the overloaded macrophages attract PMNL to the alveolar lumen as an inflammatory response. This persisting inflammation may ultimately result in hyperplastic and metaplastic changes leading to lung fibrosis and carcinogenesis. The definition of an MTD or MFTD as well as the relevance of tumors arising under particle or other overload conditions for human risk assessment is under discussion. Excessive particle overload as found in high dose long-term DEE inhalation studies is certainly no condition which can be readily extrapolated to the human situation. On the other hand, prolonged particle o00)A27.0i 3/18198 3:03pm -11-
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DRAFT Due to the comparative nature of the present study design as well as the relationship of the doses to reported environmental concentrations, the use of the same particle concentrations for both test atmospheres is considered straightforward. Animal Model Rats, mice, and hamsters have been preferred in carcinogenicity studies "because of their relatively short life span,... their widespread use in pharmacological and toxicological studies, their susceptibility to tumor induction, and the availability of sufficiently characterized strains" (OECD Guideline 451, 1986). The same recommendation was made by Lewis et al. (1989), particularly for aerosol inhalation studies. For head-only exposure, rats are technically more suitable than mice and hamsters. This fact and our long experience with the rat in inhalation studies recommend its use in this long- term inhalation study. The mouse could be considered as a second species for possible future work. In order to facilitate the detection of a low prevalence of induced tumors in the test groups, the spontaneous tumor prevalence in target organs should be as low as possible. The spontaneous prevalence of nasal cavity tumors seems to be negligible (i.e., <1 %) in the three rat strains considered. However, approximately 4-fold spontaneous lung tumor prevalences were 0002427.01 3118/98 3:03 pm - 14 -
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DRAFT to be particularly appropriate for the proposed long-term inhalation study. The suitability of the Wistar rat was corroborated in a recent historical control study. In addition, besides the Fischer 344 rat, the female Wistar rat was used in previous DEE inhalation studies (Heinrich et al., 1986, 1995). Exposure Regimen The head-only or nose-only exposure modes are most appropriate for rat inhalation studies with aerosols (Pauluhn, 1984; Phalen et al., 1984; Hahn, 1993). These modes diminish test substance deposition on the far of the animals and subsequent dermal or oral uptake by grooming, which was observed following whole-body exposure (Wolff et al., 1982; Iwasaki et al., 1988; Mauderly et al. 1989; Chen et al., 1995). In a mainstream cigarette smoke inhalation study, the two exposure modes were compared: plasma and urinary nicotine concentrations were 5- to 6-fold higher in whole-body compared to nose-only exposed rats when normalized to the nicotine concentrations in the test atmospheres (Mauderly et al., 1989). In whole-body exposure, the filtering of the test atmosphere by the fur may impair reproducible uptake by inhalation. In addition, the test atmosphere is in contact with the animal excretion products. In previous studies we found that nicotine and reactive test atmosphere components such as formaldehyde are efficiently trapped by urine and feces. 0002427.01 3118J98 3:03pm -16-
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640 P. Voncken et al. % OF FSS Qan maerqu. EBwnui cenLn K.eam carV6 fQiwpueen Ccc. rnen g1painleC wallpaper maG.nq amne Figure 1. Influence of aging and malerials on FSS. 15, - FSS ~ RASS F10- ~ Q I ~ ,n,..~ j,l~ ~l f W z J U z 5 O 0 s ev-N•A'V 'TAI~Vvll CL a ~w~ a4WA 6IlA 0 : 0 10 20 30 40 50 60 70 80 90 DAY Figure 2. TPM concentrauon.
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DRAFT reported for the Fischer 344 rat (Goodman et al., 1978; Solleveld et al., 1984; Haseman et al., 1985) compared to the Sprague Dawley (MacKenzie and Gamer, 1973; Prejean et al., 1973) and Wistar rat (Vandenberghe, 1990; Kroes et al., 1988; Deerberg et al., 1980, 1982; Rehm et al., 1984; Ueberberg and Luetzen, 1979; Takizawa and Miyamoto, 1976; Boorman and Hollander, 1973; Bombard et al., 1986). Fischer 344 rats have been used in carcinogenicity studies performed by the NTP, a preference which was based on the availability of historical data rather than scientific reasons (Gregory, 1992). Test animals should be exposed to the test material for a major portion of their life span (OECD Guideline 451, 1986). Survival in all groups should be not less than 50 % at 24 months for rats in order for a negative test result to be accepted. In past years, the longevity of laboratory rat strains has decreased substantially, possibly due to breeding targets for rapid growth (White, 1992). The Sprague Dawley rat, which has been used in our previous MS and SS inhalation studies, is no longer considered to fulfill the above requirement (British Society of Toxicological Pathologists, 1992; White, 1992; Mariani et al., 1992). In addition, the increasingly high body weight and associated large size of adult Sprague Dawley rats do not recommend their use in a long- term head-only inhalation study. Based on the reported low spontaneous lung tumor prevalence, the sufficient longevity and the body weight development suitable for head-only exposure, the Wistar rat seems 0002427.01 3A8198 3:03pm -15-
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DRAFT by the same laboratory, cell proliferation was observed in bronchi and bronchioli at 1Ag TPM/1 (Rajini and Witschi, 1994). The same effects as well as cell proliferation in the nasal epithelia were seen during the initial part of the chronic study (4 /.cg TPM/1). Obviously, the dose for the chronic study was selected based on the response in the pilot study of biomarkers considered to be related to carcinogenesis. Taking this approach for the dose selection, i.e., subchronic responses in biomarkers related to carcinogenesis, a RASS dose of 10 µg TPM/1 for the present study would seem to be justified based on the biomarker responses noted above. In a second study, Witschi et al. (1997) used a TPM concentration of roughly 90 µg/1. In the long-term study on female rats performed by Heinrich et al. (1995), the body weight of the high dose DEE group started to deviate significantly from the control on study day 200 (7 ug TPM/1, 18 hours/day, 5 days/week, weekly particle dose: 630 hours x,ug/l). The final body weight difference was 17 %. Nikula et al. (1995) found a body weight gain reduction of approximately 10 % for both sexes following about 180 days of inhalation (6.5 µg TPM/1, 16 hours/day, 5 days/week weekly particle dose: 520 hours x{.cg/1). This body weight gain reduction did not further increase for the male rats but increased to approximately 20 % for the female rats following approximately 500 days of inhalation. Thus, these long-term DEE inhalation studies which were both positive for lung tumors do not meet the historical MTD definition (10 % body N weight gain reduction within 90 days), but are in line with the minimally toxic dose concept (body al 4. weight differences at chronic time points). m' •a 0002427.01 3/l&98 3:03pm . -10-
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638 P. Voncken et al. Experiments In a series of 1-day experiments, the influence on smoke composition of aging alone (empty room) and aging in combination with materials found in rooms was investigated. The following materials were used individually or alto- gether: wool curtain (22 m=), wool carpet (11 m'), wooden book shelf, painted wallpaper (31 m'). To checkthe long-term stabillty of the smokecomposidon, FSS and RASS (all materials present in the room) were generated over 90 days, 7 days/week, 6 hours/day followed by air-flushing for 18 hours. Smoke Analyses Total particulate matter (TPM) was determined gravimetrically using glass fiber filters. Carbon monoxide was monitored continuously by nondispersive infrared photometry of the gas phase. Nicotine was determined by gas chroma- tography, and aldehydes and ammonia were determined by high-performance liquid chromatography after derivatization. Hydrogen cyanide was determined by headspace gas chromatography. For each 1-day experiment, these param- eters were determined at least three times consecutively. During the 90-day experiment the parameters were determined at regular intervals. Results One-Day Experiments Table 1 gives the analytical data for FSS and RASS generated to an equal CO concentration of approximately 30 ppm. In Figure l, the changes in composi- tion of RAS S relative to FSS are shown for those com ponents affected by aging and materials. Aging alone reduced nicotine and TPM to approximately 30% and 70% of their respective FSS concentrations, whereas other components were not affected or were only slightly affected. The individual material together with aging resulting in the most pronounced overall reductions was a wool curtain, with the frnal smoke component concentrations rangi ng between 109o an d 60% of those seen for FSS. Aging with all materials resulted in the strongest reductions, with the final smoke component concentrations ranging between 5% and 45 % of those seen for FSS. Of all com ponents, nic otin e decreased most strongly due to aging alone and ammonia most strongly by aging together with materials. 90-Day Stabiliry Over a period of 90 days, FSS and RASS (all materials present in the room)
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DRAFT to hemoglobin. For RAS S, nicotine metabolites in urine will serve as a specific monitor. If possible, TSNA-derived hemoglobin adducts will also be investigated. In addition, the green autofluorescence of alveolar macrophages is considered for use as an estimate for the steady-state lung particle dose. The feasibility of this parameter has not yet been fully validated. . The determination of the lung burden of inhaled particles or the pulmonary clearance efficacy are mandatory in chronic inhalation studies using particle-containing aerosols (Lewis et al., 1989). For DEE lung burden, nonlinear time-response relationships but linear dose-response relationships were found (McClellan, 1986; Heinrich et al., 1992). The nonlinearity has been attributed to particle overload which impairs pulmonary clearance. For SS-exposed rats, particle overload has not been published. The oxidative modification of deoxyguanosine by forming 8-OHdG is a rather frequent event due to the permanent cellular oxidative stress (Fraga et al., 1990). Therefore, efficient repair mechanisms are set in operation which, in conjunction with the impaired catabolism of the modified base, result in the urinary excretion of 8-OH-dG. Guanidine oxidation reportedly leads to base mispairing, resulting in G:C to T:A transversions (Cheng et al., 1992). In some experimental systems, a correlation between the presence of 8-OH-dG in DNA and tumor development was observed (Floyd, 1990). This endpoint has never been investigated in long-term DEE inhalation 0002427.01 3/ls/9s s:os p. - 20 -
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DRAFT studies to date. In the present study, both the excretion of 8-OH-dG in urine during inhalation as well as the respiratory tract tissue level of this base modification will be investigated. Bulky DNA adducts were observed in a subchronic SS inhalation study at 10 µg TPM/1 in lungs, heart, and larynx tissue using the 32P-postlabeling technique (Lee et al., 1993; Brown et al., 1995). This effect was not observed at lower concentrations. This type of adduct could not be found in ETS-exposed nonsmokers (Scherer et al., 1993) but has been described for smokers (Phillips et al., 1990). DNA adducts were also detected in rat lungs following subchronic DEE inhalation (Bond et al., 1990). Although the 32P-postlabeling technique seems to be the most sensitive method to detect DNA adducts, it lacks specificity. In the present study, DNA adducts will be evaluated. For this purpose, we prefer to use specific mass spectrometric methods which still have to be established in our laboratory. If the latter is not possible, samples will be extramurally analyzed by the 32P-postlabeling technique. Classical histopathology remains a basic endpoint for use in the present study. Apart from the microscopic confirmation and classification of tumors, persistent hyperplastic, metaplastic, and dysplastic tissue changes are considered as essential indicators of preneoplastic and neoplastic lesion induction. In subchronic inhalation studies on SS, hyperplasia and metaplasia of nasal and laryngeal epithelia were found in the rat (von Meyerinck et al., 1989; Coggins et al., 1993; Teredesai and Pruhs, 1994). Due to the general reversibility of these findings after cessation of the SS 0002427.01 3/18198 3:03Pm -21-
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DRAFT conditions due to a complete loss of clearance. No information is available for SS-induced pulmonary tumorigenicity in rats, but Witschi et al. (1997) followed a similar exposure regimen for their A/J mice, i.e., inhalation followed by a postinhalation observation period. For the present study, an inhalation period of 24 months followed by a postinhalation period of 6 months is planned. Size of Experimental Groups Fifty animals per sex and exposure group are considered to be sufficient to adequately evaluate a long-term bioassay (OECD Guideline 451, 1986). Assuming a spontaneous tumor prevalence of 2 %, the 51 rats per group and sex planned for the present study would be sufficient to detect an increased tumor probability of 16 %(Ist order error a = 0.05, 2nd order error (3 = 0.10). This is in the range of the expected lung tumor prevalence in the high dose DEE group based on the comparison of our planned weekly high DEE particle dose with published results (Karagianes et al., 1979; Iwai et al., 1986; Ishinishi et al., 1986; Vallyathan et al., 1986; Mauderly et al., 1987; Brightwell et al., 1989; Heinrich et al., 1986, 1992, and 1995; Nikula et al., 1995). Combining the results from both sexes would even allow detection of an increased tumor probability of 10 %. Mechanistic Endpoints aoo2n2zo1 3118/98 3:03pm -18-
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DRAFT Thus, to enhance the controllability of the test atmosphere administration to the rat as well as the route of the test substance uptake by the rats, the head-only exposure mode will be used in the present study. In inhalation studies, "long-term exposures are usually patterned on projected industrial experience, giving the animals a daily exposure of 6 hours . . . for 5 days a week (intermittent exposure)" (OECD Guideline 451, 1986). In all published DEE inhalation studies with rats, whole-body exposure was used. The daily exposure duration lasted up to 19 hours/day (Heinrich et al., 1986). Head-only exposure should not last longer than 7 hours/day due to the restraint of the rats and other technical reasons. Recently published inhalation studies on man-made vitreous fibers used head-only exposure for 6 hours/day, 5 days/week, 24 months (Smith et al., 1987; Hesterberg et al., 1993). In order to maximize our weekly doses, the present study will be conducted for 6 hours/day and 7 days/week. Depending on the longevity of the rat strain used, it is recommended to terminate carcinogenicity studies after 24 or 30 months of exposure (OECD Guideline 451, 1986). Studies with DEE-exposed rats demonstrated that nearly 80 % of the exposure-related tumors were observed later than 24 months of inhalation (Mauderly et al., 1987). However, for the expression of tumors in this late stage of the rats' lifespan, a continuation of the inhalation period over 24 months does not seem to be necessary, since the particle load will not be substantially cleared under these 000242701 3/IS/9s 3:03 pm - 17 -
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DRAFT cytotoxicity. Increased LDH activity was found in the lavage of DEE-exposed rats (Henderson et al., 1988) and may either be ascribed to alveolar macrophage lysis or to damage to the alveolar epithelium subsequent to particle overload. The determination of lung lavage LDH activity will enhance the interpretation of data in the present study. 0002427.01 3/18/98 3:03pm -25-
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DRAFT (Carbone and Ivlinna, 1992), was not considered for the present study since the rat seems to be less sensitive to ras mutations by agents positive in the mouse and hamster such as N-nitrosamines (Belinsky et al., 1990). Activation of cell proliferation, in conjunction with changes at the DNA level, is considered to be essential for initiation and tumor development. Cell division is necessary for conversion of adducts or DNA strand breaks to mutations or gaps, and also allows for mitotic recombination. However, the direct correlation between increased cell proliferation and development of neoplasia in target organs or morphological sites has been questioned (Yoshida et al., 1993). Cell proliferation, which does not give rise to formation of neoplasia, may simply be induced by the cytotoxicity of the test material in the absence of initiation. On the other hand, persistent cell proliferation may increase the probability of converting spontaneous DNA lesions to neoplastic changes. In subchronic inhalation studies on aged and diluted SS, an increased incorporation of BrdU into the rat nasal respiratory epithelium was found at a concentration of 10 Mg TPM/1(Brown et al., 1995). Following a shorter period (5 days) of inhalation, the effect was already seen at I µg/1. A similar pattern of response was found for the A/7 mouse at concentrations of 1 and 4 Fcg TPM/1(Rajini and Witschi, 1994; Witschi et al., 1995), whereas the C57BI/6 mouse did not respond. Following DEE inhalation, a transient initial increase in rat lungs was described by Wright (1986). Following chronic DEE inhalation, increased cell proliferation measured by 3H- thymidine incorporation was detected in the bronchi and bronchioli as well as at particular sites in 0002427.01 3/I8/98 3 :03pm -23-
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DRAFT inhalation, these changes have been considered an adaptive response to the irritating activity of the test atmosphere (Burger et al., 1989). Mutations are a prerequisite for initiating carcinogenesis, and most probably also play a role in epigenetically induced carcinogenesis such as by particle overload (Driscoll et al., 1996). Apart from the latter study, there is limited expertise in the detection of early mutations in rat long- term studies. The single-strand DNA conformation assay can be used to detect unknown mutations. Recently, we have been able to increase the sensitivity of this assay by several orders of magnitude. The attempt to further increase the assay sensitivity was limited by the fidelity of the DNA polymerase used in the polymerase chain reaction to amplify DNA probes. The present assay sensitivity is not considered to be sufficient enough to detect early mutations to fulfill the function of an intermediate biomarker. However, the assay will contribute to tumor differentiation in the final part of this study. Emphasis will be given to mutations in the p53 tumor suppressor gene. Mutations of this gene can be found in about half of all human cancer cases. The location and characteristics of these mutations may reveal clues about their etiology. The predominant base changes inp53 in human lung cancers (G:C to T:A transversions) were suggested to be indicative of causal lesions on the nontranscribed DNA strand by polycyclic aromatic hydrocarbons (Harris, 1993). Possible correlations ofp53 mutations with cigarette smoking have recently been discussed (Suzuki et al., 1992; Spruck et al., 1993; Habuchi et al., 1993; Brennan et al., 1995). The mutational activation of the protooncogene ras, another endpoint frequently associated with lung tumor development w. N .. 0002427.01 3/18/98 3:03 pm - 22 -
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DRAFT In evaluating the carcinogenic risk of test materials to humans or establishing relative potencies of toxicity and carcinogenicity between species or test materials, increasing emphasis is being placed on mechanistic investigations (NTP Board of Scientific Counselors, 1992; IARC Working Group, 1992; EPA, 1996). Of special interest are preneoplastic changes that might be present before tumor manifestation or at concentrations lower than those which would be required for tumor development. These mechanistic investigations may comprise studies on pharmacokinetics, including target organ dose monitoring; genotoxicity including mutations and repair mechanisms; cell proliferation; cell differentiation; immunosuppression; and inflammatory or fibrotic processes. Especially for particle inhalation studies, deposition and/or clearance studies are recommended (Lewis et al., 1989). In the present study, in addition to classical pathology, several endpoints are planned to be investigated which are thought to be mechanistically related to chronic disease and chemical carcinogenesis in the respiratory tract. The reasons for choosing these endpoints is the current mechanistic understanding of the processes under investigation as well as in-house scientific expertise. The extent of these investigations is limited by the available number of rats in a long-term head-only inhalation study. 0 Biomonitoring for both RASS and DEE will include the determination of the m w o' = ~ carboxyhemoglobin proportion in the blood and of the steady-state content of aminobiphenyl adducts w. N 0002427.01 3/18/98 3:03pm -19-
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DRAFT rat lungs (McClellan et al., 1986). Cell proliferation will be investigated in the present study using BrdU incorporation. Cytokeratins have been used for the differential characterization of preneoplastic and neoplastic changes in epithelial tissues (Broers et al., 1988; Moll et al., 1988; Lindberg and Rheinwald, 1989; Schaafsma et al., 1990; Smedts et al., 1990). In contrast to the well-defined human cytokeratin expression patterns, those of the rat have been less extensively characterized. Alterations in cytokeratin expression were found to precede the histological expression of squamous metaplasia in several epithelial tissues in vitamin A-deficient rats (Gijbels et al., 1992). Recently, rat lung tumor types could be differentiated using monoclonal antibodies to human cytokeratins (Kal et al., 1993). Inflammatory and fibrotic processes have often been associated with particle overload and carcinogenesis (Heinrich et al., 1986; Henderson et al., 1988; Muhle et al., 1990b; Morrow, 1992; Oberdorster, 1995). The PMNL proportion of lavagable bronchoalveolar cells as a sign of persistent acute inflammation was found to increase time- and dose-dependently following chronic exposure to DEE or toner. Lactate dehydrogenase (LDH) activity is a cellular enzyme which is commonly determined extracellularly, e.g., in the supematant of lung lavage, as a measure for cell lysis or 0002427.01 3/18198 3:03 pm -24- I
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DRAFT Broers, J.L.V., Ramaekers, F.C.S., Klein Rot, M., Oostendorp, T., Huijsmans, A., van Muijen, G.N.P., Wagenaar, SjSc., Vooijs G.P., Cytokeratins in different types of human lung cancer as monitored by chain-specific monoclonal antibodies, Cancer Res. 48: 3221-3229 (1988). Brown, B.G., Bombick, B.R., McKarns, S.C., Lee, C.K., Ayres, P.H., Doolittle, D.J., Molecular endpoints in rodent inhalation studies, Exp. Toxicol. Pathol. 47: 183-191 (1995). Burger, G.T., Renne, R.A., Sagartz, J.W., Ayres, P.H., Coggins, C.R.E., Mosberg,A.T., Hayes, A. W., Histologic changes in the respiratory tract induced by inhalation of xenobiotics: Physiologic adaption or toxicity? Toxicol. App. PharmacoL 101: 521-542 (1989). Carbone, D.P., Minna, J.D. (Eds): The molecular genetics of lung cancer, Adv. Int. Med 37: 153- 171 (1992). Chen, B.T., Benz, J.V., Finch, G.L., Mauderly, J.L., Sabourin, P.J., Yeh, H.C., Snipes, M.B., Effect of exposure mode on amounts of radiolabeled cigarette particles in lungs and gastrointestinal tract of F344 rats, Inhalation Toxicol. 7: 1095-1108 (1995). Cheng, K.C., Cahill, D.S., Kasai, H., Nishimura, S., Loeb, L.A., 8-Hydroxyguanine, an abundant form of oxidative DNA damage, causes G-> T and A-> C substitutions, J. Biol. Chem. 267(1): 166 (1992). Coggins, C.R.E., Ayres, P.H., Mosberg, A.T., Ogden, M.W., Sagartz, J.W., Hayes, A.W., Fourteen- day inhalation study in rats, using aged and diluted sidestream smoke from a reference cigarette. I. Inhalation toxicology and histopathology, Fundam. Appl. Pharmacol. 19: 133-140 (1992). Coggins, C.R.E., Ayres, P.H., Mosberg, A.T., Sagartz, J.W., Hayes, A.W., Subchronic inhalation study in rats using aged and diluted sidestream smoke from a reference cigarette, Inhalation Toxicol. 5: 77-96 (1993). Deerberg, F., Rapp, K.G., Pittermann, W., Rehm, S., Zum Tu_morspektrum Han: WIST-Ratte, Z. Vesuchstierkd. 22: 267-280 (1980). Deerberg, F., Rapp, K.G. Rehm, S., Mortality and pathology of Han: WIST rats depending on age genetics, Exp. Biol. Med. 7: 63-71 (1982). Driscoll, K.E., Carter, J.M., Howard, B.W., Hassenbein, D.G., Pepelko, W., Baggs, R.B., OberdSrster, G., Pulmonary inflammatory, chemokine, and mutagenic responses in rats after subchronic inhalation of carbon black, Toxicol. AppL Pharmacol. 136: 372-380 (1996). 0002427.01 3/18/98 3:03 pm - 27 -
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DRAFT Klingenberg, H., Schinmann, D., Lies, K.-H., Dieselmotorabgas -- Entstehung und Messung, VDI Berichte Nr. 888: 119-131 (1991). Kroes, R, Garbis-Berkvens, J.M., de Vries, T., van Nesselrooy, J.H.J., Histopathological profile of a Wistar rat stock including a survey of the literature, J. Gerontol. 36: 259-279 (1988). Kiihn and Bireft, TRK-Wert fiir Dieselmotoremissionen, In: Merkblatter Gefahrliche Arbeitsstoffe, 61, Erg. Lfg. 9192, pp. 93-96. Lee, C.K., Brown, B.G., Reed, E.A., Coggins, C.R.E., Doolittle, D.J., Hayes, A.W., 90-Day inhalation study on rats, using aged and diluted sidestream smoke from a reference cigarette -- DNA adducts and alveolar macrophage cytogenetics, Fundam. Appl. Toxicol. 20: 393-401 (1993). Lehmann, E., Berufliche Exposition gegeniiber Dieselabgas, VDIBerichte Nr. 888: 133-142 (1991). Lewis, T.R., Green, F.H.Y., Moorman, W.J., Burg, J.A.R., Lynch, D.W., A chronic inhalation toxicity study of diesel engine emissions and coal dust, alone and combined, In: Ishinishi, N., Koizumi, A., McClellan, R.O., Stoeber, W. (Eds.): Carcinogenic and mutagenic effect of diesel engine exhaust, Amsterdam: Elsevier, pp. 361-380 (1986). Lewis, T.R., Morrow, P.E., McClellan, R.O., Raabe, O.B., Kennedy, G.L., Schwetz, B.A., Goehl, T.J., Roycroft, J.H., Chhabra, R.S., Establishing aerosol exposure concentrations for inhalation toxicity studies, Toxicol. Appl. Pharmacol. 99: 377-383 (1989). Lindberg, K., Rheinwald, J.G., Suprabasal 40kd keratin (K19) expression as immunohistologic marker of premalignancy in oral epithelium, Am. J. Pathol. 134: 89-98 (1989). MacKenzie, W.F., Garner, F.M., Comparison of neoplasms in six sources of rats, J. Natl. Canc. Inst. 50: 1243-1247 (1973). Mariani, M.F., Eileraas, M., Argentino-Storino, A., Hill, M., Brightwell, J., Survival rate and spontaneous pathology occurring in Sprague-Dawley CD rats in 24-month studies, Abstracts of the 6th Int. Cong. of Toxicology, Rome, 1992, Toxicol. Lett. Suppl.: 1-356 (1992). Mauderly, J.L., Jones, R.K., McClellan, R.O., Henderson, R.F., Griffith, W.C., Carcinogenicity of diesel exhaust inhaled chronically by rats, In: Ishinishi, N., Koizumi, A., McClellan, R.O., Stoeber, W. (Eds.): Carcinogenic and mutagenic effect of diesel engine exhaust, Amsterdam: Elsevier, pp. 397-409 (1986). m 0002427.01 3/18/98 3:03pm - -31-
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DRAFT References Adlkofer, F., Scherer, G., Wenzel-Hartung, R., Brune, H., Thomas, C., Exposure of hamsters and rats to sidestream smoke of cigarettes: Preliminary results of a 90-day-inhalation study, In: Perry, R., Kirk, P.W.W. (Eds.): Indoor and ambient air quality, London: Selper, pp. 252-258 (1988). Baker, R.R., Proctor, C.J., The origins and properties of environmental tobacco smoke, Environ. Int. 16: 231-245. Belinsky, S.A., Devereux, T.R., Anderson, M.W., Role of DNA methylation in the activation of proto-oncogenes and the induction of pulmonary neoplasia by nitrosamines, Mutat. Res. 233: 105- 116 (1990). Benner, C.L., Bayona, J.M., Caka, F.M., Tang, H., Lewis, L., Crawford, J., Lamb., J.D., Lee, M.L., Lewis, E.A., Hansen, L.D., Eatough, D.J., Chemical composition of environmental tobacco smoke. 2. Particulate-phase compounds, Environ. Sct. Technol. 23: 688-699. Bomhard, E., Karbe, E., Loeser, E., Spontaneous tumors of 2000 Wistar TNO/W.70 rats in two-year carcinogenicity studies, J. Environ. Pathol. Toxicol. Oncol. 7: 35-52 (1986). Bond, J.A., Mauderly, J.L., Wolff, .K., Concentration-dependent and time-dependent formation of DNA adducts in lungs of rats exposed to diesel exhaust, Toxicology 60: 127-135 (1990). Boorman, G.A., Hollander, C.F., Spontaneous lesions in the female WAG/Rij (Wistar) rat, J. Gerontol. 28: 152-158 (1973). Brennan, J.A., Boyle, J.O., Koch, W.M., Goodman, S.N., Hruban, R.H., Eby, Y.J., Couch, M.J., Forastiere, A.A., Sidransky, D., Assocation between cigarette smoking and mutation of the p53 gene in squamous-cell carcinoma of the head and neck, N. Engl. J. Med. 332: 712-717 (1995). Brightwell, J., Fouillet, X., Cassano-Zoppi, A: L., Gatz, R., Duchosal, F., Neoplastic and functional changes in rodents after chronic inhalafion of engine exhaust emissions, In: Ishinishi, N., Koizumi, A., McClellan, R.O., Stoeber, W. (Eds.): Carcinogenic and mutagenic effect of diesel engine exhaust, Amsterdam: Elsevier, pp. 471-485 (1986). Brightwell, J., Fouillet, X., Cassano-Zoppi, A: L., Bernstein, D., Crawley, F., Duchosal, F., Gatz, R., Perezel, S., Pfeifer, H., Tumours of the respiratory tract in rats and hamsters following chronic inhalation of engine exhaust emissions, J Appl. Toxicol. 9 (1): 23-31 (1989). British Society of Toxicological Pathologists (Eds.), Newsletter, September 1992. 0002427.01 3118/98 3:03 pm - 26 -
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DRAFT Mauderly, J.L., Jones, R.K., Griffith, W.C., Henderson, R.F., McClellan, R.O., Diesel exhaust is a pulmonary carcinogen in rats exposed chronically by inhalation, Fundam. Appl. Toxlcol. 9: 208-221 (1987). Mauderly, J.L., Bechthold, W.E., Bond, J.A., Brooks, A.L., Chen, B.T., Cuddhy, R.G., Harkema J.R., Henderson, R.F., Johnson, N.F., Rithidech, K., Thomassen, D.G., Comparison of 3 methods of exposing rats to cigarette smoke, Exp. Pathol. 37: 194-197 (1989). McClellan, R.O., Health-effects of diesel exhaust: A case-study in risk assessment, Am. Ind Hyg. Assoc. J. 47: 1-13 (1986). McClellan, R.O., Bice, D.E., Cuddihy, R.G., Gillett, N.A., Henderson, R.F., Jones, R.K., Mauderly, J.L., Pickrell, J.A., Shami, S.G., Wolff, R.K., Health effects of diesel exhaust, In: Lee, S.D., Schneider, T., Grant, L.D., Verkerk, P.J. (Eds.): Aerosols. Research, risk assessment and control strategies, Chelsea, MI: Lewis Publishers, pp. 597-615 (1986). Moll, R., Achtstatter, T., Becht, E., Balcarova-Stander, J., Ittensohn, M., Franke, W.W., Cytokeratins in normal and malignant transitional epithelium, maintenance of expression of urothelial features in traditional cell carcinomas and bladder carcinoma cell culture lines, Am. J. Pathol. 132: 1124-1144 (1988). Morrow, P.E., Contemporary issues in toxicology. Dust overloading of the lungs: Update and appraisal, Toxicol. App1. Pharmacol. 113: 1-12 (1992). Morrow, P.E., Hasemann, J.K., Hobbs, C.H., Driscoll, K.E., Vu, V., Oberdorster, G., The maximum tolerated dose for inhalation bioassays: Toxicity vs. overload, Fundam. Appl. Toxicol. 29: 155-167 (1996). Muhle, H., Bellmann, B., Creutzenberg, 0., Fuhst, R., Kilpper, R., Koch, W., MacKenzie, J.C., Morrow, P., Mohr, U., Takansaka, S., Mermelstein, R., Subchronic inhalation of toner in rats, Inhalation Toxicol. 2: 341-360 (1990a). Muhle, H., Creutzenberg, 0., Bellmann, B., Heinrich, U., Mermelstein, R., Dust overloading of lungs: Investigations of various materials, species differences, and irreversibility of effects, J. of Aerosol Med 3 (Supplement 1): 111 (1990b). National Toxicology Program (NTP), Specifications for the conduct of studies to evaluate the toxic and carcinogenic potential of chemical, biological and physical agents in laboratory animals for the National Toxicology Program, Research Triangle Park, NC: National Toxicology Program (1991). - 0002427.01 3/18/98 3:03pm -32-
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DRAFT Henderson, RF., Barr, E.B., Cheng, Y.S., Griffith, W.C., Hahn, F.F., The effect of exposure pattern on the accumulation of particles and the response of the lung to inhaled particles, Fundam. Appl. Toxicol. 19: 367-374 (1992). Henderson, R.F., Pickrell, J.A., Jones, RK., Sun, J.D., Benson, J.M., Mauderly, J.L., McClellan, R.O., Response of rodents to inhaled diluted diesel exhausts: Biochemical and cytological changes in bronchoalveolar lavage fluid and lung tissue, Fundam. Appl. Toxicol. 11: 546-567 (1988). Hering, S.V., Comparison of sampling methods, Aerosol Sci. Technol. 12: 200-213 (1990). Hesterberg, T.W., Miller, W.C., McConell, E.E., Chevalier, J., Hadley, J.G., Bernstein, D.M., Thevenaz, P., Anderson, R., Chronic inhalation toxicity of size-separated glass fibers in Fischer 344 rats, Fundam. Appl. Toxicol. 29: 464-476 (1993). International Agency for Research on Cancer, Working group on mechanisms of carcinogenesis and the evaluation of carcinogenic risks, Cancer Res. 52: 2357-2361 (1992). Ishinishi, N., Kuwabara, N., Nagase, S., Suzuki, T., Ishiwata, S., Kohno, T., Long-term inhalation studies of effects of exhaust from heavy and light duty diesel engines on F344 rats, In: Ishinishi, N., Koizumi, A., McClellan, R.O., Stoeber, W. (Eds.): Carcinogenic and mutagenic effect of diesel engine exhaust, Amsterdam: Elsevier, pp. 329-348 (1986). Iwai, K., Udagawa, T., Yamagashi, M., Yamada, H., Long-term inhalation studies of diesel exhaust on F344 spf rats. Incidence of lung cancer and lymphoma, In: Ishinishi, N., Koizutni, A., McClellan, R.O., Stoeber, W. (Eds.): Carcinogenic and mutagenic effect of diesel engine exhaust, Amsterdam: Elsevier, pp. 349-360 (1986). Iwasaki, M., Yoshida, M., Ikeda, T., Tsuda, S., Shirasu, Y., Comparison of whole-body versus snout-only exposure in inhalation toxicity of fenthion, Jpn. J. Vet. ScL 50(1): 23-30 (1988). Kal, H.B., van Berkel, A.H., Broers, J.L.V., Klein, J.C., Munheere, E.P., Roholl, P.J.M., Zurcher, C., Ramaekers, F.C.S., Cytokeratins expressed in experimental rat bronchial carcinomas, Int. J. Cancer 53: 506-513 (1993). Karagianes, M.T., Palmer, R.F., Busch, R.H., Phelps, D.W., Frazier, M.E., McClanahan, B.J., Pelroy, R.A., Inhalation hazard to coal miners: Biological effects of chronic inhalation of coal mine dust and/or diesel engine exhaust in rodents, In: Battelle Pacific Northwest Laboratory Annual Reportsfor 1979 to the US. Department ofEnergy, P.1. Biomed. Sci. Pn1-3300, Uc-48: 1-3 (1979). 0002427.01 3/18/98 3:03pm -30-
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DRAFT Eatough, D.J., Benner, C.L., Bayona, J.M., Richards, G., Lamb, J.D., Lee, M.L., Lewis, E.A., Hansen, L.D., Chemical composition of environmental tobacco smoke. 1. Gas-phase acids and bases, Environ. Scr. Technol 23: 679-687 (1989). Eatough, D.J., Hansen, L.D., Lewis, E.A., The chemical characterization of environmental tobacco smoke, Environ. Technol. 11: 1071-1085 (1990). Elbers, G., Muratyan, S., Problematik verkehrsbezogener Aul3enluftmessungen von Partikeln (Dieselruli), VDI Berichte Nr. 888: 143-170 (1991). Environmental Protection Agency (EPA) (Eds.), Control of air pollution from new motor vehicles and new motor vehicle engines: Certification and test procedures, Code of Federal Regulations, Vol. 40, Part 86, 1990. Environmental Protection Agency (EPA) (Eds.), Respiratory health effects of passive smoking: Lung cancer and other disorders, Washington, D.C.: Office of Research and Development (1992). Environmental Protection Agency (EPA) (Eds.), Health assessment document for diesel emissions: External review draft, EPA/600/8-90/057, Office of Research and Development, Washington (1994). Environmental Protection Agency (EPA) (Eds.), Proposed guidelines for carcinogen risk assessment, EPA/600/P-92/0003C, Office of Research and Development, Washington (1996). First, M., Constituents of sidestream and mainstream tobacco smoke and makers to quantify exposure to them, In: Gammage, R.B., Kaye, S.V (Eds.): Indoor air and human health, Chelsea, MI: Lewis, pp. 195-203 (1985). Floyd, R.A., The role of 8-hydroxyguanine in carcinogenesis, Carcinogenesis 11(9): 1447-1450 (1990). Fraga, C.G., Shigenaga, M.K., Park, J-W., Degan, P., Ames, B.N., Oxidative damage to DNA during aging: 8-hydroxy-2'-deoxyguanosine in rat organ DNA and urine, Proc. Natl. Acad Scf. 87: 4533- 4537 (1990). Gijbels, M.J.J., van der Ham, F., van Bennekum, A.M., Hendrix, H.F.J., Roholl, P.J.M., Alterations in cytokeratin expression precede histological changes in epithelia of vitamin A-deficient rats, Cell Tissue Res. 268: 197-203 (1992). Goodman, D.G., Ward, J.M., Squire, R.A., Chu, K.C., Linhart, M.S., Neoplastic and nonneoplastic lesions in aging F344 rats, Toxicol Appl Pharmacol. 48: 237-248 (1978). 0002427,01 3/l8/98 3:03pm -28-
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DRAFT National Toxicology Program Board of Scientific Counsellors, NTP Advisory Review, Federal Register 57 (No. 138): 31721 (1992). Nelson, P.R., Heavner, D.L., Collie, B.B., Maiolo, K.C., Ogden, M.W., Effect of ventilation and sampling time on environmental tobacco smoke component ratios, Environ. Sci. Technol. 28(10): 1909 (1992). Neurath, G.B., Petersen, S., Dunger, M., Orth, D., Pein, F.G., Gas-particulate phase distribution and decay-rates of constituents in aging environmental tobacco smoke, Environ. Technol. 12: 581-590 (1991). Nikula, K.J., Snipes, M.B., Barr, E.B., Griffith, W.C., Henderson, R.F., Mauderly, J.L., Comparative pulmonary toxicities and carcinogenicities of chronically inhaled diesel exhaust and carbon black in F344 rats, Fundam. Appl. Toxicol. 25: 80-94 (1995). Oberdorster, G., Lung particle overload: Implications for occupational exposures to particles, Regul. Toxicol. Pharmacol. 21: 123-135 (1995). OECD Guideline 451, Carcinogenicity studies, In: OECD guidelines for testing of chemicals, Paris: Organization for Economic Co-operation and Development (1986). Pauluhn, J., Head-only and nose-only exposure, BGA-Schriften 5 (1984). Phalen, R.F., Mannix, R.C., Drew, R.T., Inhalation exposure methodology, Env. Health Perspect. 56: 23-34 Phillips, D.H., Schoket, B., Hewer, A., Bailey, E., Kostic, S., Vincze, L, Influence of cigarette smoking on the levels of DNA adducts in human bronchial epithelium and white blood cells, Int. J Cancer 46: 569-575 (1990). Pott, F., Dieselmotorabgas -- tierexperimentelle Ergebnisse zur Risikoabschatzung, VDI Berichte Nr. 888: 211-244 (1991). Prejean, J.D., Peckham, J.C., Casey, A.E., Griswold, D.P., Weisburger, E.K., Weisburger, J.H., Spontaneous tumors in Sprague Dawley rats and Swiss mice, Cancer Res. 33: 2768-2773 (1973). T Proceedings of Symposium on Particle Lung Interactions, Overload related phenomena, J. Aerosol. Med. 3: S-1 - S-207 (1990). 0002427.01 3/18/98 3:03pm -33-
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Gregory, A.R., Cancer risk -- Does anyone really care? Regul. Toxicol. PharmacoL 15: 271-277 (1992). Guerin, MR., Jenkins, R.A., Tomkins, B.A., The chemistry of environmental tobacco smoke: Composition and measurement, Boca Raton: Lewis Publishers (1992). Habuchi, T., Takahashi, R., Yamada, H., Ogawa, 0., Kakehi, Y., Ogura, K., Hamazaki, S., Toguchida, J., Ishizaki, K., Fujita, J., Sugiyama, T., Yoshida, 0., Influence of cigarette smoking and schistosomiasis on p53 gene mutation in urothelial cancer, Cancer Res. 53: 3795-3799 (1993). Hahn, F.F., Chronic inhalation bioassays for respiratory tract carcinogenesis, In: Gardner, D.E., (Ed.): Toxicology ofthe lung, 2nd ed., New York: Raven Press (1993). Harris, C.C., p53: At the crossroads of molecular carcinogenesis and risk assessment, Science 262: 1980 (1993). Haseman, J.K., Huff, J.E., Rao, G.N., Arnold, J.E., Boorman, G.A., McConnell, E.E., Neoplasms observed in untreated and corrt oil gavage control groups of F344/n rats and (C57B1/6n x C3h/HEN)fl (B6C3fl) mice, J. Natl. Cancer Inst. 75: 975-984 (1985). Health Effects Institute, Diesel exhaust: A critical analysis of emissions, exposure, and health effects. A special report ofthe Institute's diesel working group, Cambridge (1995). Heinrich, U., Muhle, H., Takenaka, S., Ernst, H., Fuhst, R., Mohr, U., Pott, F., Stoeber, W., Chronic effects on the respiratory tract of hamsters, mice and rats after long-term inhalation of high concentrations of filtered and unfiltered diesel engine emissions, J. Appl. Toxicol. 6: 383-395 (1986). Heinrich, U., Fuhst, R., Mohr, U., Tierexperimentelle Inhalationsstudien zur Frage der tumorinduzierenden Wirkung von Dieselmotorabgasen und zwei Teststauben, In: Bundesminister fiir Forschung und Technologie (Eds.): Auswirkungen von Dieselmotorabgasen auf dfe Gesundheit, Arbeitsschwerpunkt im Rahmen des Programmes "Umweltforschung und Umwelttechnologie," Forderschwerpunkt "Umweltbelastung und Gesundheit" (1992). Heinrich, U., Fuhst, R., Rittinghausen, S., Creutzenberg, 0., Bellman, B., Koch, W., Levsen, K., o_ Chronic inhalation exposure of Wistar rats and two different strains of mice to diesel engine exhaust, ~,- carbon black, and titanium dioxide, Inhalation Toxicol. 7: 533-556 (1995). .t~ :- w °- rv m ..` 0002427.01 3/18198 3:03 pm - 29 -
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DRAFT Witschi, H., Oreffo, V.I.C., Pinkerton, K.E., Six-month exposure of strain A/J mice to cigarette sidestream smoke: Cell kinetics and lung tumor data, Fundam. App. ToxicoL 26: 32-40 (1995). Wolff, R.K., Griffis, L.C., Hobbs, C.H., McClellan, R.O., Deposition and retention of 0.1 mm 67 aggregate aerosols in rats following whole body exposures, Fundam. App. ToxicoL 2: 195-200 (1982). Woskie, S.R., Smith, T.J., Hammond, S.K., Schenker, M.B., Garshick, E., Spiezer, F.E., Estimation of the diesel exhaust exposures of railroad workers: I. Current exposures, Am. J. Ind. Med 13: 3 8 1 - 394(1988). Wright, E.S., Effects of short-term exposure to diesel exhaust on lung cell proliferation and phospholipid metabolism, Exp. Lung Res. 10: 39-55 (1986). Yoshida, Y., Tatematsu, M., Takaba, K., Iwasaki, S., Ito, N., Target organ specificity of cell proliferation induced by various carcinogens, Toxicol. Pathol. ISSN 21 (5): (1993). Zaebst, D.D., Clapp, D.E., Blade, L.M., Marlow, D.A., Steenland, K., Hornung, R.W., Scheutzle, D., Butler, J., Quantitative determination of trucking industry workers' exposure to diesel exhaust particles, Am. Ind. Hyg. Assoc. J 52: 529-541 (1991). aoaz427.o1 3/18/98 3:03prn -36-
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DRAFT Rajini, P., Witschi, H., Short-term sidestream smoke on respiratory epithelium in mice: Cell kinetics, Fundam. Appl. Pharmacol. 22: 405-410 (1994). Rehm, S., Deerberg, F., Rapp, K.G., A comparison of life-span and spontaneous tumor incidences of male and female Han: WIST virgin and retired breeder rats, Lab. Anim. Sci. 34: 458-464 (1984). Schaafsma, H.E., Ramaekers, F.C.S., van Muijen, G.N.P., Lane, E.B., Leigh, I.M., Robben, H., Huijsmans, A., Ooms, E.C.M., Ruiter, D.J., Distribution of cytokeratin polypeptides in human transitional cell carcinomas, with special emphasis on changing expression patterns during tumor progression, Am. J. Pathol. 136: 329-343 (1990). Scherer, G., Daube, H., Adlkofer, F., DNA adducts as biomarkers for exposure to genotoxic substances in tobacco smoke, J. Toxicol. Environ. Health 40: 460-460 (1993). Smedts, F., Ramaekers F.C.S., Robben, H., Pruszczynski, M., van Muijen, G.N.P., Lane, E.B., Leigh, I.M., Vooijs, G.P., Changing patterns of keratin expression during progression of cervical intraepithelial neoplasia, Am. J. Pathol. 136: 657-668 (1990). Smith, et al., Long-term health effects in hamsters and rats exposed chronically to man-made vitreous fibres, Ann. Occup. Hyg. 31(48): 731-754 (1987). Solleveld, H.A., Haseman, J.K., McConnell, E.E., Natural history of body weight gain, survival, and neoplasia in the F344 rat, J. Natl. Cancer Inst. 72: 929-940 (1984). Spruck, C.H., Rideout, W.M., Olumi, A.F., Ohneseit, P.F., Yang, A.S., Tsai, Y.C., Nichols, P.W., Horn, T., Hermann, G.G., Steve, K., Ross, R.K., Yu, M.C., Jones, P.A., Distinct patttern of p53 mutations in bladder cancer: relationship to tobacco usage, Cancer Res. 53: 1162-1166 (1993). Sterling, E.M., Collett, C. W., Ross, J.A., Assessment of non-smokers exposure to environmental tobacco smoke using personal-exposure and fixed-location monitoring, Indoor Built Environ. 5: 112- 125 (1996). Suzuki, H., Takahashi, T., Kuroishi, T., Suyama, M., Ariyoshi, Y. Takahashi, T., Ueda, R., p53 Mutations in non-small cell lung cancer in Japan: Association between mutations and smoking, Cancer Res. 52: 734-73 6 (1992). N O ~ o~ ~ Takizawa, S., Miyamoto, M., Observations on spontaneous tumours in Wistar Furth strain rats, HiroshimaJ. Med Sct. 25: 89-98 (1976). . w N w 0002427.01 3/18/98 3:03 pm - 34 -
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DRAFT Teredesai, A., PrUhs, D., Histopathological findings in the rat and hamster respiratory tract in a 90- day inhalation study using fresh sidestream smoke of the standard reference cigarette 2R1, In: Dungworth, D.L., Mauderly, J.L., OberdSrster, G. (Eds.): Toxic and carcinogenic effects of solid particles in the respiratory tract, Washington: ILSI Press, ILSI Monographs, pp. 629-635 (1994). Tobacco and Health Research Institute, The reference cigarette, Lexington: The University of Kentucky Printing Services (1990). Ueberberg, H., Lfltzen, L., The spontaneous rate of tumours in the laboratory rat: Strain Chbb: THOM (SPF), Arzneim. Forsch./Drug Res. 29: 1976-1979 (1979). Vallyathan, V., Virmani, R., Rochlani, S., Green, F.H.Y., Lewis, T., Effect of diesel emissions and coal dust inhalation on heart and pulmonary arteries of rats, J. Toxicol. Environ. Health 19: 33-41 (1986). Vandenberghe, J., Life-span data and historical data in carcinogenicity testing in Wistar rats Crl:(WI)BR, Charles River Deutschland (1990). von Meyerinck, L., Scherer, G., Adlkofer, F., Wenzel-Hartung, R., Brune, H., and Thomas, C., Exposure of rats and hamsters to sidestrearn smoke from cigarettes in a subchronic inhalation study, Exp. Pathol. 37: 186-189 (1989). Voncken, P., Stinn, W., Haussmann, H.-J., Anskeit, E., Influence of aging and surface contact on the composition of cigarette sidestrearn smoke -- Models for environmental tobacco smoke, In: Dungworth, D.L., Mauderly, J.L., Oberdorster, G. (Eds.): Toxic and carcinogenic effects of solid particles in the respiratory tract, Washington: ILSI Press, ILSI Monographs, pp. 637-641 (1994). Watts, W.F., Assessment of occupational exposure to diesel emissions, In: Health Effects Institute (Ed.): DiesQl exhaust: A critical analysis of emissions, exposure, and health effects. A special report of the Institute's diesel working group, Cambridge, pp. 107-123 (1995). White, W.J., Issues in Toxicology: Longevity in rats, 6th Charles River Short Course, Bad Zwischenahn, October 22-23, 1992. Witschi, H., Espiritu, I., Peake, J.L., Wu, K., Maronpot, R.R., and Pinkerton, K.E., The Carcinogenicity of environmental tobacco smoke, Carcinogenesis 18: 575-586 (1997). Witschi, H.P., Pinkerton, K.E., Pulmonary carcinogenicity of cigarette sidestream smoke in A/J mice, Toxicologist 30(1 Part 2): 1036 (1996). 0002427.01 3/18/98 3:03pm -35-
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Page 1 Twelve-Month Inhalation Study on Room-Aged Cigarette Sidestream Smoke in Rats Hans-Jurgen Haussmann, Birgit Gerstenberg, Werner Gocke, Peter Kuhl, Georg Schepers, Regina Stabbert, Walter Stinn, Ashok Teredesai, Franz Tewes INBIFO Institut fur biologische Forschung, Koln, Germany Erwin Anskeita, Piter Terpstra CRC Contract Research Center, Zaventem, Belgium room-aged sidestream smoke inhalation This study was supported by Philip Morris USA. The authors wish to thank Donald Hanselmann for critically reviewing the manuscript and the staffs at INBIFO and CRC for their excellent technical assistance. Requests for reprints should be sent to Dr. Hans-JUrgen Haussmann INBIFO Institut fiir biologische Forschung Fuggerstrasse 3 D-51149 Koln Germany Telephone: ++49 - 2203 - 3031 Telefax: ++49 - 2203 - 303 362 a present address: INBIFO Institut fur biologische Forschung, Koln, Germany
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Page 7 MATERIALS AND METHODS Experimental Design Female rats were whole-body exposed to RASS at concentrations of 6 and 12 µg TPM/I (WB6 and WB12) or to filtered, conditioned fresh air (WBO, sham-exposed group) for 12 h per day, 5 days per week, for 12 months (Table 1). For the comparison of exposure modes, another group of rats was head-only exposed for 5 days per week for 12 months to the high RASS concentration (H012), but only for 7 h per day, which is considered the maximum daily exposure duration for this exposure mode. Due to laboratory capacity restraints, the inclusion of a head-only sham-exposed control group was not possible. However, this is not considered to seriously impact the comparison of the two exposure modes for most of the end points investigated. The two exposure modes were compared based on the TPM concentration as well as on the daily TPM dose for those end points presumably unaffected by tube restraint. Interim investigations of pulmonary inflammation and oxidative DNA damage, end points not previously included in subchronic SS inhalation studies, were performed after 6 months of inhalation for WBO and WB12 only. The study was performed in conformity with the American Association for Laboratory Animal Science Policy on the Humane Care and Use of Laboratory Animals (1991). Experimental Animals Female outbred Wistar rats (Crl: (WI)WU BR), bred under specified pathogen-free conditions, were obtained from Charles River (Sulzfeld, Germany). Wistar rats were used in the present study because of their potential suitability for long-term inhalation bioassays in terms of longevity, moderate body weight development, low rate of spontaneous tumors, particularly in the lungs, and sensitivity to rat respiratory tract carcinogens (Woutersen et al.,
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Page 4 Environmental tobacco smoke (ETS) was classified as a class A (known human) carcinogen by the US Environmental Protection Agency (US EPA, 1992). The agency based this decision on the analogy to mainstream cigarette smoke (MS) as well as on the results of epidemiological studies. It acknowledged that lifetime animal inhalation studies were lacking, which implies that further experimental toxicology is necessary to evaluate the claimed biological plausibility of US EPA's classification. Since this classification, three long-term inhalation studies on A/J mice were reported using different sidestream smoke (S5) surrogates for ETS (Witschi et al., 1995a, 1997a,b). The first study, six months of exposure to relatively fresh SS at a concentration of 4 µg total particulate matter (TPM)/I, did not show a difference in the rate of lung tumors compared to controls. In the second and third study, five months of exposure to a mixture of SS and MS at concentrations up to 87 µg TPM/i plus a 4-month postinhalation period, increased rates of pulmonary adenomas in the exposed mice were reported. However, apart from the highly toxic dose levels used, there were several inconsistencies in the latter studies, such as the large variation in the spontaneous lung tumor rate and the lack of smoke exposure-related non-neoplastic lesions in the lungs, which leave some questions open about the relevance of this experimental design. For rats, no long-term SS inhalation study has been reported to date although the rat is the most frequently used species in subchronic inhalation studies (Witschi et al., 1995b). The present chronic study extends the current scope of rat inhalation studies on ETS surrogates. With an inhalation period of 12 months, an investigation of the potential progression of respiratory tract histopathological changes or occurrence of new changes from subchronic to chronic inhalation is made possible. In addition, the investigation of inechanistic end points has been included in this study in line with EPA's proposed guidelines for risk i
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t-uussMr..w ET p,L,- FIG. ~Connnued The induction factors reported by Gairola (l98') and Pas- quini et al. (1987) were similar irrespective of the route of administradon, i.e., SS inhalation or intraperiioneal SS con- densate injection. respectively. In addition, no adaptive or progressive chan,ps were seen for the induction of the pulmo- nary cytochrome Pa5O-dependent metabolic activiiies with oroloneed SS inhalation (Gebremichael etai., 1995: Pinkerton et al., 1996). Thus. the induction of the pulmonary B(a)P metabolism is considered a stable biomarker for the pulmonary concentration of inducers followin.g both short-term and pro- G longed SS exposure at relatively low experimental SS concen- trations. - - For the nasal eeirhelia. the results of the present study snots' a hi.~her baseline activity for B(a)P metabolism in the NOE than in the NRE. This is in agreement with the site-specific distribution of dre B(a)P metabolism described by Bond and Dahl (1989). Following FSS or R.ASS inhalation, there was a distinct but slisht induction of the B(a)P metabolism in the VRE- while no e*.`ect was seen in the NOE. Since this sistri- bution of SS-reiared changes parallels those obse:red his-
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108 rcacssNLA-.vN ET ..u.. In the VOE, the baseline values of the sham-exposed group determined after 90 days were 1151 = 79, 44 =!. 773 - 33. and 42 = 3 nmoll(a protein X h) for 3-OH-. 9-OH-. 7,3-diol-. and 9.10-diol-BIaZ respectivelv. and thus about two-fold hieher than in VRE. No relevant SS-related eifec:s were seen in this tissue. - [n the lungs. the formation of alI metabolites except 1.5- dio6s(a)P was do.se-dependentlv induced, the highest factor of induction being seen for 9.10-diol-BimP iTable :j. The induo- tion was up to a factor of $ higher than in the NRE. For all induced metabolites. the induc:ion was higher in the FSS- than B in the RASS-e.enesed aoups on the basisof the CO concen- tration. The induction was similar for both S5 types when compared on vhe 'oasis of the TPN( concenuation. At the end of the pos:inhalation period, no diiferences be- tween sham and S5-exposed groups were round. - - DISCLSSIODi The S5 .onczniations used in the oresent study ranged from - 6 to 19 ppm CO and from 0.6 to 3.' ug TPSUliter. These T?S[ _- concentranons'.vere up to two orders oi ma;pitude higher than
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Page 5 assessment (US EPA, 1996) to obtain information on potential long-term/tumorigenic effects of SS using this experimental design. Pulmonary inflammation was assessed by investigating the composition of bronchoalveolar lavage cells, while oxidative DNA damage was assessed by determining tissue and urinary 8-hydroxy-deoxyguanosine (8-OHdG) levels. Other end points, such as cytokeratin expression, an epithelial cell differentiation marker, were also investigated and will be reported elsewhere (cL, Schlage et al., 1997). The results of this study should help in the design and interpretation of possible long-term bioassays with regard to exposure levels, exposure mode, and/or mechanisms involved in chronic toxicity and possibly carcinogenicity, and thus contribute to a quantitative risk assessment of ETS (cf., Witschi et al., 1995b). Since ETS can not be reproducibly generated as required for chronic laboratory experiments, ETS surrogates have been developed for laboratory studies, e.g., aged and diluted SS (Coggins et al. 1993; Ji et al., 1994) and, more recently, room-aged SS (RASS) (Voncken et al., 1994; Haussmann et al., 1998). Although the carbon monoxide (CO) concentration - as a proxy for the number of cigarettes smoked per air volume - may be the most relevant basis to evaluate experimental SS-related effects, the TPM concentration was used in the present study to enable a comparison with environmental or other experimental SS studies. Concentrations in the range used in the present study (6 and 12 µg TPM/I) have shown to be effective in subchronic studies on rodents in producing a spectrum of histopathological (von Meyerinck et al. 1989), biochemical (Ji et al., 1994), genotoxic (Lee et al., 1993), and cell proliferative changes (Witschi et al., 1995a) and were, therefore, considered suitable for a chronic RASS inhalation study. The RASS concentrations employed in this study were approximately 100-fold higher than the maximum of the average concentrations of respiratory suspended particles (RSP) reported for ETS (Guerin et al., 1992; US EPA, 1992; Jenkins et al., 1996).
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Page 11 The analytical methods used to determine TPM, CO, nicotine, nitrogen oxides, aldehydes, ammonia, and the particle size distribution as well as those for temperature and relative humidity in the exposure chambers were performed as previously described (Haussmann et al., 1998). The other analytes were determined as follows: 3-Ethenyl-pyridine and nicotine were determined in parallel using 2-ethenyl-pyridine as internal standard. Solanesol was determined in particulate matter after trapping on Fluoropore membrane filters (pore size: 1 µm; Millipore, Eschborn, Germany) by reversed phase HPLC (Lichrospher RP-sefect B, 5 µm, 125 x 3 mm, Merck, Darmstadt, Germany) and UV detection (HP 1090, Hewlett Packard, Waldbronn, Germany). Isoprene, toluene, 1,3-butadiene, and benzene were trapped in methanol at -78 2C and determined by gas chromatography/mass spectrometry (GC/MS; Hewlett Packard 5890A/5970B) with a DB-5.625 column (30 m x 0.25 mm, J and W, Fisons, Wiesbaden, Germany). Phenols were determined in the particulate phase after trapping on Cambridge type glass fiber filters (Gelman, Ann Arbor, MI, USA), extractioh, and silylation using GC/MS with a DB-5.625 column. Polycyclic aromatic hydrocarbons were extracted from a TPM-loaded glass fiber filter with methanol/water followed by back extraction with hexane, clean-up by solid phase extraction (Bakerbond amino, Baker, Gross-Gerau, Germany), and analyzed by GC/MS with a DB-17 column (30 m x 0.25 mm, J and W). N-nitrosamines were trapped in citrate/phosphate buffer with ascorbic acid and on glass fiber filters connected in series. The combined dichloromethane extracts were washed with a sodium hydroxide solution and cleaned by adsorption chromatography on aluminum oxide. The N-nitrosamines were determined by GC with a DB-5 column 30 m x 0.53 mm (ICT, Bad Homburg, Germany) and a thermal energy analyzer (TEA 543, Thermo Electron Corporation, via Isconlab, Heidelberg, Germany). For the determination of the metals, SS was collected on membrane filters with 0.22 µm pore size (GSW P 02500, Millipore). After the filters were digested with
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Page 8 1986; Kroes et al, 1988, Gupta et al., 1990; Vandenberghe, 1990; Bomhard and Rinke, 1994; Heinrich et al., 1995; Sanders and Lundgren, 1995). Female rats were used, because they showed a higher sensitivity in several long-term aerosol inhalation studies (Brightwell et al., 1989; Nikula et al., 1995), while in subchronic SS inhalation studies no difference was reported for the histopathological changes between male and female rats (Coggins et al., 1992). Wistar rats have not been used for the evaluation of SS-induced histopathological changes in the respiratory tract to date. The respiratory tracts of 5 randomly selected rats were histopathologically examined on arrival; no abnormal findings were observed. Serological screening of 10 to 15 rats performed on arrival and after 6 and 12 months of inhalation did not detect antibodies to rat-related viruses, such as hantavirus, lymphocytic choriomeningitis virus, murine adenovirus, parvovirus H-1, pneumonia virus of mice, rat coronavirus/sialodacryoadenitis virus, rat virus, reovirus 3, rodent orphan parvovirus, Sendai virus, and Theiler's murine encephalomyelitis virus, to the bacteria Clostridium plliforme, Mycoplasma pu/monis, and cilia-associated respiratory bacillus, nor to the protozoon Encephalitozoon cuniculi. The rats were individually identified using subcutaneous transponders (IMI-1000, Plexx, Elst, Netherlands; data acquisition by DAS-4001, Uno, Zevenaar, Netherlands). Following a 7-day acclimatization period before exposure, they were randomly allocated to the two RASS groups and the sham exposure group for whole body exposure (WBO and WB12: 96 rats/group; WB6: 48 rats) as well as to the high RASS concentration group for head-only exposure (HO12: 48 rats). The age of the rats at the start of the inhalation period was N) i o' between 55 and 70 days. The mean body weight at that tirrie was 110g (SD: 10 g). ~'4 ~ W The rats were barrier maintained in an animal laboratory unit with controlled hygienic ~a . conditions. The laboratory air (filtered, fresh air) was conditioned. Positive pressure was 0
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Page 9 maintained inside the laboratory unit. Room temperature and relative humidity were maintained at 22 2C (SD: 1'-C) and 61 %(SD: 9 %), respectively. The light/dark cycle was 14 h/10 h. The whole-body exposed rats were exposed and housed in wire mesh cages (2 rats/cage), whereas the head-only exposed rats were housed between exposures in transparent polycarbonate cages (2 rats/cage) on sterilized softwood granulate (Braun & Co., Battenberg, Germany). A sterilized, fortified pellet diet (MRH FF, Eggersmann, Rinteln, Germany) from cage lid racks and sterilized water from bottles with sterilized sipper tubes were supplied ad libitum in each cage. Food was not available to the rats during the daily exposure periods. During exposure, drinking water was not available to the head-only exposed rats. Chemical analyses of food, water, and bedding material confirmed compliance with the requirements set forth by the National Toxicology Program (1991). Good hygienic conditions within the animal housing and exposure rooms were maintained as evidenced by the results of the bacteriological examinations of the laboratory surfaces and air as well as of the rat diet and drinking water. RASS Generation The University of Kentucky reference cigarette 1 R4F (MS yields per cigarette: 10.8 mg TPM, 0.80 mg nicotine, and 11.6 mg CO; Tobacco and Health Research Institute, 1990) was used for SS generation as previously described for another reference cigarette (Haussmann et at., 1998). The cigarettes were smoked in basic accordance with the International Organization for Standardization as generally applied to MS generation. Room-aging was performed by continuously passing diluted SS at a rate of 56 m3/h through a 28-m3 experimental aging room with non-inert surfaces, resulting in RASS of a mean age of 0.5 h. In the aging room were materials usually found in residences and/or offices, such as wallpaper painted with a latex-based white paint (29 mZ), vinyl floor tiles (11 mz), and a polycarbonate
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ROOM-.aGED SIDESTREAM SMOKE ENHW.TiON FIG. ~Continaed metabolites analyzed was dose-dependently induced in the lungs, with different maximum induction Factors. Only the formation of=,5-diol-B(a)P. the most abundant metabolite, was not inducible by SS inhalation. This difference is most probably atmbutable to the involvement of different cyto- chrome P450 isoenzymes in the formation of the hve metab- olites analvzed. For example. subc4ronic inhalation of SS in rats at a concentration of I u_ TP~,dliter resulted in an in- creased expression of the cytochrome P1J0 isoenzvme l.-yl in nonciliated bronchiolar epithelial ~Claral as well as alveolar type II cells tJi rt A. 199d1, which was accompanied by an D 111 induction of Lat-associated pulmonary metabolic activities (Gebremichael et al, 1995). However. the cytochrome P!50 3Bl.associated activity-was nct inducible in this study. Sirni- larly, chronic inhalation of SS in.alJ mice at a cancentration of 4 ,us TP!vl/liter resulted in an induction of cytochrome P450 1?.1 in pulmonary endothetiai cells with no effect on Lsoen- zymes '_B1, 2E1, and 2F'_ (Pinkenon er af.._ 1996). Tnus. immunohistochemical and metabolic data tit tonether since cytochrome P150 I al is considered to play a major role in the metabolic activation of B(a1P i Doera er al., 1990: Voigt rt <il.. 1993).
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Page 3 was a slight but not consistent increase in the nasal respiratory and olfactory epithelia as well as in urinary 8-hydroxy-deoxyguanosine excretion. In summary, there was little indication for progression or occurrence of new effects from 3 or 6 months to 12 months of PASS inhalation. There were also no signs of inflammation or oxidative DNA modification in the lungs. Chronic head-only exposure to PASS was shown to be technically feasible and is generally considered preferable for smoke inhalation studies over whole-body exposure to avoid artificial changes in smoke composition and the non-inhalative uptake of smoke constituents.
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Page 2 The present study extends the current scope of rat inhalation studies on surrogates of environmental tobacco smoke. The 12-month inhalation period enabled an investigation of the potential progression or occurrence of new morphologic effects from subchronic to chronic inhalation. In addition, pulmonary inflammation and oxidative DNA damage were investigated. Female Wistar rats were whole-body exposed to room-aged cigarette sidestream smoke (RASS) generated from the reference cigarette 1R4F at 6 and 12 pg total particulate matter/1 for 12 h/day, 5 days/week, and 12 months. To enable an evaluation of the exposure mode, another group of rats was exposed head-only to 12 µg total particulate matter/l for 7 h/day. Whole-body exposure conditions per se resulted in changes of the RASS composition. An analysis of urinary nicotine metabolites showed that with whole-body exposure, RASS components, such as nicotine, were additionally taken up by routes other than inhalation. Independent from the exposure mode, blood carboxyhemoglobin and the hemoglobin adduct of 4-aminobiphenyl were used as biomarkers for the RASS concentration and dose, respectively. Histopathological changes were minimal to moderate reserve cell hyperplasia and slight squamous metaplasia of the respiratory epithelium as well as minimal reserve cell hyperplasia and atrophy of the olfactory epithelium in the anterior nasal cavity; slight eosinophilic globules in sustentacular cells of the olfactory epithelium in the anterior and posterior nasal cavity; pronounced squamous metaplasia and hyperplasia in the larynx at the base of epiglottis; and slight reserve cell hyperplasia in the bronchial respiratory epithelium. Most of the changes were adaptive and similar in type and degree to those seen in previous subchronic RASS inhalation studies. A flow cytometric analysis of bronchoalveolar lavage cells, i.e., alveolar macrophages, lymphocytes, and polymorphonuclear leukocytes, did not show signs of pulmonary inflammation after 6 or 12 months of inhalation. As a measure for oxidative DNA modifications, 8-hydroxy-deoxyguanosine was determined in the lungs and nasal epithelia. No change was seen for this parameter at either time point in the lungs. There
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Page 6 The aforementioned long-term SS inhalation studies (Witschi et al., 1995a, 1997a,b) as well as some subchronic SS inhalation studies (e.g., von Meyerinck et al., 1989) were conducted using the whole-body exposure mode. This is most probably related to the convenience for both experimental animals and staff. However, when using an aerosol such as cigarette smoke, this exposure mode might be subject to confounding influences, such as oral uptake during grooming. Differences between the whole-body and head-only exposure modes were reported for the uptake of smoke constituents (Mauderly et al., 1989; Chen et al., 1995). Similarly, pronounced differences in the uptake and toxicity of test atmospheres other than cigarette smoke were observed between whole-body and head-only exposure modes (Langard and Nordhagen, 1980; Wolff et al., 1982; Iwasaki et al., 1988). In the present study, therefore, the head-only exposure mode was compared to the whole-body mode with regard to various biomarkers of exposure and effect. The test substance uptake was maximized by extending the daily exposure duration as long as feasible for each exposure mode.
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R00M-.>GED SmESTRE.k`.( SMOKE L~H.aL.aT;OY 2C0-! 100-J 0~ -is 300 - 1E0 ~ i:nalancn nn2i2t~cn . 30 10 J : s;^am exPOsue -~ : F-$• ow F55, meccm -a : FZj. t`'.i5n ~ : <-r2m excCstRe ---a . F,-55, medim --_fl PASS. Mign e0 scnhzleocn O..:CO . i;G '20 "S0 'd0 . ::;Y CF S-1LGY [03 FIG. 1. Body •.verght development ,iucng the inhalation falL _roups. 43 ra[s/eroup) and pos[mhaia[ion :eriods 'sham-exeosed and hieh-dose amups, iI rztsi_rouo). (A) Sham-exoosea:onaoi and FSS amups: i 9) sham-escosed aon¢ol and @ASS =mups. 4eLnre s[andard de`:anons •'or:he :ntli•idual 2rcuos .ve:z <14o for each [ime ooine Organ weightr. Absolute weis.^.[s af :uazs wah larvnx and ;rac5es. liver. adreaal slands. :estes, and iidnevs were dete^nined. The arYan 'ueights rdatrve to body ivei¢hr.vere ealculnwd osine :he weishts oi :he exsanrzuinaied carcasses. The :ime between exsaneuinaticn and or_-an •riei2_ht detetrninaticn was kcot ;o a minimum and did not exceeu i~ min -H%sroparhaloqv. Lun,,s••vere excised.v;rh :ar,:ns and trcchea and ised hy intta[racheal insnilation with EAF'a (=0 r a:hanol.:w aee:ic aeid. 10°o iorm- afrlehvde. 1: o isaronic saline• viv. Hartsan. 1934) a[ ]0 tm water §ressure [o shieve pnysiolo;[cai disten[iun or :ne :ur,g. :ae >kin. eqes. 'ower!a`,v, and ccstni:alevcn 60 70 ao ?o -:CO -o :2o '30 ,n0 .,..Y CF STUCY brain xere reri:oved :rom'-9e zead and -5e dase vas _zemiy iushed `uita l0'', neutral Sur:ered [ormaldea•:ce solution aa:he nasopharyn~eal duc:, 1"ne Sesu •.vas nxed In 10°c aeurral-';ud'ettc toramidehvde sofunon for I dav snd. subseyuendv, in c saiur.cn ior 7:a = days Pnor':o [rimmin^_. :he ind avas iecvaned %vtih 5% nitric acid in .n ultrasonic bath. The nose ~vas tr.mmed ina :rans•ierse ierions were _ut according :e Youne ,'981.:o oatmn ::w ::mue iices at :he ialluwine I I I immediatelv ;aseer.or:o =e vocer:ncisar:eeth. ~71 at ihe incisive aaomila. The :ar•m¢eai :nns-erse ,ca:uns aere aut it :.he ame ar tpe eprJotris and u
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Page 13 In order to provide an estimate of the amount of test atmosphere taken up by the rats, respiratory frequency and tidal volume were determined on at least 10 rats/group by whole- body plethysmography, three times during the inhalation period, as previously described (Haussmann et al., 1998). Rats were allowed to adapt to the plethysmographic tubes prior to data acquisition. During the adaptation and acquisition periods, they were continuously exposed to the test atmospheres. To monitor exposure to 00, steady-state proportions of blood carboxyhemoglobin (HbCO) were determined in 5 rats/group according to Klimisch et al. (1974), three times during the inhalation period. The blood samples were collected after at least 5 h of exposure, a duration sufficient to obtain steady-state HbCO proportions (cf., Tyuma et at., 1981). Rats were removed from the exposure chambers for a short period of time, and blood samples were taken under diethyl ether narcosis by puncturing the retro-orbital sinus with glass micropipettes. To monitor exposure to aromatic amines, hemoglobin adducts of nine aromatic amines were determined in 7 to 8 rats/group following 12 months of RASS inhalation according to Kutzer et al. (1997). The hydrolyzed amines were derivatized with pentafluoropropionic anhydride (Aldrich, Steinheim, Germany) and analyzed using gas chromatography/mass spectrometry with negative chemical ionization in the SIM mode (DB-5MS column, J&W Scientific, Folsom, CA / TSQ 700, Finnigan, Bremen, Germany). For internal standardization, D5-aniline (Aldrich) and Dy-2-aminobiphenyl (IC Chemikalien, Ismaning, Germany) were used. To provide an estimate of the amount of nicotine taken up by the rats, nicotine metabolites were determined in urine collected from 6 ratslgroup over 24 h, three times during the inhalation period. During the 7-h head-only exposure period, the urine was collected using specially modified exposure tubes. During the postexposure period and for the whole-body N o ~_ ~ '._. . w m N - .P
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Page 12 nitric acid, the metals were determined by graphite furnace atomic absorption spectrometry by Henkel KGaA (Dusseldorf, Germany). Animal Exposure System The whole-body exposure chambers made of glass and stainless steel were equipped with 24 stainless steel wire mesh cages, which were mounted above stainless steel excretion pans. Each cage had a separate supply of test atmosphere. The flow rate through the chambers was 180 I/min. The position of the cages within the chamber was systematically changed on a weekly basis. Rats were exposed to RASS as well as to filtered, conditioned fresh air (sham-exposed control) for 12 h per day, 5 days per week, using two exposure chambers per group for WBO and WB12 and 1 chamber for W66. Another group of rats (H012) was head-only exposed to RASS for 7 h per day, 5 days per week. This exposure system was described previously (Haussmann et al., 1998). The position of the rats in the chamber was systematically changed on a daily basis. In-Life Observations The rats were observed daily for mortality, moribundity, signs of overt toxicity, or injuries. Detailed checks on general condition and behavior of the rats were performed on 3 rats/group per day shortly after the end of the daily exposure throughout the first 3 months, and two times per week in months 4 to 6. Starting with month 7, tumor checks were performed two times per week on all rats. The body weight of the individual rats was determined one day after their arrival, at the start of the inhalation period, and once per week during the inhalation period. Biomonitoring
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Page 10 window (2 m2) (cf., Voncken et al., 1994). The materials in the aging room were unexposed at the start of the inhalation. The painted wallpaper was replaced every 13 weeks. A ceiling fan was operated to facilitate uniform distribution of the RASS. The room was illuminated by fluorescent "daylight" lamps (Lumilux L58W/11, Osram, Munich, Germany). Two heat exchangers (approximately 60 m2 surface area) were used to keep the room temperature constant. RASS was conveyed via glass tubing to the exposure chambers. RASS generation was started approximately 2.5 h before the start of the daily exposure to achieve a steady- state test atmosphere for inhalation. During overnight, non-smoking periods, the room was flushed with filtered, conditioned fresh air at 56 m3/h. The test atmosphere for WB6 was obtained by diluting the RASS from the aging room with filtered, conditioned fresh air. Whole-body exposure normally results in slight losses of TPM, mainly due to particle deposition in the chamber and on fur. In order to obtain the same TPM concentration in WB12 and H012, the RASS from the aging room was diluted by 20 % with filtered, conditioned fresh air before entering the head-only exposure chamber. Sham-exposed rats (WBO) were exposed to filtered, conditioned fresh air under the same conditions as the whole-body RASS-exposed rats. Analytical Characterization of the Test Atmospheres At designated time intervals, a broad range of analytes was determined to characterize the test atmospheres, to evaluate the reproducibility of the test atmosphere generation, and to detect possible cross contamination in the sham-exposed group. Samples were collected within the exposure chambers at sites representative for the breathing zone of the rats. CO was continuously monitored. TPM was determined at least once per day. The other analytes were determined at less frequent intervals from weekly to twice a year.
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Page 16 population from a dot plot histogram of axial light loss vs. right angle light scatter, the green fluorescence (515 - 530 nm) was quantified in arbitrary units normalized to that of the admixed latex beads. From each sample, 15,000 counts were collected. 8-Hydroxy-Deoxyguanosine Determination As a measure for possible oxidative DNA damage, 8-OHdG was determined in 8 rats/group after 6 (WBO and WB12 only) and 12 months (all groups) of inhalation. During the last exposure day, rats were killed as described. The lungs were perfused in situ with isotonic saline to remove erythrocytes. The lungs as well as the nasal respiratory epithelium and the nasal olfactory epithelium were stored frozen at -70 °C until further processing. DNA extraction was performed according to Gupta (1984) with emphasis on fast tissue processing to prevent both DNA repair and artificial DNA oxidation. Enzymatic digestion of DNA as well as chromatographic separation (Nucleosil 100-5 C18 precolumn, CS Chromatographie Service, Langerwehe, Germany; Novapack C18, Waters Millipore; HPLC HP 1050, Hewlett Packard) and electrochemical determination (HP 1049A, Hewlett Packard) of 8-OHdG were performed according to Shigenaga et al. (1990). The amount of deoxyguanosine (dG) present in the samples was determined by its absorbance at 245 nm. As an integrative monitor for oxidative DNA damage without specification of the organs involved, 8-OHdG excretion in urine was determined in 5 to 6 rats/group at 5 and 12 months of inhalation. Twenty-four hour urine samples were collected as described for nicotine biomonitoring. The purification of 8-OHdG from the rat urine was performed according to Shigenaga et al. (1990) with some modifications. An internal standardization method was employed using 'H-labeled 8-OHdG prepared from [1',2''H]-dGTP (Amersham, Braunschweig, Germany) according to Shigenaga et al. (1990). Interfering substances in the
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Page 17 eluate were oxidized at +0.35 V before the actual 8-OHdG determination using an additional electrochemical detector. Statistical Analysis The following statistical tests were performed on the biological data: For the overall comparison of the whole-body RASS- and sham-exposed groups (WBO, WB6, and W612), the one-way analysis of variance for continuous data (Sachs, 1982) and the generalized Cochran-Mantel-Haenszel test (Koch and Edwards, 1988) for ordinal data were used with the TPM concentration as the stratifying variable. If the overall comparison showed a significant difference, the Duncan test (Duncan, 1955) and the generalized Cochran-Mantel-Haenszel test were applied to continuous and ordinal data, respectively, for a pairwise comparison between the groups. All tests were conducted at the nominal level of significance of a= 0.05 (2-tailed). Due to the large number of parameters analyzed, no correction for multiple testing was applied, which would have made the tests very insensitive. Statistical significances, therefore, have to be considered as explorative indicators rather than confirmatory evidence. RESULTS RASS Composition Throughout the 12-month inhalation period, RASS was reproducibly generated and delivered to both types of exposure chambers. The analytical characterization of the sham and RASS exposure groups is shown in Table 2. As targeted, the TPM concentrations determined in the high concentration groups were the same for both exposure modes. The concentrations of most of the particulate phase constituents paralleled the TPM concentrations in the various groups. However, the catechol concentration in H012 was almost twice as high as in WB12, for which there is no explanation. To achieve equal TPM
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Page 14 exposed groups, custom-made metabolism cages were used. All samples per rat and time point were combined, centrifuged, and stored frozen. The nicotine metabolites were determined by HPLC after derivatization with 1,3-diethyl-2-thiobarbituric acid (Rustemeier et al., 1993). The separation of all analytes in one chromatographic run at ambient temperature was enabled by modifying the derivatization conditions (0 °C and pH 3.3), the composition and pH of solvent A (54 mM 1-oentanesulphonic acid, 5 mM 1-heptanesulphonic acid, pH 5.0), and the solvent and flow program. Gross Pathology and Organ Weight Necropsy was performed without prior fasting. On the day following the last exposure at the end of the 12-month inhalation period, 8 rats/group were killed and examined as previously described (Haussmann et al., 1998). The weights of the lungs with larynx and trachea, liver, heart, adrenal glands, and kidneys were determined. Histopathology Histopathology of the respiratory tract was performed after 12 months of inhalation as previously described (Haussmann et al., 1998) with additional levels in the posterior nasal cavity (levels 3 and 4 according to Young, 1981). Processing and sectioning of the samples was performed at Huntingdon Research Centre (Huntingdon, Cambridgeshire, U.K.). The larynges of most of the rats were not reproducibly sectioned at the pre-defined levels needed for a semi-quantitative evaluation and a comparison to previous data. Thus, they could only be evaluated qualitatively. All slides were read by a veterinary pathologist in a blinded manner with experience in cigarette smoke-related changes in the respiratory tract of rodents. All pathological findings were scored according to a defined severity scale from 0 to 5 (marked effects). Mean severity scores were calculated based on all rats of a group. ~ o~
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Page 19 Signs of eye inflammation were found starting at 7 months peaking at 9 months of inhalation and were predominantly found in WBO and H012 (40 to 50 % of all rats) and less frequently in W66 and WB12 (approximately 10 %). A cross-check of the data revealed no influence of this local inflammation on the other parameters of the study. Biomonitoring RASS Uptake Statistically significant decreases in respiratory frequency were seen in the whole-body exposed rats accompanied by a tendency to increased tidal volume resulting in slight decreases in the minute volume. For example, after 5 months of inhalation, the minute volume was 180 ± 7, 168 ± 8, and 162 ± 7 ml (mean ± SE) for W B0, W86, and W B12, respectively. The respiratory minute volume for H012 rats was 130 ± 6 ml. Daily RASS TPM doses were calculated taking into account these differences in respiratory minute volume, body weight, TPM concentrations, and daily exposure durations, the results being 2.8, 6.0, and 3.1 mg/kg body weight for W86, W812, and H012, respectively. Thus, apart from the conventional comparison based on equivalent TPM concentrations (WB12 vs. H012), a further comparison based on equivalent daily TPM doses is possible between WB6 and H 012. Carboxyhemoglobin The steady-state HbCO proportions were in agreement with those expected based on the CO concentrations in the various test atmospheres (Figure 1 A). N o. o+ : w . o+ Hemoglobin Adducts Following 12 months of inhalation, a TPM dose dependent increase in hemoglobin adducts of 4-aminobiphenyl (4-AB) was found for WBO, WB6 / H012, and W B12 (Figure t B). The adduct levels for the other aromatic amines investigated (aniline, o-, m-, p-toluidine, 2-ethylaniline, 2,4-dimethylaniline, o-anisidine, and 3-aminobiphenyl) were I
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ROOZI;-AGED SIDESTRE2..v1 SS[OKE LLVH.aLAT10"i TABLE 2 Histopathologcal Findings after 90 Days of Inhalation 105 FSS RASS Parameter Sham Low Medium High Low Ytedium Hi~h Nasal cavity, Level I Reserve cell hyperplasia of 0 0 0 -0.6-0.2• 0 0 0.1 _0.1 respiratory epitheliumt 0120 0C0 0/?0 3G0 0!]0 O!?0 1/19 Larynx Base of epiglotds Squamous maaplasia of 0 02 = 0.l 0.6=0.1' L0=0.'_• 0 0.I-_ 0:1• 0.7=0.L' pseudastradfied epirhelium* 0/17 3/l9 l0/13 [i/19 0/I9 :/19 Il!0 Hyperplasia of squamous 0 - 0.2 - 0.1 0.3 Ia=0.3` 0.1.x0.; 0.'_=0.1 0.7c0.P epitheliumi 0/17 3/19 It/ts 11/I9 :A9 3/I9 1J^0 Arytenoid prajections Ventral depression Hyperplasia of 0 0.7 - 0.1 0.'--0.1 0.l -0.1 0 a1 =0.1 0.-Y=O._- cubaidal epithelium 0/13 3/16 JI3 IC0-- 0/13 Lt6 1/1', Squamaus memplasia of 0 0 0 0.1-0.1 0 0.I-J.1 0 cuboidal epttltelium 0/13 0116 0/13 IC0 0/L3 1/16 0/17 Vocal cords. lower medial re_eion Hvperplasia of 0.1 - 0.1 0.J - 0' 0.7=0.T 08=0_- 0G-0.1 0.9=0.=' i.0-0._` squamous epithefium 'Jl8 3/16 9/13 I1/70 1/13 9/t6 10/17 Traches Goblet cell hyperolasia of 0.1 - 0.1 0.? _ 0.2 D.3=0.1 0_ 0.1=0.1 0.3=0.1 0.1=0.1 resprtazory epithelium 1/17 "J1- 3/17 0/13 . 1119 3119 1/70 Lun3s Goblet cell hyperplasta of 0.3 - 0.1 0.7 = 0.1 0.1-0.1 0.'-0.= 0.3=0.] 0__0.1 05=0= respirutary epithelium 1/^-0 . 3/'_0 J^_0 9/_0 _C0 7C0 " - . 6C0 ,Vore. Histopathaloeical indin;s are given as mean score ~_ SE and incidence. ' Indicates amustlcally signincanr.diiference to sham-esposed control croup. ` Inaiea¢s stausucally signincan[ differences tietween the two SS tvp2s. RASS groups were found to be diluted at relatively constant proportions from the high- to the medium- (2.2 = 0.2) and from the high- to the low- (1.6 = 0.9) dose levels, respec- tively. The TP4 concentrations for RaSS decreased by 60 to '04c compared to FSS. The agins-related decreases found for nicotine, formaldehyde, and ammonia were numerically similar to that found for TP%S, but may no[ necessarily result from [he same mechanism since, e.g., nicotine was found only in the gas phase and was not associated with particulate matter. On avera?e. the mass median aerodvnamic diameter was sliehtly higher following room-asing (changed from 0.36 to 0.-=2 µm) with no effect on the zeome[ric standard deviation (1.3 to 3.0 of the particle size -distribution. No oxidation of nitric oxide occurred durins the a.,ine process. Acetaldehyde and acrolein were not affected by 55 room- a_einL. The relative humjdiry in the sham-exoosed group was 54 - 1°n i mean - SD); this is considered to be representative for the other exposure groups and complies with the exposure condi- tions speci¢ed by the OECD t 195ib/. The temperature within the exposure chambers vas 26 - l'C for all groups and thus also met OECD (L9SLb) specincations. -- - - In-Life Obsernatians - There was no smoke-associated mortality, nor were there _ effects on seneral condition and'oehavior o[ the rats. BodvWeignr Develooment N The body weights of the rats increased duens the inhalation ,~ and postinhaladon periods (Fi,. I). The mean body weieht or E,,7 the hi_eh-dose FSS exposure group was statistically signiti- ~' A cantly lower than that of the sham-exposed group between N inhalation days ~9 and 7:. At the end of the inhalauon pedcd. N a numet-ical body weight gain reduction of 1% for this e.eco- sure group compared to the sham-exposed group was calcu- lated (no statistically signidcant difference). No body .aei?ht effect was seen rollowing R.aSS inhalation. During the posrn- halation period• the body cveiehts of the rats in all grouos increased to the same level. indicatine a reversal of the reduc- [ion in body weight gain associated with the tube restraint durins the inhala¢on_period. . Bianonitori.n; - - - - ~ - No relevant e[fec:s on the respiratorv minuce volume ot-the raa were observed (data not shown/. The steadv-state blood
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l16 HAtisSMA.11u Er .uL of cytochrome P450 IAl and aryl h-vdrocarbon h-vdcoxylase acdviry in rat nasal mucosa. J. Hisrocnem. Cyrachem. 41, 377-885. von Meyarinck, L.. Schereq G., Adlkofer, F.. Wenzel-HarmnB, R., Brune. H., and Thomas. C. (1989). Exposure of rats and hamsters rn sidestream smoke from cigarettes in a subchronic inhaladon study. Ecp. Par6ol. 37, 186-L39. Voucken. P., Schepers, G. and Sch3fec Y.-H. (1989). Capillary gas chromaco- graphic detemtinadon of :ran.c-:i -hydroxy-cotinine simultaneously with nico- tine and codnine in urine and blood s.vnples. J. Chrorwrogr. 479, J 10--118. Voncken, P., Sttnn, W., Haussmann. H: f., and.Aiiskei[. E. (1994). Influence of aging and surface contact on the composidon of cigarerte sidestream smoke. Models for environmenml tobacco smoke. In To.ne and Carcinogenic Ef- (ects of Solid Pamcfes in rhe Respirararv Traca (D. L. Dungworth. J. L. M aud<d,v, and G. Oberd'drsrer. Eds.), pp. 637-641, Q-SI Mono.paphs. Q.SI Press. Washington. Witscht. H.- Pinke¢on. K. E-. Coeuins, C. R.- Penn. A.. and Gori. B. B. (1995a). Environmenml tobacco smoke: Experimental facts and societal issues. Fundam. AppL TosicaC 26. 3-I3. Witschi. H., Oreffo, V. [.. and Pinkeaon, K E. (1995b). SiX-month exposure of strain -VJ mice to cigarene aides¢eam smoke: Cell kinetics and lun_e tumor dau. Fxndam ,{ppl. To.vcol. 26, J2-10. .. Witscbi, H.. Espiritu. L. Peake. J. L.. Wu. K.. Muonpot. R. R.. and Pinkeaon. K. E. (1997). iPe csrdno¢eniciry of environmental tobacco smoke. Carci- nogenesis 18, 575-536. Woutersen. R..{„ pppe!man. L. YI.. Wiimer, f. W. G. Yt., Falke, H. 1., and Peron. V. 7. (1987). Su6chranic (I3-week) inhalation toxiciry study of formaldehyde in rats. l. dppi. To.xicol. 7,17-19. Youn¢, 7. L(1981). Histopatholo3ic exunination of ;he ra[ nasal cavitv_. Fundmrt. dppL Toxicaf. 1,
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Page 20 not statistically significantly different among the groups, although a notable increase (up to 6- fold) in the 3-aminobiphenyl adduct level was seen (data not shown). Nicotine Uptake For the evaluation of the nicotine uptake, five major urinary nicotine metabolites were determined, i.e., nicotine-N'-oxide, nornicotine, cotinine, trans-3'- hydroxycotinine, and norcotinine. Nicotine itself was also determined but not used in this evaluation, since control experiments showed that aerosol nicotine directly dissolved in the urine collected in the whole-body chambers. The sum of the five metabolites excreted over 24 h at 5 months of RASS inhalation is presented in Figure 2A. Similar data were obtained at 12 months of inhalation. At 3 weeks of inhalation, the total amount of metabolites excreted in the whole-body exposed groups was up to 50 % lower than at the later time points. The amount of nicotine metabolites excreted, and correspondingly the nicotine uptake, increased dose dependently in the whole-body exposed groups to levels which far exceeded that in the head- only exposed rats. This is partially due to the longer daily exposure duration and the higher respiratory minute volume for the rats in the WB groups: A theoretical uptake of inhaled nicotine was calculated using the nicotine concentrations in the test atmospheres, the exposure durations, the respiratory minute volume, the latter being calculated based on the body weights of the rats at the respective time points (Guyton, 1947), and assuming total absorption. Based on this theoretical uptake, the nicotine uptake in the whole-body exposed rats was still 2- to 3-fold higher than that in the head-only exposed rats (Figure 2B). The sum of the five metabolites excreted in H012 accounted for almost 50 % of the estimated nicotine taken up. Overall, the pattern seen for the five nicotine metabolites relative to each other was similar at all three time points as well as for the different dose levels and exposure modes (data not shown). Body Weight Development
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IG6 1.0 __~ 0.8 --i 0.6 -J 0.4 02 li 0.0 H.iC:SSNI.\.NN ET AL. ~ : FSS. Maiaecn  ; FSS. oosnnhalaticn --~ : RASS. nh&aacn . . RASS. posanha!anon 0 T~ zo 30 CO CCNC:.~VTRAIiCN !ppm) B d I -a : FSS, mhalanan . ; FSS. posbnhalatian RASS, iohalaacn ~: RASS. postinh2lation I 0 e s :o ?M CCNCS`7TRATCN !µg/!1 FIG. 3. Histopathologic2i 5ndin¢s in the larynx. Effects are shown based on Soeh dose paramerers. i e.. CO ind TPM toncentrazions. !n FSS and RASS. .(A. 8) H,vperplasia of ihe squamous <pitheiium at ;he base of apialottis (AI Effec:s based an ;he CO <or.ceatntions as dose parame:ert ~ 8) efiecs based on :he TPM conaentrumns as duse 2arame:ec Results are _iven as mean scores = SE. HbCO proportions I 1. '_. and id, above sham contrbl values (0'6°c) for the tow-, medium-, and hi_h-dose levels, reSpeC- tivelv) were in agreement with those expected from the CO concentrations in both SS types. - - The amounts of nicotine. cotinine. and trans-3'-hydroxvco- tinine found in the urine s'amples collected during and for 13 h after exposure showed an almost linear increase with increas- ing concentrations of nicotine in FSS and RASS (Fia. 2). The absolute amounts found in the urine samples did not account for the total uotake of nicotine. since oniv nicotine and two of its metabolites were determined. There is no difference in the relative proportion of nicotine and ~he two merabolites beobeen the two SS types. HbCO and nicotine metaboiite data in the sham-exposed _roup contirmed nonexposure :o 5S. - - Gross Pnthology There were no SS-re!ated gross pathological nndines. Slight ve!low-brown discoloracon of the fur was observed which was roughly dose-deoendent: the cause of this discoloration is unclear. OrganNrigitts _ . The absolute weights of the lunss with larvns and trachea. kidneys, and liver were statistical:y signincandv lower lmas- imurh effect: 11%) in the hish-dase FSS-exposed group com- pared to the sham-exoosed eroup. No effects in orean weights were seen en in the RASS-exposed rats. For organ weights rela- tive to body weight. no differences 6etween FSS- or R-sSS- exposed and sham-e :posed eroups were seen. At the end of the postinhalation peded the arzan weiehts of the FSS-esposed rats retumed to those of the sham-ezposed rats. Histopathologv it the end of the 90-day inhalation period, bniy sligct histopathological changes in-t.he upper respiratory tract in the FSS- and RaSS-exoosed groups were consistently observed in almost all rats (Table 2). In the nose at the most anterior level (level 1), slistht patchy reserve cell hype;-plasia was observed in rats of the high-dose groups on1v, the mean score for this nndins being statistically si;niticantlv hi;her for PSS eompared to RASS. This dif.`er- ence is related to the re[advelv hi^h inr.dence of this hndin_g in the high-dose FSS zroup compared to only one rat in the
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Page 21 The body weight of the rats increased throughout the inhalation period (Figure 3). After 3, 6, 9, and 12 months of inhalation, the body weight gain was statistically significantly lower by 5 to 8 % in WB12 compared to WBO; with the exception of the 12-month time point, the body weight gain in W 86 was statistically significantly lower by 4 to 6% compared to W BO (SE 1 to 2 % in all cases). The pronounced body weight effect seen in H012 is considered to be mainly due to the exposure mode (Griffith and Standafer, 1985). A direct evaluation of the RASS effect on body weight development under head-only conditions was beyond the scope of the present study since a corresponding sham-exposed head-only control group had not been included.
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Page 25 DISCUSSION The chemical characterization of the RASS used in the present study is the most comprehensive characterization of SS in an inhalation study to date. It allows a detailed comparison of RASS to ETS. The most coherent and representative recent publication on ETS composition involved volunteers in an environmental chamber who smoked the 50 top- selling US cigarette brands (Martin et al., 1997). Respirable suspended particles (RSP) as reported in this study can be considered to be solely particulate matter due to the controlled conditions employed, and would thus correspond to the definition of TPM in the present study. On the basis of TPM or RSP, the high RASS concentration in the present study was about 10- fold higher than the extreme ETS concentrations employed in the market cigarette study (Martin et al., 1997) (Figure 6). The ratios of the other S5 constituents determined in both studies are in the same order of magnitude suggesting a proportional composition of 1 R4F RASS and ETS generated from market cigarettes. In the most thorough recent ETS field study performed in 16 US cities, only those ETS constituents were determined which have generally been used as ETS markers (Jenkins et al., 1996). The comparison of the ETS concentrations of these constituents to those determined in RASS again supports the representative character of RASS for ETS (Figure 6). The RASS concentrations in the present study were 2 to 3 orders of magnitude above the ETS concentration determined by Jenkins et al. (t996) in smokers' homes, and approximately 2 orders of magnitude above maximum average RSP concentrations contributed by ETS for residential (0.1 µg/I) and office (0.06 µg/I) environments reported by the US EPA (1992). Based on these RSP concentrations and a respiratory minute volume of 7 Ilmin for a 70 kg person, the daily RSP dose taken up in residences during 16 h and in offices during 8 h can
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Page 24 of RASS inhalation indicating that a saturation or a steady-state equilibrium was already obtained after the shorter inhalation period. 8-Hydroxy-Deoxyguanosine Formation Tissue Content Both after 6 and 12 months of RASS inhalation, the nasal 8-OHdG content increased in WB12 compared to WB0 (Figure 4A and B); this increase was statistically significant in two out of four cases. The most pronounced effect was seen in the respiratory epithelium after 12 months of inhalation (+158 %). Overall, a steady-state equilibrium between formation and repair of this modification seemed to be reached already by the 6-month time point. The results for the head-only exposed group are difficult to interpret, because a tube restraint-related effect cannot be excluded. In the lungs, the 8-OHdG content did not change statistically significantly. However, a consistent trend to lower 8-OHdG levels (up to 30 %) was observed at both time points regardless of the exposure mode (Figure 4C). Urinary Excretion At 5 months of RASS inhalation, an increased excretion of 8-OHdG was observed in the whole-body exposed groups with increasing RASS concentrations (Figure 5). Such an effect was not seen at 12 months of inhalation. Again, a contribution of a tube restraint-related effect cannot be excluded for the H012 results. ro .. or m.
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ll0 HrlasSi.ti.`N ET .~,L TABLE 3 Laryngeal Epithelial Thickness at Three Sites of the Arytenoid Projections after 90 Days of Inhalation FSS R.aSS 0 ,an/site Sham Low blcdium High Low Medium Hioh Larynx Acytenoid projectians Ventral depression 7.9 - 0> 9.3 x 0.3 9.7 ± 0.3 10.0=L0 3.1=0.? 8.9x0.1 3.5c0.'- 6% 10% 27Sb' 7% 13% 3:0 Floor of the larynx 10.5 - 0.4 . 11.2 = 0.+ 11.3 = 0.1 I'_2=0.a . _10.7-0.1 1?.1=L0 C.0-0.9 1% 7% Ifi%' - 2% 15% Il:n Vocalcardst 211=L'' "'6-1? ?4.5- 1.1 '_6.1 _ 1.3 '_i= - 1.1 37.3 = 1.3 '-', 5= 1.1.. 2% 10% 17% 9 0 '-1R • -- o Vore. Epithelial thickness (µm) is aiven as mean -_ SE and percentage increase relative to (he sham-exposed cantrol group. ' Indicates statistically signilicanr difference to sham-exposed control group. _ _ T Indicates statistically signincant differences between the two 5S types. 2063643226 the base of the epielottis was similarly observed following subchronic glycerol inhalation (Renne er al., L992) and has been discussed as a commonly observed adaptive response to repeated inhalation of aerosols (Gopinath et al., 1987; Burger er al.. 1939). The lowest-observed effect level for histopatholo,gical chanses was 12 ppm CO, equivalent to 3.6 and 1.2 µg TPbU liter for FSS and RASS, respectively. The no-observed-effec[ levels were 6 ppm CO, equivalent to l.5 and 0.6 µg TPMfii.ter for FS5 and RASS, respectively. As discussed before, the relevant basis of comparison be- tween the two SS types is the number of cigarettes smoked per unit of air volume, a dose parameter which is represented in the present study by the CO concentration in the test atmospheres. On this basis of comparison. the biological activity of RASS is approximately two- to three-Pold lower than that of FSS for the histopatholo$ical findins in the anterior nose and in the larynx at the base of the epislottis. FSS and RASS are equally active for changes in the larynx at the arytenoid projections when comoared on a CO concentration basis: Previous SS inhalation s[udies with eapenmental animals have usually been based on the TPVI concentration as dose parameter. FSS and RASS were equallv active based on TPNI concentrations with one excep- tion, i.e.. histopathological findin_s at the arytenoid projections which were more pronounced for RASS than for FSS. The described differences in the biological activity of FSS and RASS mav also ;ive some clues as to the mechanism and the SS components which may be involved in inducing such effects: -Most of the histopatholooical chanees observed seem to correlate with the TPNI concentration. In the larynx, at the base of the eoi_lottis, this may be interpreted as a consequence of particle impaction on the sites where the inhaled air s[ream bends. E.ccept for the base of the epigtoctis. this correlation with the TPM concentration was no[ expected. For example, amonv the gas-phase components of SS analyzed. the alde- hydes were described to induce epithelial chanses in the nose. in particular acrolein as [he most ac[ive of [he three aldehvdes analyzed at their respective dose levels (Feron er al.. 1973; Appelman et aL, 1936; Woutersen er al., 1987). Apparently, the concentration of the gas-phase aldehydes was no[ hioh enoueh to substantially impact the SS-rela[ed morphological effects at this site. There is only one site where hiscopatholog- ical findings were no[ seen to depend on the particle concen- tration, i.e., at the arytenoid projections, namely the vocal cords. Vo explanation for this is available to date. Tne data may suggest a dependence on the SS gas phase, but a qualita- tive change of the particulate matter by room-asin2 cannot be excluded either. In order to clarify the role of particulate and gas phase, a subchronic itudv comparing the separate phases would be useful. - - Duriti's the postinhalation period. all histopaihoiouical chanses reve¢ed [o the sham control level, contirmins their adaptive nature-(cf., Burger er al.. 1989). - The B(a)P metabolism was investieated in the present study by determining the amounts of five individual metab- olites Pormed. This is different from the method employed in previous SS-retated studies in which the "aryf hydrocarbon hydroxvlase" activity was determined by analyzing the total amounts of B(a7P metabolites formed. The pulmonarv aryl hydrocarbon hydroxylase in rats was induced following subchronic inhalation (Gairola. 1987) or intraperi[6nea1 ad- ministration of SS condensatz or condensate fractions , Pas- quini er af., 1987). No direct measurement of the SSS con- centration used was made by Gairola l,1937), but based on the 1-IbCO proportions reported, it can be assumed that it was approximately fivefold hioher than those in the 4iah- dose groups of the present study. To date, no studies have been reported that investigate dose responses for the B(a)P metabolism at SS concentrations that are closer to [he real- istic human em:ironment. In :tddition, the effect of room- aeine has not been investieated previously. In the present study, the formation of four of the five 3lwP
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Page 15 Composition of Bronchoalveolar Lavage Cells As a measure for possible inflammatory processes in the lungs, a differential count of bronchoalveclar lavage cells was performed using flow cytometry. For this purpose, 8 rats/group were killed as described following 6(WB0 and WB12 only) and 12 months (all groups) of RASS inhalation. The lungs were lavaged via the trachea with 120 ml lavage medium per rat in 10 lavage cycles. The lavage medium was Dulbecco's phosphate buffered saline without calcium and magnesium (Biochrom, Berlin, Germany) supplemented with 3.25 g/I bovine serum albumin (Sigma) (pH 7.2). The viability of the lavaged cells of all groups was 95.5 % (SE: 0.4 %) as determined by the Trypan blue dye exclusion method. After fixation of the cells, they were incubated on ice for 48 h in a cell membrane permeabilization medium (according to Sander et al., 1991, with some modifications) to enable labeling of an intracellular epitope with the pan rat macrophage antibody ED1-fluoresceinisothiocyanate (FITC) conjugate (15 mg/I; Serotec, Kidlington, UK). Propidium iodide was added as a nucleic acid marker at 5 mg/I. The cells were analyzed using a Cytofluorograf 50H flow cytometer (Ortho Diagnostic Systems, Westwood, MA) in conjunction with the Cicero data acquisition and analysis workstation (Cytomation, Ft. Collins, CO). The cell populations (alveolar N macrophages (AM), lymphocytes, and polymorphonuclear leukocytes (PMNL)) were identified ° by their characteristic appearance on a dot plot histogram of green (ED1-FITC; 515 - 530 nm) °~a ca N vs. red (nucleic acid content; ? 630 nm) fluorescence. From each sample, 30,000 counts were -A .3 :-. collected. Microscopic evaluation of Pappenheim-stained (Rcmeis, 1968) smears of bronchoalveolar lavage cells confirmed the flow cytometric results. An aliquot of the above fixed lavaged cells was analyzed for spontaneous fluorescence. The system was internally calibrated by adding monodisperse (diameter: 1.95 µm) fluorescent latex beads (Duke Scientific, Palo Alto, CA) into the cell suspension. After gating the AM
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Page 23 and squamous metaplasia to be the only findings observed in this organ. Further, the results given in Table 3 are representative for the findings in each of the respective groups. These effects were pronounced and seemed to be dose-dependent. At the tracheal bifurcation, slight reserve cell hyperplasia of the respiratory epithelium was observed in only a few rats (not statistically significant; Table 3). In the pulmonary bronchial respiratory epithelium of the lungs, the degree of this finding was similar but the incidence was higher occurring most frequently in H012. For both WB6 and WB12, this finding was statistically significantly different from WBO. The number of goblet cells was similar in all groups. A slight accumulation of pigmented AM was seen in WB12 and H012. The histopathological findings in the upper respiratory tract were less pronounced in H012 compared to WB12. In the larynx at the base of epiglottis, the degree of the findings in H012 was similar to that in WB6 (equal TPM doses). In the lower respiratory tract, the H012 findings were closer to those seen in WB12, and the accumulation of pigmented AM was even seen at a higher incidence in HO12 than in WB1 2. Composition of Bronchoalveolar Lavage Cells There was a slight but statistically insignificant increase in the number of AM and thus of the total number of bronchoalveolar lavage cells with increasing TPM concentrations after 12 months of RASS inhalation (Table 4). No effect was seen for the number of lymphocytes and PMNL. Thus, no sign of a RASS-related inflammatory effect was observed. The same holds true for the comparison of WB12 and WBO following 6 months of RASS inhalation (data not shown). A green fluorescence was observed in the AM, the intensity of which increased with the RASS concentration (Table 5). For WB12, the intensity was similar following 6 or 12 months
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Page 18 concentrations in the high concentration groups, the TPM losses in the whole-body chambers were compensated by dilution of the RASS entering the head-only chamber. This was reflected by a 20 % lower CO concentration in H012 compared to WB12. Most of the other gas phase components paralleled CO, with the exception of nicotine and formaldehyde, which were 30 and 70 % lower, respectively, in WB12 than expected based on the HO12 data if a proportional dilution of all RASS components would be assumed. The carbon dioxide concentration was determined both at the inlet and outlet of the exposure chambers to assess its concentration in RASS as well as the concentration added by the exhalate of the rats, respectively. The particle size distribution was the same for all RASS exposure groups regardless of the exposure mode, with an average median mass aerodynamic diameter of 0.42 µm and a geometrical standard deviation of 1.8. The relative humidity in the sham-exposed group was 58 ± 8 % (mean ± SD); this is considered to be representative for the other exposure groups. The temperature within the exposure chambers were between 22 and 24 2C (SO: 1°C). These environmental conditions complied with the exposure conditions specified by the OECD (1981). ro. - ea - In-Life Observations W ! There was no RASS-related mortality. Shortly after the end of the daily exposure, detailed checks of the rats revealed findings that occurred more often in rats in one or both high RASS concentration groups than in those of the sham-exposed group, i.e., secretion from the Harderian glands, slight yellow-brown discoloration of the fur, and impaired gripping ability. No other RASS-associated effects on the general condition and behavior of the rats were detected. O .
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i RCO\4AGED SIDESTREAM SMOKE LNHAL.aT:OS . 107 FIG. 4. Transvesse sec::ons at dte larqn<, base of epi;loais. (A) Overview (sh:un-ezposed rat): IB, C) dnam-esoosed rat showing aormal squamous epithelium at the venvomedial site and pseudosvanfied epithefium at venrrotateral sites: iD, E) hish-dose FS5-ezposed rut. (F. G) hteh-dose RASS-e%Oosed rat. both ehowin_• hvperplasia of rhe squamous epimeiium (ventromedial site) and squamaus me:aplasia of the pseudas¢atined apirheiium ivenvolateral sitei. high-dose RASS aroua the severitv of this flndine beins similarly low for both SS types. .at the second nose leveh no changes were seen. . The larvnx was round to be the most sensitive orzan for histopatholo_ical chanses following FSS or RASS zxposure (Table ^_: Fia. 3). At the 'oase of the epi_Iottis. a dose-depen- dent diffuse sauamous metaolasia of the pseudostratided epi- thelium and hvperplasia of the squamous epithelium IFia. 4) were found. The mean scores were statisticallv sienificandv hi_her for the FSS- compared to the R-aSS-exposed groups based on the CO concentration. As was the case for nasal epithelial hyperplasia. the incidence was hie-her in the FSS sroups compared to the RASS eroups. while the severity of these hndings showed no remarkable difference. At the ventral depression (arvtenoid projections), very slight hyperplasia and squamous metaplasia were observed in few rats. The statisti- cally significant difference benveen the high-dose RASS and :he sham-exposed eroup concemine the hyperplasia at this site is considered to be incidental since a verv simila mean score and incidence were obtained :or the low-dose FSS _•roup with no indication for dose dependency. There was no difference between the two SS types at the ventral depression. At the lower medial region or the vocal cords, a dose-dependent increase in hvperpiasia of the sc!tamous epithelium was ob- served. This tindins was also seen in two rats of the sham- exposed control and is considered to be incidental. No differ- ence between the two 55 types was seen for this effect when compared on the basis of CO concentration. This is the only morpholopc effect for '.vhich, on the basis of the TP.M con- centration. RA.SS was sli_htlv more active-than FSS. There was no finding in the alveolar region of the lungs. At the tracheal bifurca[ion:-minimal doblet cell hvperplasia was seen in few rats of all exposure groups with no indication for a SS-related affec: (Table'_). Slight goblet cell hv-erplasia was also seen in the bronchial respiratory epithelium with a sii_hdv higher indde^ce in both aigh-dose ctroups ~ no statis- tical sisniticance). No diiference between ihe two SS rypes was observed. NLorphometric determination of the laryngeal epithe!ial thickness at :he araenoid orojections showed numerical in- creases at ail sttes measured in the SS-a.eposed groups com- pared to that of theiham-e.eposed group in a rouehlv dose- dependent manne: (Tabie J), although the increases were statistically sienincant in only a few cases. The only statisti- callv significant dicerexe between the two 5S types vas seen at the vocal cords. %%here RASS was more active than FSS when compared on s,e basis of the CO cancentration. At the end of the -'-day postinhalation period. no relevanr SS-related histapathological cnanves were observed. The eci- thelial chanees obser-ed at the end of the inhalation Pe::cd reverted comoletek . 8(a)P .bLernbofism In the NRE. the ;crrnadon of the bav re?ion metabolites. i.e.. 9-OH-. 7,3-diol-. ar,d 9.10-diol-B(cqP. was induced in all SS- exposed grouos. the aighest factor of induction bein_ seen for 9,10-diol-B(cilP (Table 4). The induction was rou_shlv dose- dependent and statisticallv iilniricanL .
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Page 26 be estimated to be approximately 10 and 3 µg/kg, respectively. The daily TPM dose taken up by the rats in the present study was 3 orders of magnitude higher. As a result of dilution, the concentrations of most RASS components decreased in proportion to the concentrations of either the particulate matter or gas phase markers. The major exceptions to this rule were the low concentrations of nicotine, formaldehyde, and catechol found in the whole-body compared to the head-only exposure chambers. Nicotine is known to adsorb with high affinity to all kinds of surface materials. The same lack of proportional dilution associated with the exposure mode was found for the nicotine concentration in MS (Chen et al. 1989). Formaldehyde is a highly reactive compound which, under whole-body exposure conditions, even reacts with rat excretion products and/or fur (unpublished results; Kewitz and Welsch, 1966). No explanation is available to date for the comparatively low catechol concentrations in the whole-body exposure groups. Biomonitoring via the analysis of HbCO proportions in blood, hemoglobin adducts of 4-AB, and nicotine metabolites in urine confirmed exposure of the rats as planned. Further, valuable information was obtained on the feasibility of using these biomarkers in smoke inhalation studies and on their concentration/dose-responses. After establishing a steady-state equilibrium, the blood HbCO proportion is directly proportional to the RASS CO concentration. However, the HbCO proportion does not reflect the overall dose of RASS taken up by the rats, especially when considering the differences in the daily whole-body and head-only exposure durations. The levels of the 4-AB hemoglobin adduct corresponded to the daily TPM doses for all groups. This adduct is considered to reflect the overall dose of metabolically activated 4-AB over the lifetime of the erythrocytes. However, for smoke inhalation studies, another explanation for the increase in this adduct with increasing TPM doses cannot be ruled out: The metabolic activation of 4-AB might be
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Page 22 Gross Pathology and Organ Weights No RASS-related gross pathological changes were observed. The only statistically significant change in absolute organ weights in the whole-body exposed groups was a slightly decreased absolute heart weight in WB12 (-9 ± 2 %), which was no longer apparent when normalized to the respective body weight. As for the body weights, possible organ weight effects in H012 could not be evaluated due to the lack of a corresponding sham-exposed head-only control group. Histopathology In the anterior level of the nose (level 1), patchy, slight to moderate reserve cell hyperplasia of the respiratory epithelium was observed (Table 3). This effect was concentration dependent in the whole-body exposed rats and similar in degree and incidence in H012 and W812. Slight squamous metaplasia of the respiratory epithelium was observed in WB12, this effect being seen in only a few rats in WB6 and H012. Minimal goblet cell hyperplasia was also seen. At level 2, minimal epithelial changes were seen in the RASS- exposed groups, such as reserve cell hyperplasia and atrophy of the olfactory epithelium and reserve cell hyperplasia of the respiratory epithelium. At levels 2 and 3, slight eosinophilic globules were observed in the sustentacular cells of the olfactory epithelium of the RASS- exposed groups. There were no other histopathological findings in levels 3 and 4 of the nasal cavity. The few larynges that could be evaluated as intended showed distinct squamous metaplasia of the pseudostratified epithelium and hyperplasia of the squamous epithelium at the base of epiglottis (Table 3). Those laryngeal sections that were not cut at the pre-defined m~ '° W . levels could only be qualitatively evaluated. This evaluation confirmed epithelial hyperplasia N - cn : ~'
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Page 28 into the gastrointestinal tract by about a factor of two compared to nose-only exposure. Mauderly et al. (1989) found nicotine concentrations in the plasma and urine of rats which were 5- to 6-fold higher with whole-body MS exposure than with nose-only exposure, based on equal nicotine concentrations in MS. Their corresponding urine cotinine concentrations were 2.6-fold higher. The latter factor fits well with the 2- to 3-fold higher amounts of nicotine metabolites excreted by whole body vs. head-only exposed rats in the present study, based on equal nicotine inhalation. Thus, for RASS, as is the case for other aerosols, whole-body exposure is associated with a significant uptake of test materials by routes other than inhalation. The sum of the five nicotine metabolites determined in H012 accounted for almost 50 % of the calculated inhaled nicotine dose per day. About the same percentage was obtained for the urinary excretion of these five metabolites following intravenous administration of nicotine to male Sprague-Dawley rats (Schepers et al., 1993). This percentage decreased to approximately 20 % in Aroclor 1254-induced Sprague-Dawley rats due to a different metabolic pattern in induced and non-induced rats. These data suggest that the nicotine metabolite pattern of the RASS-exposed rats was similar to that of non-induced rats, although this comparison includes an extrapolation between different rat strains and genders. This interpretation is supported by the lack of any change in the relative pattern of the five metabolites between 3 weeks and 12 months of inhalation. The above mentioned biomonitoring end points cannot be used to determine the deposition of particulate matter in the lungs. The accumulation of pigmented AM may be an indication of particles accumulating in the lungs following smoke inhalation, since this phenomenon was found in rats following inhalation of whole MS but not in those only exposed to the vapor phase of MS (Davis et al., 1975a and b; Coggins et al., 1980). However, the
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ROOM-AGED SLDESTRE.IuI SMOKE LYFGII-ATION TABLE 4 B(a)P Metabolism in the Nasal Respiratory Epithelium 113 FSS RASS Metabotite Sham Low "'[edium High Low Medium wgh 9-OH-B(a)P+ 16 -_ ! 24 = 2 24 r I 35- 2 '?= 1 23- I 23 t 2 1.5' l.5• Z.1' L4' 1.3' 7,8-Diot-B(a)P` 37 = 2 54 = 4 60 = 3 91= 6 50= 3 iS= 2 53 = 5 1.5. L6" 2.5' t.J• 1.3' l.l' 9,10-Diol-B(a)P' ?? = 1 38 s? 51 = 3 79= 6 33= 2 34 _ 2 r0 = 5 L7' 2.3' 3.5' 1.5' IS' L3' 3-OH-B(a)P 602 L 37 726 = 60 571 = 31 650x22 686=35 613=34 613 = 35 1.2 1.0 1.1 _ 1.1 1.1 1.0 4.5-DioI-B(a)P 343 s]1 398 s 36 313 = 19 350 =?1 380 x:9 339 - 27 323 = 19 1.2 0.9 L0 11 I.0' 1.0 ,Vate. y[embolic acti'ittes (nmoU(~ protein x h)) ate saven as mean = SE and factor of induction relative ro the snam-excosed consol graup. ' Indicates statistically siSniucant difiereace to shatn-exnosed conttol goup. T Indicates statlsticatlv si=nificant differences between the two S5 types. topatholoaicaily, it might be speculated that the changes in B(a)P metabolism reflect the changed distdbudon of cell types following SS inhalation- or that there is ceLl-specihc induction. Model cytochrome P450 inducers, such as phenobarbital, 3-methylcholanthrene, and B(a)P itself, which most probably do not affect the morphology of the nasal epithelia, failed to induce the nasal B(a)P metabolism (Bond, 1986; Voist er al-, 1993). Only the administration of the most potent inducers, ?,3.7,3-tetrachlorodibenzo-p-dioxin (Bond, 1986) and Aroclor 1254 (Voigt er al., 1993), resulted in an induction of the B(a)P metabolism in the nasal epithelia. The Aroclor 1254-mediated induction of the B(a)P metabolism was more pronounced in the NRE than in the NOE and did not coincide with the distribution of the cytochrome P450 lA1 induction, suggesting the involve- ment of multiple enzymes in the nasal metabolism of B(a)P. Based on the CO concentrations, the induction of the B(a)P metabolism was more pronounced following inhalation of FSS compared to RASS. Based on the 1P'9[ concentrations, there was no difference in response. mnis could be expected since the components of cigarette smoke that induce the B(a)P merabo- lism. e.g., polycyclic aromatic hydrocarbons, are found mainly in the particulate matter fraction of the smoke (Pasquini at al., 1987), and there seems to be no qualitative change in the SS particulate mate[ial by room-aeing with regard co cytochrome P450 induction. Filtered SS did not induce cvtochrome Pl";O LAI-associated metabolic activities in rnt luns (GebremichaeL er al-, 1995). The dose-dependent induction of the respiratory tract Bta)P metabolism may be useful as a biomarker of exposure to inducing agents, particularly at relatively low doses, although TABLE 5 B(a)P Metabolism in the Lungs FSS RASS M erabolire Sham Low Medium Hi;n Low Medium Hish 9-OH-B(a/P• 13 - ^ 4 117 - 30 177 = 14 235 ="2 . Si - 3. 71 = 13 135 = 1' 7.9-Dibl-B(a)P' -- _ 1 I0.5' -58 = 55 . U." 339 = 21 CS' 476 = 16 107 = 14 5.7' 1->y = 24 _ 316 =_3 1 9.t0-DioI-B(a1P- L_ 4 I L6• t79 = a_ 15.2' ]-16 - t8 '-L4' . 362 = :9.. .- ?.8' _'- x 11 S.5' 105 = 21 14 256-- 11.9' 16.3' '--1.0" 4.9' 7.0' 16.9' t ' ` - 9 - 47 -' 576 53 '14 18 39 237 Q J t2 = 10 3-OH-Bfa)P lo9 13 379 _ 57 6 - - = = . . W 3s' :.u' 5.3" - - 2.0' 2.2' 1.1' a+ , - 5-diol-B(aIP a IG6 = 311 308 = 126 1089 = 134 1a17 = 193 . - 1iC0 ='57 935 = 733 1381 = 243 -A . W l 0.7 19 1.3 L.0 _ 23 L2 tV 1V .Vote. 4leeabolic ac:wi:ies • nmoi//e protein 1< in am _tven as mean = SE and factor of induc-lon refatice :o the sham-exposed control -troup. ' Indicates stanstmailv ~r;mr.cant l.ifference :0 snum-exposed conttol ;roup. -- `Indicates etattsncally si8niticant differences ber.veen :he two SS'Ines. - --
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Page 27 dose-dependently induced, similar to the induction seen for benzo(a)pyrene metabolism in previous cigarette smoke inhalation studies (e.g., Gairola, 1987; Haussmann et al., 1998). Indeed, pretreatment of rats with polycyclic aromatic hydrocarbons was shown to shift the hepatic metabolism of 4-AB towards activated metabolites and to proportionally increase the yield of protein adducts (Orzechowski et al., 1994). In any case, even if the increase in the 4- AB hemoglobin adduct seen in the present study is not a specific marker for 4-AB uptake, it might be a marker for the uptake of compounds capable of inducing 4-AB metabolism. The source of 4-AB responsible for the formation of the adduct level found in the sham-exposed group remains to be determined. A similar 4-AB background adduct level was reported by others (Bryant et al., 1987). High background adduct levels may also be responsible for the failure to detect possible increases in hemoglobin adduct levels of other aromatic amines associated with RASS inhalation. The biomonitoring end points discussed above do not necessarily reflect the route of uptake of the respective compounds. This is especially important for whole-body exposure. Therefore, care has to be taken in determining the dose taken up by inhalation vs. the dose taken up by other routes. During whole-body exposure, aerosol is known to deposit on the fur of the rats and its constituents may be taken up either transdermally or orally by grooming (Langard and Nordhagen, 1980; Wolff et al., 1982; Iwasaki et al., 1988). This increases the total dose taken up and may exaggerate the associated toxicity. It should be kept in mind, however, that humans are exposed to ETS in a manner comparable to whote-body exposure. A certain degree of transdermal uptake of ETS constituents can thus be expected; its contribution to the total uptake remains to be determined. In rats, the gastrointestinal uptake of test material by grooming adds to the non-inhalative transdermal uptake, and this is certainly not representative for human ETS exposure. Chen et al. (1995) concluded that whole-body exposure increased the amount of cigarette smoke particles passing from the fur
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-o.r,cceccc+L saesc=s 11, 1C0-1 t6 ~ 1999) ARr.ae .n. T'C9=8; - Comparison of Fresh and Room-Aged Cigarette Sidestream Smoke in a Subchronic Inhalation Study on Rats Hans-Jiirgen Haussmann,"•` Erwin Anskeit,*'t Dorothea Becker,+ Peter Kuhl,i Walter Stinn,-I Ashok Teredesai, i Peter Voncken.: and ROdiger-A:exander Walk°'t •CRC Cun~rcct Research Cmrer, B-193? Zavenrem• @elgium.• and '*LVBlFO Insirur tir hmlagescne Forsenung, D.51119 K6la Germ_an?_ - Received -May 19. 1997: xcepted September 16, 1997 possible effect thresholds via the determination of dose- Comparison of Fresh and Room-Aged Cigarette Sidestream resnonse relationships. However. one of the most critical issues Smoke in a Subchronic Inhalation Study on Rats. Haussmann, in inves:i_aucg potentiallv toxic effects of environmental ma- H.-J., Anskeit, E., Becker, D., Kuhl, P., Stinn, W., Teredesai, A., Voncken, P., and Walk, R.-A. (1993). Toxicol. Sci. 41, 100-116. [eria!s is the selection of an appropriate experimental ;urroaate - for the test material. Two experimental types of cigarette sidestream smoke (SS) were ETS is a combustion product composed of sidestream smoke compared in a subchronic inhalation study on rars. Fresh SS (FSS) (SS) as well as exhaled mainstream smoke (.ldS) (First. 1935: was generated continuousl,v from the reference cigarette ?R1. Room- Lofroth er al.. 1989; Benner er nL• 1989; Eatou~h er a(.• 1989. aged SS (RASS) was generated by a~ng FSS for 1.5 h in a room with 1990; Baker and Proctor, 1990; Guerin at al., 199?). ETS is noninert surfaces with materials typically found in residences or highly diluted with room air and undergoes chemical and offices. Nlale Sprague-Dawley rats were head-only exposed to three dose levels of each SS type and to filtered, conditioned fresh air physical chanses in composition as a function of aeinsz, e.s.- bv (sham-exposure) for 6 h/day, 7 dayslweek, for 90 days. Room-agn, contact with various surfaces over time (Eatouqh er uL, 1990). resulted in decreased concentrations of various SS componenrs, e.,., Exhaled MS can contribute from 15 to 13y'n of the particulate total particulate matter (TP3p and nicotine, while other componenrs, matter in ETS. but ontv small amounts of the 2as-phase con- such as carbon monovde (CO), were not affected. The CO concen- s[itueats (Baker and Proctor, 1990). tradons were 6, 13, and 23 ppm for both SS tvpes. TP'vt concentra- Real environmental atmospheres are not reproduciblv available rions were between 0.6 and 3.7 µJliterand thus up to 100-fold above as required for a laboratory esceriment- most norablv~ fcr a pro- the maeimum of average concentrations of respiratory suspended loneed inhalation studv. In previous rodeat inha!ation s:udies to particles reported for environmental tobacco smoke. Slight reserve aSSess the biotescal eccviiv of ETS, diluted SS was used as a cell hyperplasia in the anterior part of the nose as well as hyperplasric - and meraplasdc epithelial chan,es in the larynx were the only ob- surrogate (a.~.. von Vleverinck r nl.. 1939; Coggins er c(.. 1997a: served dosedependent fmdiniiis. The metabolism of benzo(rt)- Joad er al.. 1993: Teredesai and Priihs• 199,1; Wiac:.i er al., pyrene-as a proxy for pol-vcvcGc aromadc hydrocarbon membo- 1995b). itw~as used fresh or mederatelv ased bv contac with lism-was induced in the nasal respiratory epithelium and in the c!ativeiv ine.^. SurPaces in whole-body exposure chambers made lunes while no effecr was seen in the nasal olfactory epithelium. The of stainless s.eel and glass and for short duration (s5 min: .wres (owest-observed effect level was 6 ppm CO or 0.6 µ~- IP!Wliter. Ylosr er al., 199d: Teaque e: al•, 199s), due to the hish number of of the effects seen were less expressed in R-ASS- than in FSS-e¢posed air-charioes per hour in these e:coasure systems. These asanfi rats when compared on the basis of the CO cohcentrations. When conditions u'e less representative of human residences or oince compared on the basis ofTP^rI, these effects were equally pronounced for both SS types, suggesting a tuajor role of particulate matter- environmenrs• where :here are materials with larme ;trace areas associated compounds. ?Jl findines reverted to sham conrrol levels and adsorption po[ential- such as curtains or carpets. in addition. follovring a42day postinhaladon period. nssa s«z>~r ~r ra~maw. mean air chan~es oi approxiala[e!y 0.5 per hour :ue charac:erstic ~Sepp5nen. !99-1 ror.residences. ••vhich would cor•acond :o-a mean ETS aee of 2 h. - Exposure to environmental tobacco smoke (ETS) has been In order to address the .zlevance of SS aging in experimental studies. (he objective of the present study was m compare reported to be asseciated with adverse health effects respiratcry ~rsc- responses in the rat to fresn SS tFSS) and Environmental Protection Asencv• 1992). Experimental tasi- - ~ - - ~ coloe)i can provide data on :his associa[ion is discussed in aroom-a~edSS ~ R.~SSj. RASS was ~_enerated by sgm; FSS for recent symposium oaer:iew by Witscai er a(. (1995a), e.g., on 1.d h (mean ase) under axperimental conditions which are more:ealistic ror the human environment than those previously ' P~sant xdd¢sr. 1NBIF0 :nsr.tur Nr bioloe_ische Forscnun_3, D-51f19 used. or whic:: even °.Ca_'_$erate realistic conditions for the Kaln.Ger:nany. . purpose of 7ae-expe.^.ment. To enable a direct compadson.- :c9n.~osn,sa `u0 - 2063643216 Cao~n;it J 19~N 'JV :.^, ivC:ay ~r ToRlcoiO;y .. >!I aghrs ai reprcdueaun n m:+ totm reer.ad. . . .-
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Page 29 pigmentation may also result from lipid peroxidation or hemoprotein-derived iron incorporation in the AM as a secondary effect due to smoke particle inhalation. The number of AM staining positive for iron/hemosiderin was found to increase in rats following subchronic SS inhalation (CO concentration: 35 ppm; Escolar et al., 1995). In the present study, no iron-specific staining was used, and the slight accumulation of pigmented AM in the high concentration groups was seen in hematoxylin/eosin-stained sections. No such pigmentation was reported for subchronic SS inhalation studies on rats (von Meyerinck et al., 1989; Coggins et al., 1993; Lee et al., 1993; Teredesai and PrUhs, 1994; Haussmann et al., 1998). It remains to be investigated whether this pigmentation is due to the higher daily particulate matter dose in the chronic compared to the subchronic studies and/or whether this is related to the chronic inhalation period. AM pigmentation was not seen in the chronic SS/MS inhalation study on A/J mice at 87 µg TPM/l (Witschi et al., 1997a). The discrepancy between the results of these two chronic inhalation studies remains to be resolved in view of a possible use of AM pigmentation as a pulmonary particulate biomonitor. To date, this discrepancy speaks for a species-specific secondary source for the pigmentation rather than for a simple endocytosis of TPM. A dose-dependent increase in the fluorescence of AM from bronchoalveolar lavage was seen in the present study. This fluorescence has not been described in SS inhalation studies to date. Increased AM fluorescence was reported for MS-exposed rats (Coggins et al., 1980; Skold et al., 1993) as well as for smokers compared to nonsmokers (Vassar et al., 1960; Skold et al., 1989). There was no progression of this effect between 6 and 12 months of RASS inhalation, suggesting either a saturation of the effect as discussed for the AM fluorescence in smokers (Skold et al., 1989) or the constitution of a steady-state equilibrium which would be dependent on the RASS concentration rather than the daily or accumulating dose. The nature of the MS-dependent fluorescence is still open, and similar causes have been discussed as for the histologically observed AM pigmentation (e.g., Skold et al., 1992).
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R00%4AGED S[DESTREA,v( SNtGKE CiH.-vLaT(ON 109 the maximum of average concentrations of RSP reported for ammonia are chemically reactive compounds. They reacted ETS, particularly in residences and offices (Oldaker et al., differently with various surface materials in a room (Voncken 1990; Guerin et aL, 1992; U-S. Environmental Protection et aL., 1994). The aas-phase components acetaldehyde and Agency, 1992; lenkins et al., 1996). The lowest concentrations acrolein were less reactive than formaldehyde and did not used here may overlap with those encountered in extreme change in spi[e of their aldehyde functional o oups. Due to the human exposure situations. The concentration range between _ low concentration of nitroccen oxides, NO remained stable 0.1 and 10 p.~g TP'vlLliter has been used widely by the scientific under the conditions of this study with no oxidauon-to N0, community for subchronic or chronic toxicolo;ical testing of being detectable. Also. CO was not affected by aging, and thus experimental ETS surrogates in rodents (e.g., von 'vleyerinck et proved to be useful as a marker in experimental studies on SS al-, 1989; Cogons er al., 1993a; Joad er al., 1993; Teredesai and the leading dosing parameter in the present study. In field and Pruhs, 1994; Witschi er al., 1995b), and enables the de- studies, however, CO is not useful as a marker for ETS since termination of lowest-observed effect levels. In only a few the majority of the indoor CO stems from sources other than cases was this concentration ranee exceeded, for example in ETS (Eatough er al., 1990). the chronic study reported by Witschi er al. (1997), in which The chemical and physical characteristics of RASS re- this concentration range was exceeded by one order of mag- mained constant over the 90-day period of inhalation, resulting nitude. However, this concentration was hishly toxic, as evi- in stable and reproducible test atmospheres throughout the denced by the body weight loss in the exposed mice. The study. Thus, no saturation of reactive surfaces or shifting conceno•ation range between 0.1 and 10 µ2 TPk1/I was used to equilibria with built-up deposits were observed. investit:ate a variety of respiratory tract end points- such as The lack of a detectable body weistt effect by SS exposure morphological (von Meyerinck er a/., 1989) or biochemical (Ji in the present study is in agreement with the results seen in er al., 1994) changes, ,;enoroxiciry (Lee er al., 1992), or in- previous studies at similar SS concentrations (von Meyerinck creased DN?A synthesis (Rajini and Witschi, 1994). Therefore, et aL., 1989: Coggins er aL., 1993a; Teredesai and Pruhs, 1994). this concentration ranse was deemed useful for a comparative The histopatholoQical findines seen in previous SS inhala- inhalation stud-v on the effects of room-aging in the rat respi- don studies of similar design were also seen in the present ratory tract. _ study. Slight hyperplasia of the respiratory epitF.etium was - No information on the effects of 5S on the nasal xenobiotics observed in the antetior par, of the nose with no findines at the metabolism has been reported to date. Therefore, to assess the next posterior level. which includes the olfactory epithelium. effect of SS inhalation on the xenobiotics metabolism in the This is consistent inrype, location, severity, and seasitivir,v respiratory tract, the formation of B(a)P merabolites in the with the findings described by von Mevennck er ad. (1989), nasal olfactory and respiratory epithelia and tunes was also Cogsins er a!. (1993a). and Teredesai and Pruhs (199=). T'ne investieated in the present study. metaplasia described by von Meyerinck er a!. (1989) w'as seen - -5S was room-aged under steadv-state dvnamic conditions. neither bv Coggins et al. (1993a) and Teredesai and Pruhs The room-a6ne-related changes in the chemical composition (1994) nor in the present study. As in the previous studies, na - of the SS confirmed previous experience about the instability statistically significant histopathological findings were seen in of SS due to the physiccchemical and chemical nature of its the lower respirstor,v tract, although there was an indication in components reviews by Baker and Proctor, 1990. and the present study of an increased incidence of 'oronchial _oblet Eatouoh et al.. I990). Details on the contribution of various cells in both high-dose groups. - materials to the overall aging effect seen in this study are given The most sensitive site for histopatholoqical changes in the bv Voncken er a!. 1994). - present study was the laryax, showing slieht hyperpLasia and - The decrease in TPM concentradons can be attributed pri- me[aplasia of a number of epithelia at different locations within -- marily to particle deposition on all surtaces in the aging room the larynx, in oarticular •:he base oi epiglottis. Similar chanees as indicated, e.a.. bv the yellowish staining of the wallpaper, were seen in a previous subchranic SS inhalation study per- N,- Dunno the asin,, orocess, the mass median aerodvnamic di- formed in :he same labaratory (Teredesai and Pruhs, 1991). ameter of the aerosol slightly increased, the seometric standard However, no morpholo2ical changes in laryngeal epithe!ia W~-- deviation remaining unchansed. This small shift is not ex- were obser:ed in the studies by von Meyerinck et a(. (1939) ~ pected to influence the particle deposition probability in the and Cog3ins er al. i 1993a), The few differences Cn the exper- - respirator,v tract of the rats (Raabe rr al.. 1997). Reports about imental designs among these studies are not considered to ~ asine-related changes in SS particle size distcibution have been account for this discrepancv in lar,inx findings. Rather, differ- inconsistent describing both initial size decrease (Ingebrethsen ences in sectioning levels mlght affect the optimal detection of and Sears. 1986) and increase (Benner e: al., 1989) upon aaing these chanses. The larvneeal •n,vpervlasia at the arytenoid pro- in relatively inett chambers. In contrast to NIS. nicotine in SS jections was conflrmed'oy morphometric analyses of the epi- is a~las-phase component ireviewed'oy Eatough ztal., 1990). thelium at this sifeT the increase in the epithe!ia1 thickness It readily adsorbs to surfaces and reevaporates upon cleansing being up to approxiniately 30'.b in the present and previous the ambient atmosphere (Piade et al., 1996). Forrnaldehyde and (Teredesai and Priihs. 199d) studies. Squamous metaplasia at
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Page 30 A possible relationship between AM pigmentation and fluorescence remains to be i nvestigated. The degree of body weight gain reduction observed for W812 can be interpreted as a sign of toxicity and demonstrates that the study design would satisfy the requirements set by the OECD (1981) or others for the highest dose level in a carcinogenicity study. With few exceptions, the histopathological changes observed in the nose were qualitatively and quantitatively similar to those observed following subchronic RASS inhalation (Haussmann et al., 1998) indicating that there was no progression of these findings. This analogy further suggests that these effects, except for the minimal atrophy in the olfactory epithelium, could be considered adaptive responses to repeated irritation as shown for subchronic studies (von Meyerinck et al., 1989; Coggins et al., 1993; Haussmann et al., 1998). In addition to the reserve cell hyperplasia seen in the anterior part of the nose in the subchronic studies, slight squamous metaplasia of the respiratory epithelium in this part of the nose was seen in WB12. The appearance of this effect in WB12 might be due to the higher daily TPM dose compared to the subchronic studies and/or the chronic inhalation period. Some additional epithelial changes in nose level 2, although not seen in subchronic SS inhalation studies, are not considered relevant due to their very low incidence and degree. The eosinophilic material observed in olfactory epithelial sustentacular cells at levels 2 and 3 has not been reported in cigarette smoke inhalation studies. The significance of this finding is not clear. In the larynx, the hyperplastic and metaplastic changes were the most pronounced. Those seen in H012 were in line with those observed in the previous subchronic head-only exposure study (Haussmann et al., 1998) on the basis of an assumed linear concentration-response relationship (Figure 7A). However, the degree of the changes seen in W66 and WB12
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Page 32 The composition of the bronchoalveolar lavage cells did not indicate an inflammatory response to chronic RASS inhalation, although there was a slight but statistically insignificant increase in the number of AM, a response similar to that seen in rats following MS inhalation (Gairola, 1986; Bjermer et al., 1993; Miller et al., 1996). Also for MS, Mauderly et al. (1989) reported an increase in the concentration of PMNL in the bronchoalveolar lavage fluid of rats, whereas no statistically significant responses were found by others for this species (Gairola, 1986; Bjermer et al., 1993; Miller et al., 1996). Mice were considered to be more sensitive to MS inhalation with regard to PMNL responses (Gairola, 1986). However, no histopathological indication of an inflammatory response was seen by Witschi et al. (1997a) when A/J mice were chronically exposed to a mixture of SS and MS at a TPM concentration as high as 87 µg/l. On the other hand, pronounced effects on rat bronchoalveolar lavage cell composition indicating a strong inflammatory response were found following inhalation of aerosols including insoluble particles,- such as Diesel engine exhaust, at particulate matter concentrations far below those used in cigarette smoke inhalation studies (Henderson et al., 1988). Pulmonary inflammation following particle inhalation has been associated with lung carcinogenesis in experimental animals (Dungworth et al., 1994). An increase in the formation of 8-OHdG has been investigated in the present study as a marker for oxidative stress since this end point has been associated with carcinogenesis (Floyd, 1990) and with cigarette smoking (Asami et al., 1996, 1997). The observed increase of the 8-OHdG levels in the nasal epithelia at the highest RASS concentration was only in part statistically significant and consistent following 6 or 12 months of inhalation. The biological relevance of this effect remains to be investigated. With regard to its relation to carcinogenesis, it is interesting to note that Witschi et al. (1997a,b) have observed increased cell proliferation and epithelial lesions in the nose but did not specifically report nasal tumors I
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Page 31 exceeded those observed in H012 and also those seen in the previous subchronic head-only exposure studies. Normalizing these larynx effects to daily TPM doses reveals a dose- response relationship with no distinctTon between subchronic or chronic inhalation periods for the squamous metaplasia (Figure 7B) as well as the hyperplasia. Thus, there seems to be no progression with prolonged inhalation. An association of most of the histopathological changes with the particulate matter phase of the SS aerosols was assumed in the previous subchronic study (Haussmann et al., 1998). The upper respiratory tract results of the present study support this interpretation and place emphasis on the daily particulate matter dose rather than on concentration (WB12 vs. H012 / W86). This contrasts with gaseous irritants, such as formaldehyde, which induced epithelial hyperplasia and metaplasia in the anterior part of the nose depending on the concentration rather than on the daily inhaled dose (Wilmer et al., 1989). The base of epiglottis was the most sensitive site of the respiratory tract in this and the subchronic study (Haussmann et al., 1998). The hyperplastic and metaplastic changes seen there were not seen in SS inhalation studies performed by other laboratories (von Meyerinck et al., 1989; Coggins et al., 1993). The only relevant histopathological finding in the lower respiratory tract following chronic RASS inhalation was slight reserve cell hyperplasia in the bronchial respiratory epithelium, which was not seen in the subchronic SS inhalation studies (von Meyerinck et al., 1989; Coggins et al., 1993; Teredesai and Pruhs, 1994; Haussmann et al., 1998). It remains to be investigated whether this effect is due to the chronic inhalation period and/or the higher concentration or dose in the present study compared to the previous ones. Morphometric analyses to assess the pulmonary changes as reported by Escolar et al. (1995) were not performed in the present study.
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Page 35 seen on the levels of the 4-AB hemoglobin adduct (RASS, this study) or those of pulmonary DNA adducts (MS, Mauderly et al., 1989) detected by the postlabeling method. The role of particulate matter-related smoke constituents in the pulmonary carcinogenicity in A/J mice has recently been questioned by Witschi et al. (1997b), who reported that the carcinogenic potential of a HEPA-filtered mixture of SS and MS would be similar to that of the unfiltered smoke. However, the susceptibility as well as the relevance of this animal model remains to be clarified (Maronpot et al., 1986), especially at the dose/toxicity level employed. in summary, for histopathological changes, pulmonary inflammation, or oxidative DNA damage, there was little indication of progression or occurrence of new effects following an extended inhalation period of 12 months. The two slight histopathological changes which were not seen in the subchronic studies, i.e., squamous metaplasia in the anterior nasal respiratory epithelium and the bronchial reserve cell hyperplasia, might well be due to the higher concentration and daily dose in the chronic compared to the subchronic studies. However, it cannot be ruled out that these effects are due to the chronic inhalation period. Chronic head- only exposure to cigarette smoke was shown to be technically feasible and is considered preferable to whole-body exposure in order to avoid artificial changes in smoke composition and the considerable non-inhalative uptake of smoke constituents. I
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Page 37 Chen, 8. T., Bechtold, W. E., Barr, E. B., Cheng, Y.-S., Mauderly, J. L., Cuddihy, R. G. 1989. Comparison of cigarette smoke exposure atmospheres in different exposure and puffing modes. InhaL Toxrcol. 1:331-347. - Chen, B. T., Benz, J. V., Finch, G. L., Mauderly, J. L., Sabourin, P. J., Yeh, H. C., and Snipes M. B. 1995. Effect of exposure mode on amounts of radiolabeled cigarette particles in lungs and gastrointestinal tracts of F344 rats. lnhal. ToxicoL 7:1095-1108. Chow, C. K. 1993. Cigarette smoking and oxidative damage in the lung. Ann. N. Y. Acad. Scr. 686:289-298. Coggins, C. R. E., Fouillet, X. L. M., Lam, R., and Morgan, K. T. 1980. Cigarette smoke induced pathology of the rat respiratory tract: a comparison of the effects of the particulate and vapour phases. Toxicology 16:83-101. Coggins, C. R. E., Ayres, P. H., Mosberg, A. T., Ogden, M. W., Sagartz, J. W., and Hayes, A. W. 1992. Fourteen-day inhalation study in rats, using aged and diluted sidestream smoke from a reference cigarette, I. Inhalation toxicology; histopathology. Fundam. AppL Toxicol. 19:133-140. Coggins, C. R. E., Ayres, P. H., Mosberg, A. T., Sagartz, J. W., and Hayes, A. W. 1993. Subchronic inhalation study in rats, using aged and diluted sidestream smoke from a reference cigarette. Inhal. Toxicol. 5:77-96. o* - W,- mf ~ Davis, 8. R., Whitehead, J. K., Gill, M. E., Lee, P. N. Butterworth, A. D., and Roe, F. J. C. w Q; 1975a. Response of rat lung to inhaled vapour phase constituents (VP) of tobacco smoke alone or in conjunction with smoke condensate or fractions of smoke condensate given by intratracheal instillation. Br. J. Cancer31:462-468. I
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Page 34 compared to W812: Aqueous catechol solutions were capable of generating 8-OHdG in vitro (Leanderson and Tagesson, 1990), and a combined intraperitoneal administration of catechol and hydroquinone or phenol resufted in an increased level of 8-OHdG in mouse bone marrow (Kolachana et al., 1993). Using the whole-body exposure mode, the rats could be exposed for a daily duration longer than that considered feasible for head-only exposure, thereby maximizing the daily RASS dose. This also enabled evaluations on the impact of the daily RASS dose vs. the RASS concentration as demonstrated, e.g., for the histopathological changes in the larynx. For most of the end points investigated in this study, the exposure mode per se did not influence the RASS-induced changes, which were mainly local effects in the respiratory tract. It did, however, impact the evaluation of the body weight gain, which was essentially the only sign of systemic toxicity in the present study. In addition, the exposure mode might have also influenced the formation and excretion of 8-OHdG. For cigarette smoke carcinogenicity studies, whole-body exposure must be considered to be a substantial confounding factor: (1) The composition of the cigarette smoke aerosol, either RASS (present study) or MS (Chen et al., 1989), was found to artificially change in the whole-body situation. (2) There is a considerable non-inhalative uptake of smoke components which, based on the nicotine biomonitoring data for RASS (present study) and MS (Mauderly et al., 1989; Chen et al., 1995), might exceed the inhalative uptake by far. NNK, a compound structurally similar to nicotine, is a rodent lung carcinogen regardless of the route of administration, including dermal application (LaVoie et al., 1987), with a preferential formation of adenomas and adenocarcinomas (Hoffmann et al., 1996). Furthermore, pulmonary DNA adducts were found after topical application of MS condensate (Randerath et al., 1988) or Diesel engine exhaust extracts (Gallagher et al., 1990) to mouse skin. The extent of non-inhalative uptake might be different for nicotine and other compounds, since no influence of the exposure mode was
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Page 42 Lee, C. K., Brown, B. G., Reed, E. A., Coggins, C. R. E., Doolittle, D. J., and Hayes, A. W. 1993. Ninety-day inhalation study in rats, using aged and diluted sidestream smoke from a reference cigarette: DNA adducts and alveolar macrophage cytogenesis. Fundam. Appl. Toxrcol 20:393-401. Liu, J., Wang, X., Shigenaga, M. K., Yeo, H. C., Mori,_A., and Ames, B. N. 1996. Immobilization stress causes oxidative damage to lipid, protein, and DNA in the brain of rats. FASEB J 10:1532-1538. Loft, S., Vistisen, K., Ewertz, M., Tjonneland, A., Overvad, K., and Poulsen, H. E. 1992. Oxidative DNA damage estimated by 8-hydroxydeoxyguanosine excretion in humans: influence of smoking, gender and body mass index. Carcinogenesis 13:2241-2247. Maronpot, R. R., Shimkin, M. B., Witschi, H. P., Smith, L. H., and Cline, J. M. 1986. Strain A mouse pulmonary tumor test results for chemicals previously tested in the National Cancer. Institute carcinogenicity tests. J. Natl. Cancerlnst 76:1101-1112. Martin, P., Heavner, D L., Nelson, P. R., Maiolo, K. C., Risner, C. H., Simmons, P. S., Morgan, W. T., and Ogden, M. W. 1997. Environmental tobacco smoke (ETS): a market cigarette study. Environ. Int 23:75-90.
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Page 33 using SS/MS concentrations up to one order of magnitude higher than those in the present study. In contrast to the results obtained for the nasal epithelia, in the lungs no statistically significant effect in the levels of 8-OHdG was found following 6 and 12 months of RASS inhalation; indeed, the levels tended to decrease. This might be plausible in view of several adaptive antioxidant responses reported for lungs following cigarette smoke exposure, e.g., the accumulation of vitamins E and C or increased activities of glutathione peroxidase or superoxide dismutase, as reviewed by Chow (1993). Furthermore, the repair of oxidative DNA modifications might be induced as a consequence of chronic exposure as reported for the repair activity in the leukocytes of smokers (Asami et al., 1996). However, the above results do not preclude possible oxidative changes in particular pulmonary cell types not detectable by analyzing the homogenate of the whole organ. At the dose level used in the present study and within the limitations of the method, these data do not support the hypothesis of a free radical-mediated oxidative stress in the lungs of SS-exposed rodents (Witschi et al., 1997b). The determination of the urinary excretion of 8-OHdG enables an overall assessment of oxidative DNA modifications and repair in the whole body (Shigenaga et al., 1990). A 50 % increase of the 8-OHdG excretion was reported for smokers (Loft et al., 1992). In the present study, a dose dependent increase of 8-OHdG excretion in the whole-body exposed rats was seen following 5 months of RASS inhalation. After 12 months, however, this effect was no longer seen. It is not known whether this can be explained by adaptive processes in the course of chronic RASS inhalation. Further, a distinction between effects related to the head- only RASS exposure and those related to the oxidative stress possibly associated with a certain degree of immobilization (Liu et al., 1996) imposed by the head-only exposure mode is not possible. Another point to consider is the higher catechol concentration in H012 O '"
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Page 36 REFERENCES American Association for Laboratory Animal Science 1991. Policy on the Humane Care and Use of Laboratory Animals. Lab. Animal Sci. 42:17. Asami, S., Hirano, T., Yamaguchi, R., Tomioka, Y., Itoli, H., and Kasai, H. 1996. Increase of a type of oxidative DNA damage, 8-hydroxyguanosine, and its repair activity in human leukocytes by cigarette smoking. Cancer Res. 56:2546-2549. Asarni, S., Manabe, H., Miyake, J., Tsurudome, Y., Hirano, T., Yamaguchi, R., Itoh, H., and Kasai, H. 1997. Cigarette smoking induces an increase in oxidative DNA damage, 8- hydroxydeoxyguanosine, in a central site of the human lung. Carcinogenesis 18:1763- 1766. Bjermer, L., Cai, Y., Nilsson, K., Hellstriim, S., and Henriksson, R. 1993. Tobacco smoke exposure suppresses radiation-induced inflammation in the lung: a study of bronchcalveolar lavage and ultrastructural morphology in the rat. Eur. Respir. J. 6:1173- 1180. Bomhard, E., and Rinke, M. 1994. Frequency of spontaneous tumours in Wistar rats in 2-year studies. Exp. Toxic. Pathol. 46: 17-29. . Brightwell, J., Fouillet, X., Cassano-Zoppi, A.-L., Bernstein, D., Crawley, F., Duchosai, F., ~ 0 Gatz, R., Perczel, S., and Pfeifer, H. 1989. Tumours of the respiratory tract in rats and hamsters following chronic inhalation of engine exhaust emissions. J Appl. Toxicol. 9:23- m - nJ ~ 31. Bryant, M. S., Skipper, P. L., Tannenbaum, S. R., and Maclure, M. 1987. Hemoglobin adducts of 4-aminobiphenyl in smokers and nonsmokers. Cancer Res. 47:602-608.
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Page 43 Mauderly, J. L., Bechtold, W. E., Bond, J. A., Brooks, A. L., Chen, B. T., Cuddihy, R. G., Harkema, J. R., Henderson, R. F., Johnson, N. F., Rithidech, K., and Thomassen, D. G. 1989. Comparison of 3 methods of exposing rats to cigarette smoke. Exp. Pathol, 37:194- 197. Miller, K., Hudspith, B., Cunninghame, M., Prescott, C. and Meredith, C. 1996. Effect of cigarette smoke exposure on biomarkers of lung injury in the rat. Inhal. Toxicol. 8:803- 817. National Toxicology Program 1991. Specifications for the conduct of studies to evaluate the toxic and carcinogenic potential of chemical, biological and physical agents in laboratory animals for the National Toxicology Program (NTP). Research Triangle Park: NTP. Nikula, K. J., Snipes, M. B., Barr, E. B., Griffith, W. C., Henderson, R. F., and Mauderly, J. L. 1995. Comparative pulmonary toxicities and carcihogenicities of chronically inhaled Diesel exhaust and carbon black in F344 rats. Fundam. Appl. Toxicol. 25:80-94. OECD Organization for Economic Co-Operation and Development 1981. Guideline for Testing of Chemicals 451, Carcinogenicity Studies. Paris: OECD Orzechowski, A., Schrenk, D., Schut, H. A. J., and Bock, K. W. 1994. Consequences of 3- methylcholanthrene-type induction for the metabolism of 4-aminobiphenyl in isolated rat hepatocytes. Carcinogenesis 15:489-494. Randerath, E., Mittal, D., and Randerath, K 1988. Tissue distribution of covalent DNA damage in mice treated dermally with cigarette 'tar': preference for lung and heart DNA. Carcinogenesis 9:75-80. I
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Page 38 Davis, B. R., Whitehead, J. K., Gill, M. E., Lee, P. N. Butterworth, A. D., and Roe, F. J. C. 1975b. Response of rat lung to inhaled tobacco smoke with or without prior exposure to 3,4-benzpyrene (BP) given by intratracheal instillation. Br. J. Cancer 31:469-484. Duncan, D. B. 1955. Multiple range and multiple F-tests. Biometrics 11:1-42. Dungworth, D. L., Mauderly, J. L., and Oberdoerster, G. 1994. Toxic and carcinogenic effects o(solid particles in the respiratory tract. Washington, D.C.: ILSI press. Escolar, J. D., Martinez, M. N., Rodriguez, F. J., Gonzalo, C., Escolar, M. A., and Roche, P. A. 1995. Emphysema as a result of involuntary exposure to tobacco smoke: morphometrical study of the rat. Exp. Lung Res. 21:255-273. Floyd, R. A. 1990. The role of 8-hydroxyguanine in carcinogenesis. Carcinogenesis 11:1447- 1450. Gallagher, J. E., Jackson, M. A., George, M. H., and Lewtas, J. 1990. Dose related differences in DNA adduct levels in rodent tissues following skin application of complex mixtures from air pollution sources. Carcinogenesis 11:63-68. Gairola, C. G. 1986. Free lung cell response of mice and rats to mainstream cigarette smoke exposure. Toxicol. Appl. Pharmacol. 84:567-575. Gairola, C. G. 1987. Pulmonary aryl hydrocarbon hydroxylase activity of mice, rats and guinea pigs following long term exposure to mainstream and sidestream cigarette smoke. Toxicology 45:177-184. Griffith, R. B., and Standafer, S. 1985. Simultaneous mainstream-sidestream smoke exposure systems II. The rat exposure system. Toxicology 35:13-24.
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Page 40 Hoffmann, D., Rivenson, A., and Hecht, S. S. 1996. The biological significance of tobacco- specific N-nitrosamines: smoking and adenocarcinoma of the lung. Critical Rev. Toxicol. 26:199-211. Iwasaki, M., Yoshida, M., Ikeda, T., Tsuda, S., and Shirasu, Y. 1988. Comparison of whole- body versus snout-only exposure in inhalation toxicity of fenthion. Jpn. J. Vet. Sci. 50: 23- 30. Jenkins, R. A., Palausky, A., Counts, R. W., Bayne, C. K., Dindal, A. B., and Guerin, M. R. 1996. Exposure to environmental tobacco smoke in sixteen cities in the United States as determined by personal breathing zone air sampling. J. Exposure Anal. Environ. Epidem. 6:473-502. Ji, C. M., Plopper, C. G., Witschi, H. P., and Pinkerton, K. E. 1994. Exposure to sidestream cigarette smoke alters bronchiolar epithelial cell differentiation in the postnatal rat lung. Am. J. Respir. Cell. Mol. Biol. 11:312-320. Kewitz, H., and Welsch, F. 1966. Ein gelber Farbstoff aus Formaldehyd und Kynurenin bei hexaminbehandelten Ratten. Naunyn-Schmiedeberg's Arch. Pharmakol. Exp. Pathol. 254:101-108. Klimisch, H. J., Meissner, K., and Wernicke, H. 1974. Quantitative determination of carbon monoxide in blood by gas chromatography. Z. Klin. Chem. Klin. Biochem. 12:535-538. w , ON Koch, G. G., and Edwards, E. 1988. Clinical efficiency trials with categorial data. In W; N V Biopharmaceutical statistics for drug development, ed. K.E. Pearce, pp. 403-457, New ro,- York, Marcel Dekker.
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Page 46 US Environmental Protection Agency 1996. Proposed guidelines for carcinogen risk assessment. Federal Register 61(79):1 7960-1 801 1. Vandenberghe, J. 1990. Life-span data and historical data in carcinogenicity testing in Wistar rats Crl.• (WI)BR. Sulzfeld, Germany: Charles River Deutschland. Vassar, P. S., Culling, C., and Saunders, A. M. 1960. Fluorescent histiocytes in sputum related to smoking. Arch. Pathol. 70:649-652. Voncken, P., Stinn, W., Haussmann, H.-J., and Anskeit, E. 1994. Influence of aging and surface contact on the composition of cigarette sidestream smoke. Models for environmental tobacco smoke. In Toxic and carcinogenic effects of solid particles in the respiratory tract, eds. D.L. Dungworth, J.L. Mauderly, and G. Oberdorster, 629-635. Washington, DC: ILSI Press. von Meyerinck, L., Scherer, G., Adikofer, F., Wenzel-Hartung, R., Brune, H., and Thomas, C. 1989. Exposure of rats and hamsters to sidestream smoke from cigarettes in a subchronic inhalation study. Exp. Pathol. 37:186-189. Wilmer, J. W. G. M., Woutersen, R. A., Appelman, L. M., Leeman, W. R., and Feron, V. J. 1989. Subchronic (13-week) inhalation toxicity study of formaldehyde in male rats: 8-hour intermittent versus 8-hour continuous exposures. Toxicol. Lett. 47:287-293 Witschi, H., Oreffo, V. I. C., and Pinkerton, K. E. 1995a. Six-month exposure of strain NJ mice to cigarette sidestream smoke: cell kinetics and lung tumor data. Fundam. Appl. Toxicol. 26:32-40. rt €n _ w na ' v
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Page 44 Romeis, B. 1968. Mikroskopische Technik. Munchen, Germany: Oldenbourg Verlag. Rustemeier, K., Demetriou, D., Schepers, G., and Voncken, P. 1993. High-performance liquid chromatographic determination of nicotine and its urinary metabolites via their 1,3-diethyl- 2-thiobarbituric acid derivatives. J. Chromatogr. 613:95-103. Sachs, L. 1982. Applied statistics., New York, NY: Springer Verlag. Sander, B., Andersson, J., and Andersson, U. 1991. Assessment of cytokines by immunofluorescence and the paraformaldehyde-saponin procedure. Immunol. Rev. 119:65-93. Sanders, C. L., and Lundgren, D. L. 1995. Pulmonary carcinogenesis in the F344 and Wistar rat after inhalation of plutonium dioxide. Radiat. Res. 144:206-214. Schepers, G., Rustemeier, K., Walk, R: A., and Hackenberg, U. 1993. Metabolism of S- nicotine in noninduced and Aroclor-induced rats. Eur. J. Drug Metab. Pharmacokinet. 18:187-197. Schlage, W. K., Biilles, H., Friedrichs, D., Kuhn, M., Teredesai, A., and Terpstra, P. 1997. Tobacco smoke-induced alterations of cytokeratin expression in the rat nasal cavity following chronic inhalation of room-aged sidestream smoke. Abstract presented at the 6`" European Meeting of Environmental Hygiene, Graz, Austria; manuscript submitted. Shigenaga, M. K., Park, J: W., Cundy, K. C., Gimeno, C. J., and Ames, B. N. 1990. In vivo oxidative DNA damage: measurement of 8-hydroxy-2'-deoxyguanosine in DNA and urine by high-performance liquid chromatography with electrochemical detection. Methods in enzymology 186:521-530.
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Page 47 Witschi, H., Pinkerton, K. E., Coggins, C. R. E., Penn, A., and Gori, G. B. 1995b. Environmental tobacco smoke: experimental facts and societal issues. Fundam. Appl. Toxicol. 24:3-12. Witschi, H., Espiritu, I., Peake, J. L., Wu, K., Maronpot, R. R., and Pinkerton, D. E. 1997a. The carcinogenicity of environmental tobacco smoke. Carcinogenesis 18:575-586. Witschi, H., Espiritu, I., Maronpot, R. R., Pinkerton, D. E., and Jones, A. D. 1997b. The carcinogenic potential of the gas phase of environmental tobacco smoke. Carcinogenesis 18:2035-2042. Wolff, R. K., Griffis, L. C., Hobbs, C. H., and McClellan, R. O. 1982. Deposition and retention of 0.1 µm 6'Gaz03 aggregate aerosols in rats following whole body exposure. Fundam. Appl. Pharmacol. 2:195-200. Woutersen, R. A., Appelman, L. M., Van Garderen-Hoetmer, A., and Feron, V J. 1986. Inhalation toxicity of acetaldehyde in rats, 3. Carcinogenicity study. Toxicology 41:213- 231. Young, J. T. 1981. Histopathologic examination of the rat nasal cavity. Fundam. Appl. Toxicol. 1:309-312.
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Page 41 Kolachana, P., Subrahmanyam, V. V., Meyer, K. B., Zhang, L., and Smith, M. T. 1993. Benzene and its metabolites produce oxidative DNA damage in HL60 cells in vitro and in the bone marrow in vivo. Cancer Res. 53:1023-1026. Kroes, R., Garbis-Berkvens, J. M., de Vries, T., and van Nesselrooy, J. H. J. 1988. Histopathological profile of a Wistar rat stock including a survey of the literature. J. Gerontol. 36:259-279. Kutzer, C, Branner, B., Zwickenpflug, W, and Richter, E. 1997. Simultaneous solid-phase extraction and gas-chromatographic mass-spectrometric determination of hemoglobin adducts from tobacco-specific nitrosamines and aromatic amines. J. Chromatogr. Sci. 35:1-6. Langard, S., and Nordhagen, A.-L. 1980. Small animal inhalation chambers and the significance of dust ingestion from the contaminated coat when exposing rats to zinc chromate. Acta Pharmacol. Toxicol. 46:43-46. LaVoie, E. J., Prokopczyk, G., Rigotty, J., Czech, A., Rivenson, A., and Adams, J. D. 1987. Tumorigenic activity of the tobacco-specific nitrosamines 4-(methylnitrosamino)-1-(3- pyridyl)-1-butanone (NNK), 4-(methylnitrosamino)-4-(3-pyridyl)-1-butanol (iso-NNAL) and N-nitrosonornicotine (NNN) on topical application to SENCAR mice. Cancer Lett. 37:277- 283. Leanderson, P., and Tagesson, C. 1990. Cigarette smoke-induced DNA damage: role of hydroquinone and catechol in the formation of the oxidative DNA-adduct, 8- hydrocydeoxyguanosine. Chem. Biol. Interact. 75:71-81.
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Page 39 Guerin, M. R., Jenkins, R. A., and Tomkins, R. A. 1992. The chemistry of environmental tobacco smoke: Composition and measurement. Chelsea, MI: Lewis Publishers. Gupta, R. C. 1984. Nonrandom binding of the carcinogen N-hydroxy-2-acetylaminofluorene to repetitive sequences of rat liver DNA in vivo. Proc. Natl. Acad. Sci. USA 81:6943-6947. Gupta, M. P., Khanduja, K. L., Koul, I. B., and Sharma, R. R. 1990. Effect of cigarette smoke inhalation on benzo[aJpyrene-induced lung carcinogenesis in vitamin A deficiency in the rat. Cancer Lett. 55:83-88. Guyton, A. C. 1947. Measurement of the respiratory volumes of laboratory animals. Am. J. Physiol. 150:70-77. Haussmann, H: J., Anskeit, E., Becker, D., Kuhl, P., Stinn, W., Teredesai, A., Voncken, P.; and Walk, R: A. 1998. Comparison of fresh and room-aged cigarette sidestream smoke in a subchronic inhalation study on rats. Toxicol. Sci. 41:100-116. Heinrich, U., Fuhst, R. Rittinghausen, S., Creutzenberg, 0., Bellmann, B., Koch, W., and Levsen, K. 1995. Chronic inhalation exposure of Wistar rats and two different strains of mice to Diesel engine exhaust, carbon black, and titanium dioxide. Inhal. Toxicol. 7:533- 556. Henderson, R. F., Pickrell, J. A., Jones, R. K., Sun, J. D., Benson, J. M., Mauderly, J. L., and McClellan, R. O. 1988. Response of rodents to inhaled diluted Diesel exhaust: biochemical and cytological changes in bronchoalveolar lavage fluid and in lung tissue. Fundam. Appl. Toxicol. 11:546-567. r a ° o+ 'd .p ,
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Page 48 TABLE 1. Experimental Groups Group Code TPM Target Concentration (µg/I) Exposure Mode, Daily Exposure Duration Daily TPM Target Dose (µg/I x h) Total Number of Rats Initially Exposed WBO 0 whole-body, 12 h 0 96 WB6 6 72 48 WB12 - 12 144 96 H012 12 head-only, 7 h 84 48
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Page 49 TABLE 2. Concentrations of Selected Smoke Components in the Test Atmospheres Exposure Groups Parameter Unit of Measure n WB0 WB6 WB12 H012 TPM pg/1 257 to 259 n.d. 5.9 ± 0.5 11.9 ± 0-6 12.1 + 0.7 carbon monoxide ppm 259 n.d. 27 ± 2 51 ± 2 42 + 2 carbon dioxide (chamber inlet) " 11 to 12 270 ± 130 390 ± 150 510 ± 160 480 + 150 "(chamber outlet) " 11 to 12 1060 ± 90 1400 ± 240 1410 ± 260 2000 + 650 nicotine pg/I 56 to 60 n.d. 0.94 ± 0.38 1.95 ± 0.57 2.35 + 0.56 3-ethenyl pyridine " 52 to 53 - 0.20 ± 0.05 0.42 ± 0.06 0.37 + 0.05 ammonia " 11 1.3 ± 0.7 2.8 a 5.2 ° 4.5 + 1.2 solanesol " 13 - 0.06 ± 0.01 0.12 ± 0.02 0.10 + 0.02 formaldehyde ppm 12 - 0.10 ± 0.03 0.17 ± 0.04 0.50 + 0.0/ acetaldehyde " 12 - 0.67 ± 0.05 1.31 ± 0.06 1.11 + O.Ob acrolein " 12 - 0.13 ± 0.01 0.24 ± 0.01 0.22 + 0.01 nitric oxide " 11 0.01 C 0.84 ± 0.08 1.68 ± 0.09 1.42 + 0.13 nitric oxides 11 0.03 ± 0.05 0.87 ± 0.10 1.73 ± 0.10 1.47 + 0.13 1,3-butadiene pg/I 5 to 6 - 0.07 ± 0.03 0.14 ± 0.06 0.13 + 0.'07 isoprene " 5 to 6 - 1.1 ± 0.2 2.5 ± 0.6 2.1 + 0.7 benzene " 5 to 6 - 0.16 ± 0.02 0.33 ± 0.05 0.29 + 0.04 toluene " 5 to 6 - 0.47 ± 0.05 0.90 ± 0.15 0.73 + 0.10 median; quartiks: 13 and a.l/ pgll median; quartilcs: 4.7 cmd 6.2 pgll uredian; 4uaiulc%: 0.00 anJ 0.0.3 ppin tazf;vs6s0z
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Page 50 TABLE 2. Concentrations of Selected Smoke Components in the Test Atmospheres (conf.) Exposure Groups Parameter Unit of Measure n WBO WB6 WB12 H012 phenols phenol ng/l 4 - 2.8 ± 0.5 6.9 ± 0.7 6.6 ± 0.9 o-cresol " 4 - 0.48 ± 0.06 1.19 ± 0.10 1.12 + 0.13 m-cresol " 4 - 0.53 ± 0.08 1.32 ± 0.14 1.37 + 0.17 p-cresol " 4 - 1.1 ± 0.2 2.7 ± 0.3 2.6 ± 0.3 catechol " 4 - 7.4 ± 2.2 16.7 + 4.1 29.7 + 4.9 resorcinol " 4 - 024 ± 0.02 0.44 ± 0.04 0.43 +_ 0.05 hydroquinone " 4 - 32 ± 5 69 ± 8 71 ± 7 polycyclic aromatic hydrocarbons fluoranthene pg/I 2 - 170/120 370/270 510/440 pyrene " 2 - 160/120 320/260 430/370 benzo(a)anthracene " 2 - 140/ 90 260/190 270/210 chrysene " 2 - 320/220 610/490 560/510 benzo(-)fluoranthene " 2 - 100/ 80 200/170 170/160 benzo(a)pyrene " 2 - 65/ 55 132/123 112/121 indeno(1,2,3,-cd)pyrene " 2 - 25/ 28 53/ 53 42/ 50 dibenzo(-)anthracenes " 2 - 6/ 7 14/ 14 11/ 11 benzo(ghi)perylene " 2 - 16/ 19 39/ 37 30/ 33 zaz8ti9E9az
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Page 45 Skold, C M., Eklund, A., Hallden, G., and Hed, J. 1989. Autofluorescence in human alveolar macrophages from smokers: relation to cell surface markers and phagocytosis. Exp. Lung Res. 15:823-835. Skold, C M., Eklund, A., Hed, J., and Hernbrand, R. 1992. Endocytosis of cigarette-smoke condensate by rabbit alveolar macrophages in vitro measured as fluorescence intensity. Eur. Respir. J. 5:53-58. Skold, C. M., Andersson, K., Hed, J., and Edlund, A. 1993. Short-term in vivo exposure to cigarette-smoke increases the fluorescence in rat alveolar macrophages. Eur. Respir. J. 6:1169-1172. Teredesai, A., and Priihs, 0. 1994. Histopathological findings in the rat and hamster respiratory tract in a 90-day inhalation study using fresh sidestream smoke of the standard reference cigarette 2R1. In Toxic and carcinogenic effects of solid particles in the respiratory tract, eds. D.L. Dungworth, J.L. Mauderly, and G. Oberdorster, pp. 629-635. Washington, DC: ILSI Press. Tobacco and Health Research Institute 1990. Research Cigarettes. Lexington, KY: University of Kentucky Printing Services. Tyuma, I., Ueda, Y., Imaizumi, K., and Kosaka, H. 1981. Prediction of the carbonmonoxyhemoglobin levels during and after carbon monoxide exposures in various animal species. Jap. J. Physiol. 31:131-143. US Environmental Protection Agency 1992. Respiratory Health Effects of Passive Smoking: Lung Cancer and Other Disorders. US EPA/600/6-90/006 F.
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Page 54 TABLE 4. Bronchoalveolar Lavage Cell Composition Following 12 Months of RASS Inhalation Exposure Groups Cell Type WB0 WB6 WB12 H012 AM 5.3 ± 0.5 5.6 ± 0.6 6.4 ± 0.4 6.4 ± 0.6 lymphocytes 0.074 ± 0.009 0.100 10.013 0.079 ± 0.006 0.136 ± 0.019 PMNL 0.075 ± 0.017 0.048 ± 0.014 0.036 ± 0.007 0.040 ± 0.014 Note- Bronchoalveolar lavage cells are given in absolute numbers (10fi), mean ± SE (no statistically significant differences). 48ZEb9G90Z
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Page 53 TABLE 3. Histopathological Findings Following 12 Months of RASS Inhalation (cont.) Exposure Groups Parameter WBO WB6 WB12 H012 trachea bifurcation reserve cell hyperplasia of 0 0 0.4 ± 0.2 0.1 ± 0.1 respiratory epithelium 0/7 0/7 3n 1 /7 lungs bronchi, respiratory epithelium reserve cell hyperplasia 0 0.6 ± 0.3 ' 0.9 ± 0.3 * 0.9 ± 0.1 0/8 4/8 5/8 7/8 goblet cell hyperplasia 1.1 ± 0.5 0.3 ± 0.2 1.1 ± 0.5 0.9 ± 0.5 4/8 2/8 4/8 3/8 alveoli accumulation of pigmented 0 0 0.6 ± 0.4 0.9 ± 0.1 macrophages 0/8 0/8 3/8 7/8 Note. Histopathological findings are given as mean score ± SE and incidence. Due to the sectioning problems, only few larynges per group could be evaluated, thereby precluding statistical analysis. * For whole-body exposed groups: statistically significantly different from WBO. [lt
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101 HAL-SSVGiN7! ET AL. TABLE 1 Chemical Characterization of Test Atmospheres: Sham-Exposed Control and High-Concentration FSS and RASS Groups Parameter Y Sham FSS RASS TPh[ (µ(pliter) 91 0 3.7 = 0.7 2.6 = 0.3 Carbon monoxide (ppm) 39 <1.5 27.3 - I._' 13.7 = I.7 Carbon dioxide (ppm) 7-13 302 = 75 4i'_ = 63 50?=18 Nicotine (µs_iliter) 51 <0-05 2.21 = 0.+3 0.52 = 0.10 Yitmc oxide (ppm) 12 _- 0.0I 0.63 -0.09 0.69 - 0.1a Nitrogen oxides (ppm) C 0.02 - 0.02 - 0:63 = 0.08 0.69 = 0.13 Fonnaldehyde(ppm) l^--13 - <0.02 . Ua-0.05 0.16 = 0.01 Acetaldehyde tppm) l'_-13 <0.04 0.63 - 0.06 0.-1=0.05 Acrolein(ppm) If-I7 <0.02 - -- - -0.10-0.01 0.10 _ 0.01 Ammonia (y.Jliter) 12 - - 3.71 = 0.65 . i.39=0.37. .Vore. In the sham-exposed control _roup. the raw data sho,ved a median TP9I concentration of 0._9 yg/liter 125 and 7j'-c quartiles: 0.19 and 0.43 µarliter) which •uas subtracted from the nw TPlI means ror ilI proups. The nitric oxide concentntion in the sham-exeosed _rcuo is __rven as ;he mgdian (]5 .md'j~'c quartiles: 0.00 and 0.0'_ ppm). __ Carbon dioxide was analyzed using vondispersive Infrared photometrv (Qltra- mat ~E) of the gas phases For arconne determmanons, samples were drawn on suifunc acid-impresnared diatomite m6es (Extrelut, bkrck. Darmsmdt- Ger- many). Extraction was periorr.ed :vith n-butvlacerate conrainine 5S5 [v/v) oiethy!amine. Nicotine was analyzed by capillary gas chromatautraphy 1HP5390, Hewlett Packard. lValdbronn. Ge:many) with a DB-5 column ( Um X 0.25 mm. 1 and'.tl• C.trlo Erba. Hotheim. Germany) usinu a nitro3en- ohosohonu detector. Nitrogen oxides .vere determined bv chemoluminescence tn the gas phase of the :est atmospheres after catalytic reduction and reaction with ozone (VO/N0,analyzer CLD -COAL Teaan. Hambrecitikon. Switzer- land). The a!dehydes were determined Sy reverse-phzse HPLC (Hvpersil ODS. 5 µm. 250 x4 mm. He.vleu Packard) and 1:V detection I HP1050 Multiple Wavelength Deteaor. F!e•.vleu Paakard) of the 2-[-dini[rophenvlhydrazine (DN'PH) drivatlves aiter tnppins n acid DVPHIace[oaitrile solution. Ammo- nia was detennined by :iquid chromroemphv /Lichrosorb RP-13. 10 µm. ._O.x 1S mm. Merck) and iuarescence detection i6j0-105. Perkin Elmer, Cberlinzen. Gertnanv) of 9uarescamme denvatives after traooinrz an ulPuric ac:d-imoreenated diatomne. For :he cetermmation uf the par,ic!e .aize distri- bunan, the particles were prer.oitared on a tilter etcp in a spinnine spiral duct (S[bher and Flachsbart. 1969) followed bv a tluorametric determination or the particulate mat[e: elured •`ram seouennally ca[ I1[er pieces. ine particle size distribution was calculated usin_e 'inear re_¢ression andlysis after probu trans- 'onnatmn iFinnev. 197I1. .. .. The temperature in the exposure chamoers '.vns monitored mnnnuouslv usm, a disnal [hermeme:e: ~Tasto[herm DTOO. )}fPAC Eleknonic. Fmnkiurt. Gemmal The relative numidity was de:ermined osvchrometmcail-v (Therm =2a6. Ahlbom. Vumber¢, Germany7 in the .nmosphere of the sham-exoosed group. .vhich also served as a proxv ior :he il¢red. eonahaoned rresh air used to generate and dilute the 55. .Lnimal ecposure system. ine rats '.vere head-only esposed to the xst atmospheres for 6 Ndav. - days/•seek or 90 .lays, The ;iead-unly exposure made was used to ensure reproduc:ble inhalanon of 9ie`est atmospheres and to minimize uptake Sv noninhalation vutes. e.z.. by dermal absorption ar :nuesnon .'o!lowmg preening of :he fur i Mauderly « ui.. 1989). The axpbsure chamoers consrs[ine of zlass. stainless steei. and brass i[NBIFO. x[a2onic cross-section. 303 cm=; 4e:2hr. 7_ em) were equipped with custom-made ylass tubes tar animal exposure :hat ve:e conical at :he `rant end to-n[ the rat skull and saaled with rubber stonpers at :he rear end. The ra[s were restrained in the front oaa ai the :ube :vnh their Seatis protmdino into the stream of the rest atmospnere. which pas.,eu nrow,n :he cepasure chamber .[[ a rate of approx- imately ICO Iiters/min care,candim; :o aoproxtmateiy 2 li tervrmin .< raq. Tne rubes tiited ;liahdy aaudally in nrder to minimize aonraet or :"e rnt •.vuh '.es urine. T:m _Iass tube ixc va :•uneU ~ccorGing tn the Sodv •.vight or the rats. The position of the n¢ in the txnosure chambers was systemaucally chan_^ed an a daiiv basis. Sham-exoased rars'vere exposed to •iiared, conditioned fresh air under the same conditions u:he test aamospnere-ezcosed crts. Durin_s the postinhala[ion period. 13 rats of rhe high dose and the sham- exposed groups were kept in polycarbonate cases, 2 per caee. Diet and drinkin2 water were available to the m[s ad !ibitum. !n-[ife observations. The rats were observed for mortality. signs of overs rosicitv. or injuries '.vhen they wece transferred from their cuees to rhe exposure cnambers and when beiue transferred back ro their canies. More detailed checks on ge^eral condition and behavior of the rars were eerrarmed on three'ra[s per grouo and day shortly aiter the end of the daily exoosure. Body 'we_i;hr dererminations. The body weight of individual rats was determined one day ]ft2r amval of the rars- at the start of the tnhelatton ?edod. and once oer week dunnr the inhalanon and postinhaiadon penads. Bimnoziarin,. In order to provtde an cstimate of the amount of :esr atmosphere taken uu Sv ;he ricrs. rspiramr,r frequencies and voiumes :vece determined on at least six rats per grouo ,iunn¢ exposure by •.•.hcle-bouy -_ p!ethysmoumphv in :ce exposure tubes ICoireins er oL, 1981). The ditkrenual pressure signals tb'aiidyne 11P=j. HSE blar::h-Hureste[ten. Genmmn'mere dimtized and analvzed usio_ the 3atvent oro_-ram develooed bv S. A. 9uca. Stavimariet Surroik. CnaeU Yinsdom. To monitor excoiure to the test atmosoderes. ¢eadv-;taze :rooor.ar.s ar blood carcoxv-hemoeiobin iHbCOI '.vere determined ance in three rats :er ;roup according :a Slimisch er oi. t 197.t1. 3lood samoles were tak:.9 af;rr at !easr 5 h of escasure .`rom-ra¢ vithdrawn or a short period of :imeirem ^e exoosure :ubes bv ouncanno the retro-urbital sinus with +!ass mir,ociceues. To provide a rouen estimate of the amount of niconne taken up bp t`e rats. unne '.vas callec[ed t:cm riva :ats per vroup dunne the 6-h expusere =ened usins soeciall_v modiced e.enosvre :ubes and dunne the rollawinei3 iasmu custom-made menbaiism c1§es. Tne two samples per rat were ;omcmed :o determine aicotine.. ~nnme.ind :rans-j'ivdroxvcoeinine hy;as ahrcmata_e- rapi[v (Vbneicen er al.. 1959). . . .Vecropsy and gross pathology. The mts .vere no[ iasted before necrotis;:. On the aay rtiilowin3 the last exposure. 20 rats per group were anesheo¢ed bv intrapentaneal in)eaian ai iodium ?eneobatbi[al 00 mglk. body •.v; and subseyueady ,acncced Sv e.csane~i nu[ion Sv ;ranssec[ing the +bdominai aora. The carcasses were :vei~heu and )ubjered :o a complete gross ermma[wn under>uperrisiun or a ~ater:nary pa[hoiuyat u¢h speciai a¢en[ian paid :u;^e respirtar,; -racr. TSe ame proce•.lurc was roiluwea tor !'- rats per _roup ei :he htuh-dou and sham-<sco~ed =rnuo' at :he ead of the pns[inhalauon =ened. 2063643218 I
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Page 57 oxide, TOL: toluene, FA: formaldehyde, EP: 3-ethenyl-pyridine, BEN: benzene, BUT: 1,3 butadiene, SOL: solanesol, CAT: catechol). Figure 7 Squamous metaplasia of the pseudostratified epithelium in the larynx, base of epiglottis, in the present and the previous subchronic inhalation study (Haussmann et al., in press) (mean scores ± SE): A: Dependence on the TPM concentration, B: Dependence on the daily TPM dose calculated from the respiratory minute volume averaged over the duration of the inhalation period (n = 4 to 10 rats/group).
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H.AL'SSMAiv'U ET AL. 40 -~ 0~ O : shem exposure 0 : FSS ~ : RASS mcotine - : ccunir.e - : hyt:roxycotir.ine 4 0.0 0.5 t0 1.5 20 25 NICOT)NE CCNCNTRAT;CN IN SICES iBE4M SMOKE Gi9/I) FIG. 2. Amounts of nicotine and its main metabolites recovered fram unne samples colleanng dur,n=:he 5-h exposure period and the 19It between exposures as a func:ton of the 5S nicotine concenuatians (means = SE). the arytenoid projections according to Lewis (1931). The uachea was trimmed and longitudinally cut at :he tracheal bifurcation. A frontal section of the left lung includins the left matn bronchus and its major branches was cut according to Lamb and Reid (L969r. The :issues were oracessed.embedded in Paraolast. vt.n 5 to fi um thickness. and stained with hemro.cylin and eosin (HE), In addidon. the sect:ons oi the :racSea and lun¢ were stained with .{Ician blue/ceriodic acid Schift s reaeeat m demanstrate srobler cells. Alt slides were rcad by a ve:ermary Pmholoeist with experience in dsarette smoke-related changes in the respiramry tmct of rodenrs without knowled°e of the treatmenr aroups. All itstoparholo2tcal nndines were scored according to a derined ;eventy scale .`rom 0:o 5 tmarked eifects). Mean se•rcritv scores were calculated based on all rats !n a=roup. .bforphamerrical analvris of !arvnz. The thickness of the larynsseal eni- thelium '.vas determined .vithoue 'mowledse of the trta[mene arouos on a standardized HE-stained section at the ar,rtenold projections. which included the ventral depression. dear ot ;he Iarmx, and vocal crords. At each of these sttes. :he cpiehelial thickness was measured at 10 soecined locations directly on the microscopic ;mage usins a Leica 1licrovid system iHensheim• Garmany). Ana[yris of the B(a)P metabolism. The micrasamal BIaIP metabolism was assessed by duorescence detection of i Btu1P metabalites after reverse- phase HPLC separation. T'ne activity was normalized usins the micrwomal protein content- Reference materials 'or the 8fa1P :netaholites f7-hydroxy-. 9-hydrozv. 1S-diaf., 7.3-diol-. and 9.10-diol-B(a1P) were obtained :iam :he U.S. National Cancer Institute Chemical Carcinogen Repashor+ Midwest Research Insnmte. Kansas Citv, bt0). . . Rats were euthanized as descihed above. The rieht Iune and the aasal resoiratory and olfa.::crv epuheiia NRE. NOE) 'Nere removed and stored at -70`C. Lunn and nasal :n:c:oscmes were isolared by diiferentlal ultracentri- iueation aecordins to Grover erA , 19711 with minor madineatrons. blicrosomal suspenaions i lunxs. 100:o 600 p_ ?rorein: NRE. 100 ta'-00 µg protnn. YOE. auprox. IJO µs cratein'I were incubated (or 60 min at J7°C in Tris/HCI buffer (50 mSL pH 7.6) in [he presence of B(a)p (30 µtq, vADP- (770 µbq. elucose-6-phasphate (2.5 mbq, glucose-6-phosphate dehydrosenase (I unit/mU, MsC!_ (5 m,bq, and'eDTA (^_10 µM) (three replicates per rat and eissue). The incubation was stopped by adding methanol conminin3 henz(a)antl:racene as an internal standard. After cen- trifugation. the supernatant was directly injected into the HPLC. Mono and dihydroxv B(a)P metabolites were separated using a Hewlett Packard HPLC 1090 with a`lucleosil 100-5 C-1S precolumn (5 um, 4 em x+ mm. Knauer KG. Oherursel. Germany) linked to a Vovapak RP-l3 column (5 µm. 15 cm x 3.9 mm- MilliparelWaters, Eschborn. Germany). The sal- vents iar step gradieat eluuan were solvent A(l0 mM KH_PO.,, ?H 1.3) and solvent 8(aceronitriU. Peak detection and quantitation were performed using a Hewlett Packard IOa6 A duorescence detector equipped •.vnh a 5-al rlow cell. EmcinciaNemission wavelen°rhs were as follows (in the order of elueion from the column): 137/;11 nm for 9-and7-OH-B(a)P: '13/v00 am for 4.5- and 7.3-diol-B(n)Pt 'S7/-'04 am iar 9,10-diol-B(a)P: and '-37/s-f nm for benzta)anthncene and BfaIP. The protein canceatnnons of rhe micosomal suspensions were determmr.i accarding :oLewry er aL 1195 1). as madiced by Pererson i t933), using an automated micmrnesad with Sovine serum albumin a5 a itandard. Duoiicate -de[etminations were certormed. Sratirricu7 analrses. For :he comparisons of the FSS- and @ASS-espased -eroups with the sham-exposed groua. respectively, the [ollowing statistical tests were periormed: ;or the overail comparison. the one-way analysis ur variance for continuous data and the eeveralized Cuchmn-:b(antel-Haensz<1 test (Koch and Edwards. 1988) for ordinal dara were used with the CO concenuauon os the itratifving variable. If the overall comparisan showed a sienificant differenca then ior a pairwise comoarison the Duncan [est (puncm. 1955) was applied 'or continuous da[a and the generalized Cxhran-Mantel- Haenszel xs[ t'dr ordinal dra. Forhe compar.son af r'SS-with RiSS-exposed noups, rhe twa-wavanaLysis af variance far continuous data and the Coc:iran- Mantel-Haenszel test ior ordinal data were used. All tests were conducted at the nominal level or sisnihcance ai n= 0.05 . (two-railed). Due m t.he laree number oi parameters analyzed. no correc;ion for multiple testing was apolied. which would have made tte tests very :nstnsitivt. Statistical sigatncances. thereiore. Save :a be ,:onside;ed-as cxplorative indi- eamrs nther than contirmatorv evidence. No coref::on for ruln_ale zsnn_° was applied. RESULTS reSr ArPtoSf.itfre5 . . The test atmospheres were-eenerated reproducioly threugh- out the 90-day inhalation period. As :areeted, the CO concen- trations of FSS and R.aSS were equal for each of the three dose levels. The CQO concentrations (mean = SD) for the .ow-, medium-, and hih-dose levels of : SS were ;S = 0.4. 1^.6 = 0.6. and'-7.3 =!.'- ppm, respectively. The respective vaiues [or R.aSS were ~.~ = 0.1..12.'- = 0.6, and "_3.' = I.? 7pm. The TP.1,I concent:ations for the low-, medium-, and hish-dose levels of FSS were 1J - 0.6. 3.6 = l?. and 3.7 = 0.7 µgiliter. respectively. The respective values for RASS were-0.6 = 0.3. l_' = 0.7. and 16 = 0.J_µgfliter. The time course of the daily mean TP`vI concentrations in the high-dose eroups was re- ported separate!y (Voncken er al.. I994). The analytical char- acterization of ihe hi_h-dose test atmospheres as well as that of the sham-exposed group is-presented in Table 1. The individual smoke components in the medium- and low-dose FSS and 2063643220
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Page 51 TABLE 2. Concentrations of Selected Smoke Components in the Test Atmospheres (cont.) Exposure Groups Parameter Unit of Measure n WB0 WB6 WB12 H012 N-nitrosamines N-nitrosodimethylamine ng/I 2 n.d. 0.30/n.d. 0.58/n.d. 0.57/n.d. N-nitrosopyrrolidine " 2 n.d. n.d. n.d. 0.14/0.12 N-nitrosonornicotine " 2 n.d. n.d. n.d. 0.21/0.25 N-nitrosoanatabine " 2 n.d. n.d. n.d. n.d. N-nitrosoanabasine " 2 n.d. n.d. n.d. n.d. NNK " 2 n.d. 1.26/1.12 2.08/1.98 2.33/2.17 cadmium pg/I 2 n.d. 280/310 470/590 460/520 Note. - Data are given as mean ± standard deviation (except when n= 2). WBO: whole-body sham-exposed control group; WB6 and WB12: whole-body exposure groups at 6 and 12 µg TPM/l, respectively; HO12: head-only exposure group at 12 pg TPM/I. =': not determined. - n.d.: not detectable.' - Detection limits: TPM: 0.1 µg/1, GO: 1.5 ppm, nicotine: 0.05 pg/I, N-nitrosodimethylamine: 0.16 ng/I, N-nitrosopyrrolidine: 0.11 ngll, N-nitrosonornicotine: 0.21 ng/l, N-nitrosoanatabine and N-nitrosoanabasine: 0.19 ng/l, NNK: 0.36 ng/l, cadmium: 10 pg/I. - Chromium, nickel, lead, and zinc: no RASS-dependent increase above blind filter contents of these metals. £8ZEv9E90'~
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79931s7oexs NICOTINE METABOLITES (nmol(24 h) c31 0 cn o ~ Q °0 °0 °0 °0 °0 I I I I I I D Hi
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Page 52 TABLE 3. Histopathological Findings Following 12 Months of RASS Inhalation Exposure Groups Parameter WB0 WB6 W812 H012 nasal cavity, level I respiratory epithelium reserve cell hyperplasia 0 0.8±0.2' 1.9±0.1' 1.6 +0.3 0/8 6/8 8/8 7/8 squamous metaplasia 0 0.1 ± 0.1 1.0 ± 0.3 * 0.3 ± 0.2 0/8 1/8 5/8 2/8 goblet cell hyperplasia 0 0 0.4 ± 0.4 0.1 ± 0.1 0/8 0/8 1/8 1/8 nasal cavity, level 2 respiratory epithelium reserve cell hyperplasia 0 0.1 ± 0.1 0.1 ± 0.1 0 0/8 1/8 1/8 0/8 olfacto ithelium e ry p reserve cell hyperplasia 0 0 0.1 ± 0.1 0 0/8 0/8 1/8 0/8 atrophy 0 0 0.3 ± 0.2 0.3 f 0.2 0/8 0/8 2/8 2/8 eosinophilic globules 0.1 ± 0.1 1.1 ± 0.5 1.0 ± 0.5 1.3 ± 0.5 1/8 3/8 4/8 4/8 nasal cavity, level 3 olfactory epithelium eosinophilic globules 0 0.6 ± 0.4 0.9 ± 0.5 1.5 ± 0.6 0/8 2/8 3/8 4/8 larynx base of epiglottis squamous metaplasia of 0 2.8 ± 0.3 4.0 ± 0.0 2.6 ± 0.2 pseudostratified epithelium 0/1 4/4 3/3 5/5 hyperplasia of 0 3.3 ± 0.3 4.0 ± 0.0 2.8 ± 0.2 squamous epithelium 0/1 4/4 3/3 5/5
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Page 55 Table 5. Alveolar macrophage fluorescence (relative units). Exposure Groups Inhalation Period WBO WB6 W612 H012 6 months 100 + 5 - 315 + 14` - 12 months 100 + 5 211 + 1 9' 271 + 15' . 260 + 16 Note: Results are expressed as percentages of WBO (means ± SE). 'For whole-body exposed groups: statistically significantly different from WBO."
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RECOVERY (% of Inhaled nicotine) cn 0 cn 0 0 0 0 I I I , W £bZEt79E9QZ _ _ -----
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2063643294 AOfllS 00i7 J0 Xt/0 09E 006 09Z OOZ 09l 00l 09 9- I I I I I I I I Ii r- 0 ZIOH : •~-- ZLBM : 13 99M = ~- 09 08M: O--- ~-001 I- 09l ~ 00E°
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2063643295 I SHINOW ZL I ~ SHINOW 9 ~ ZI LOH I L ZIeM I 98M I 08M I ~ ZlBM I 08M ~ I Ef I r- 0'0 ~ ~ v
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6 -l ~ 2~~ 0 -' o:wBo ~ : WB6 n:we12 ~ : H012 r 0 T 10 -r 20 IT ID 30 40 50 60 CO CONCENTRATION (ppm) 10 ,
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114 H?.usSNtA.VN ET AL. independent from the route of exposure. Conclusions as to its toxicological relevance are limited by the experimental model used and the complexity of the toxification pathway Ieading to the ultimate animal carcinogen. For example, the micrasomal fraction used in this study to assess the B(a)P metabolism does not account for most phase II detoxificadon pathways which might be induced in parallel to cytochrome P450. A better approach to assess the relevance of the described induction would be obtained by detertnining DNA adducts in target organs, although their specific determination would require higher B(a)P doses than those taken up by the rats in this study. The Lowest TPM concentration effective in inducing the pulmonary B(a)P metabolism, i.e., 0.6 µ,,v/liter, is consistent with the lowest reported concentration effective in inducing the cytochrome P450 lAl, i.e., I µs TP-MAiter (Ti et af., 1994: _ Gebremichael er aG., 1995). No differences between sham- and SS-exposed eroups were seen at the end of the postinhalation period. The lack of a persistent induction after cessation of SS exposure strongly suggests an effective clearance of the lungs from materials inducing the B(a)P metabolism. In the present study, the amount and/or surface area of materials present in the SS-agino room were exaogerated com- pared to those typically found in residences or office environ- ments in order to investigate in princfple the effects associated with room-aging. Correspondingly, the mean age of ETS in human indoor environments other than residences, e.s., offices - or public buildings, is probably shorter than the mean aqe used to generate RASS in the present study (Seppdnen. 1995). However. the results of this s:udv show that room-aQing in ~eneral reduces the biological activity of FSS. This may impact the risk evaluation based on experimental studies using more or less fresh SS. In this context. RASS is considered a more realistic experimental surrogate for ETS than FSS. ' ?.CK,vo W LEDGME`+-'I' This study was iupported by Philip Morris C.S.d. Tne authors thank Donald Hanselmann for critically reviewing he manuscipt. REFERENCES American Association for Laboratory Animal Science (1991). Policy on the humane care and use of laboratory animals. Lab..amna Sci. 42. l7. Appelman. L. ti[.. Wouteaen. R. A.. Feron. V. J.. Hooftmn. R. N., and Vottea W. R. E t 1986). Effect of vanabie versus rixed <ioosure fevels an rhe toxicity of aceta:dehyde in rats. J. .ippl. Tae¢ol. 6. 331-J'-6. Ayres. P. H.. Mosber¢, .A. T.. and Coegins. C. R. E. (199u). Design.:ronsrnse- don and operation of an inhalation system for exposing laboratory animals to env¢anmental :ooacco smake..{m. (nd. Avq. .assoc. J. 55, 306-310. Baker. R. R.. and Procmr. C. J. 11990). T.;e on;ins and properties of environ- menraL [obacco smo:e. Env/ron. inc t6.'_71'= a5. _ Benner. C. L.. Bayona. 1. Y(.. Ceka, F. M.. Tans, H.. Le.vis. L.- Crawrbrd, 1.. Lamb. 1. D.. Lee, YL L.. Lewis. E. A.. Hansca. L. D.. and Eatou¢h. D. 1. ,1989). CSemical composinon of envtronmearal ;obacco smokc. =. Partic- ulate-phase zompuunds. Enwrnn. Sci. Tecnnol. 23. 688-699. Bond, J. A. (1986). Bioactivadon and Siotransformation of xenobiadcs in rat nasal tissue. In Toxicology af the .Varal Passages (C. S. Barrow, Ed.), pp. 219-261. Hemisphere. Washington. Bond. 1. A., and Dahl- A R. (1989). Netabolism of xenabiotics in the rupiratory mtcC In /ntermediarv%nobione.Netabalism in Animals: .bferh- odology, .tfechmeisms and Sigrupcance (D. H. Huaon, 1. Caldwell, and G. D. Paulsan, Eds.). pp. 41-64. Taylor & Francis, London. Burger, G. T., Renne- R. A.. Sagu2 J. W., Ayres. P. H., Coggins, C. R. E., btosberg, A. T., and Hayes, A. W. (1989). Histologic changcs in the respiratory trsct induced by inhalation of xeaobiotics: Physiologic adapra- don or roxiciry? To.ncaL AppL Pharmacol. 101, 52I-5J2. Comns- C. R. E.. Duchosal. F..-Ytusy, C., and Ventrone. R. (1981). Mea- surement of respiratory partems in rodents using whole-bqdy plethysmag- mphy and a pneumornchograph. Lab. Anim 15, 137-140. _. Coggins-C. R. E.. Ayres. P. H.. Mosberg, A. T., Ogden-Vf. W., Sagara.J. W„ and Hayes. A. W.(1992). Fourteen-day inhalation study in rats. usint aged and diluted sidestream smoke iom a reference cigarette. Fundam. Appl. Pharmaco(. 19, 133-I-0. Coggins. C. R. E., Avres. P. H.. `dosber¢, A. T., Sagqrt3. 1. W„ and Hayes. A. W. (1993a). Subehronic inhalation study in rats, using aged and diluted sidesrream smoke nrom a reference cioarette. bdraL To.ricol. i, 77-96. Co_gins. C. R. E.. Ayres. P. H.. and YLosber,. A. T. (1993b). Comparative inhalation study in raU usins cieare¢es containing tobacco expanded with chloroduoracarbon-II tCFCII) or hydro-Buoracarbqn-l]3 (HCFC-1'-3). Inhal. Taxicol. 5, 97-115. Dogm- S., Doehmer. 1.. Giatt, H., YLqelders, H.. SiexR P., Friedbers:, T.. Seidel. A.. and Oesch. FR t t990). Stable expression of rat cytochrome P,50 IAI cDNA in V79 Chinese hamster cells and their use in mums_eiciry . tesrin?. Hol. Phannacol. 37- 608-fi 13. Duncan. 0. B. (195). Nultiple ranee and multiple F-tesrs. 8iamerrics 11, 1-i'-. Eatoush. D. 1.. Benner. C. L., Bavona- J. M.. Richards, G.. Lamb. J. D.. L-e. M. L.. Lewis- E. A.. and Hansen. L. I). (1989). Chernical-eqmposition of environmental tobacco smoke. I. Gus-phase acids and bases. Envirou. Sci. --TrclmoL 23, 679-637. Earouen. D. 1_ iiansen- L.. D.. and Lewis, E. A.(I990). The chemie-L - charactenzation of eavironmenral mbacco smoke. Environ. Tecluml. 11- 107I-1085. Feran. V. 1.. Kmvsse. A.. TiL H. P.. and [mmel. H. R. f1973).. Repeated exposure to acroletn vapor: Subacute studies in hamsters, rats and rabbits. To.tico(ogv 9, 47-57. Finney. D. 1. 0971). ProbirAnnlpsis. Cambad_se Cniversiry Press. Cambndee. UK. First. M. (1985). Constituents of sidestceam and mainstream tobacco smoke and markets to cuantirv esoosure .o them. In Indoor Air nndHuman Heairh (R. B. Gammage and S. V, Kaye. E'Gs.). pp. 19:-Z07. Lewis. Chelsea. llt. Funtham. E.. Cor.ea. P.. Revnaids, P.. Wu-Williams• A.. Buffler. P. A.. Greenbere. R. S-. Caen. V. W.. qlterman. T., Boyd. P.. Austin. D. F.. and Liff, 1. (1994). Environmemal :obacco smoke and Lung cancer in aonsmok- ing woman. J. Am. 1[rd. dssac. '-72- C53-I759. Garsala. C. G. ~. (987). P"Imonar,: ard nydrocarban hydroxyLsse ac:iyic, af mice. rats and 2uinea oigs following !ong eeml exposure to mainstream and sidestream cigareue smoke. Toxicology 45, 177-13-4. • . :. Gebremicheai..A.. Cians, A: Yl.. Buckprtt. A. R.. P!oopen C. G., and Pin:c- erron, K. E. f 19951. Postnatal deve:opment of cytqchrome Pd501A1 and Q '_8L in rat !une and Iiver. Effect of aged and diluted sidestream smoke. ~_ Tasicol. Appi. Pharmacof. 135-]=6-_'53. _ _ Q; _ German Law on Chemtcals 11990). Grundsdtze der Guten Laborpre.eis. An- w; hang I zu > 19a Abs. 1 der Yeui:usung Jes Chemikaliengesctzes vom L+. ~; . ~,f9rz 1990. enndes;esercpluh L• 13), 521-5a8. W I_,
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2063643296 [ SHINOW Zl ~ i SHINOW 9 [ [ ZIOH j [ ZlBM [ 98M ~ 08M ~ I ZlBM I 08M _ I i I I a r 0'0
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Page 56 FIGURE LEGENDS Figure 1 Biomonitoring of RASS exposure: A: Blood carboxyhemoglobin determined at three time points (means ± SE), B: Hemoglobin adduct of 4-aminobiphenyl after 12 months of RASS inhalation; daily TPM dose calculated from the respiratory minute volume averaged over the duration of the inhalation period (means ± SE; `: for whole-body exposed groups: statistically significantly different from WBO). Figure 2 Biomonitoring nicotine uptake: A: sum of five nicotine metabolites (nicotine-N'- oxide, nornicotine, cotinine, trans-3'-hydroxycotinine, norcotinine) excreted in 24-h urine in month 5 of the inhalation period (means ± SE), B: Data from A, normalized to the inhaled nicotine dose (based on the nicotine concentration in the test atmospheres, the respiratory minute volume calculated according to Guyton (1947), and the body weight of the rats; means ± SE). Figure 3 Body weight development (means). Figure 4 Tissue levels of 8-hydroxy-deoxyguanosine: A: nasal respiratory epithelium, B: nasal olfactory epithelium, C: lungs (means ± SE; *: for whole-body exposed groups: statistically significantly different from WBO). Figure 5 Urinary excretion of 8-hydroxy-deoxyguanosine (means ± SE; ': for whole-body exposed groups: statistically significantly different from WBO). Figure 6 Comparison of RASS in present study (HO12) with ETS either generated at an extreme concentration under experimental conditions (Martin et al., 1997) or as determined in a large field study (Jenkins et al., 1996) (abbreviations: CO: carbon monoxide, TPM: total particulate matter, AMM: ammonia, NIC: nicotine, ISO: isoprene, AA: acetaldehyde, NO, nitric
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2063643298 ~ SHINOW ZL ~ I SHINOW 9 ~ ZLOH ~ ~ Zl9M I 98M J 08M ~ ZI LOH I ~ ZLBM I 99M I 08M ~ f I 0 i- ooz
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5-I 0 O : WBO o : WB6 o : WB12 ,w : H012 0 2 4 _ 6 DAILY TPM DOSE (mg/kg body weight)
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w 10 t' A J 1111 1111 1111 :1:111' 1111 '1111 1111 - IIl I'1111 1 U1 14 1 111 /!-Al r712-Month I / I/ V M ':f E]' J 1A _ A Ll .'I 1J.P L7 I I I I L7 :1 0 1 1-1 1 1`1 I 1`I 1 1`f-1 P-'/ ruSSSwetr 10 r v I / I IL•MLLYILMhY/L_YIL...YII-VIL-YIC-MLYII~/ILVIL1/ILJ/IExparim.ETS C 1 CO TPM AMM NIC 66ZE1r9E90Z ISO AA NO TOL FA EP BEN BUT SOL CAT Smoke Components 11
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2063643297 i sHiNOw ZL iI sHurvOw s i ZI lOH I~ ZlBM I 98M I 08M ~ I ZlBM I 08M ~ r 0'0 I I I ~- Z'0 0 'r 9,0 ~
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A : sham-exposed controls : chronic RASS inhalation WB6 : chronic RASS inhalation WB12 chronic RASS inhalation H012 : subchronic RASS inha(ation HO : subchronic FSS inhalation HO a 9 12 15 0 3 6 TPM Concentration (f.cg/I)
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0 : sham-exposed controls ,m, : subchronic FSS inhalation HO w : subchronic RASS inhalation HO ,w : chronic RASS inhalation H012 v: chronic RASS inhalation WB12 -n, : chronic RASS inhalation WB6 T 1 r 0 2 3 4 5 6 Daily TPM Dose (mg/kg body weight)
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ROOM-AGED SIDESTRF.ASi SMOKE LtiHrV.->TION Gopin3th- C., Prentice. D. E., and Lewis. D. J. ( I937). dtla: of Ezperimental Tosicologie Pathology. pp. 25-23. Sf1? Press. Lancaster, UK. Grover, P. L.. Hewer. A., and Sims. P. (1971). Metabolism of polycvclic hydrocarbons by rat-lung preparations. Biacirem PharnracoL 23. 323-332. Guerin. M. R.. Jenkins. R. A., and Tomtins, R. A. (1992). The Clremisrry of Environmental Tobacco Smoke: Composition and.Neasurement Center for Indoor Air Research. Lewis. CSelseo. Yf1. Harrison, P. T. C. (1984). An erhanol-acedc acid-formed saline fisative for routine use with special application to the fixation of non-perfused tu lung. Lab. Anim 18, 325-331. Ingebrethsen. B. J.- and Sears. S. B. (1985). Particle size distribution measure- ments of sidestream cigarette smoke. 39rh Tobacco Clmmisu' Research Conference, Montreal. Quebec. Canada. - International Organization for Standardization (1973). Tobacco and Tobacco Products -Atmaspheres for Condirioning and Testing, 2nd ed. [Inrernatienal Standard 150 34021 International Organization for Standardization (1986). Cigarerres=Roraine- .Inalyticnl Ciqarerre Smoking Nachine-Depnirions and Srandard Cndi- rinn,r. 2nd ed. [International Standard ISO 33031 Jenkins, R. A.- Palauskv. A.. Counts. R. W., Bavne, C. K.. Dindal, A. B., and Guerin. NL R. (19961. Exposure to emironmental tobacco smoke in sixteen cities in the l:'nited States as determined by personal breachin¢ zone air sampling. J. Prpostne dnal. Envirar. Eordem. 6. 47]-50'-. !i, C. SI.. Plouper. C. G.. Witschi. H. P.. and Pinkerton, K. E. (199d). Exposure to sidestream cigarette smoke alters bronchialar epitheliaf cell dilferentration in the postnatal rat luns. dm. J. Respir. Cell. :Nol. Biol. 11, 312-320. load, 1. P.. Pinkerton. K. E., and Bric. J. b(. i 1993). Effects of sidestream smoke exposure on pulmonary and ainvay reacnviry in developing rats. Prdiarr Wdmnnol. 16. 331= 33. Klimisch. H. I.. Nleissner, K.. and :b'ermcke. H. (1971). Quantitative deter- mination of carbon monoxide in blood by _sas chromatooraph-v. Z.Klin. Chem. K(ur. Binc'hem. 12. >3J-S•3. ' . - Koch, C. G.. and Edwards. E. (19331..Cinical :rficiency trials with cate=orial data. In Biopirnrmercnrrcal Stansrics ;or On iq Dereinpmenr f K. E.-pecrce. Ed.). pp. =03-1j7. Dekker. New York. Lamb. NI. B.. and Reid. L. t19h91. Goble( cell mcrease in rat bronchial eprthelium after exposure to asarerte and cigar tobacco smoke, Br.. Ved. J. 1, 33-35. _ - Lee. C. K.. Brown. B. G.. Rccd. 8. A., Rahn, C. A.. Coir_ins. C. R. E. Doolittle. D. 1.. and Hayes. A. W. r 199'_). Four.een-day inhalation studv in rats, using aged and diluted sidestream <meke rrom a rere}eaceii_are¢e: [Y. DNA addocts and alveolar macroohase cytoeer.esis. F.mdam..IpPL Tu.riml. 19,111-116. - - Lewis. D. J. 11931). llitotic indics of rat Iar:n_geal epithe!ia. l. Arrne-172. -t19--t_3. - Li5[mth. G.. Bunon. R. YL. Foreaand. L.. Hammond. K. S.. Seila. 7. L.. Zweidinzer. R. B.. and Lewtas. J./1939). Cnaracrerizanon ofem'ironmearal :abacco smoke. Envvnn. Sc•i. TxcinoL 23. 610-611. Lowry. D. H.. Rosebrough. N. J.. Far,. A. L., and Randal!. R. L~1951). Protein megsurement .vuh the Folin Phenol reagent. 1. Binl. Cnea. 193, :65='.>. Mauderlv. J. L.. Bechmid. W. E.. Bond. 1. A.. Brooks. A. L.. Chen. B. T.. Cuddihy. R. G.. Harkema. J. R.. Heauersan. R. E. Johnson. N. F.. RithidecS. K.. andThomassen. D. 0. (1989, . Comparisan of 3.nechods of exposin_ rats to cigarette smoke. E.p. Purfmi. 37. !9,t-I97. 1luramnrsu. T.. Weber. A.. }Iuramat.su. 5.- and Akermann. F. 119531. An experimental study on :rritanon 3r,d aeanyancs due so pasc;ve smokin_s. lat. :Ireh Occup. Enarnn. Nealth SL OECD 1 I9.4 W. Prine:nle, oi Gaod L:bor_mry P-ev:fce: Annez : C31 70, (o I(5 OECD Guidelines for Te.rnng of C.lemimis. Organization for Economic Cooperation and Developmen4 Par.s. OECD (1981b1. Subchronic Inhalation Toxicity: 90-day Study. In OECD Guidelines for Testing of Chemicals. Organization for Economic Coopera- tion and Develooment. Paris. Oldaker, G. B.. Periem, P. F.. Conrad. F. C., Conner, J. bL- and McBdde. R. L. -([990). Results of surveys of environmental tobacco smoke in offices and restaur.rnts. Inr. .arcir. Occup. Environ_ Health 62, 99-I04. Pasquini, R.. Sforzolini. G. S.. Savino, A.. Angeli. G.. and Monarca- S. (1987). Enzyme induction in rat Iung and liver by condensates and fractions from main-stream and side-i¢eam cigarette smoke- Envirou Res. 44, 302-311. Peterson, 0. L. (1983). De[emunadon of rotal prorein. In Methods in En,-;- mology: En`yme.Structare tC. H. W. Hits and S. N. Timasheff. Eds.l. Vol. 91, pp. 95-I19. Academic P;ess, New York. Piade, 1. 1., D'Andres. S., and Sanders. E. B. (1996). Sarption phenomena of nicotine and etheaylpyr,dine vaoours an different materials in a test cham- ber. In (ndoor4ir '95. Proceedings ql dte %rh Internarionul Conferenc'e on fndoor.iirQualityand Climate. Vol. 1IK. Ikeda.md T. [wata. Eds.j. on. 33-38. Institute of Public Health, Tab+a. _ ' - Pinkerton. K. 6.. Peake. J. L.. Espirim. L. Goldsmith. SL, and Wi¢chi, H. (1996). Quantitative histioloey and cvrocSrame P-a:0 immunocytochemts- try of the lung parenchyma (ellawing 6 months of cxposu2e ai strain A/J mice to cigarette sidestrcam smoke. Jnlml. To.ncol. 3, 977-9a5. Raabe, 0. G.. Yeh. H.-C.. Vetcton. G. !.. Phalen. R. F.. and Ve:asquez D. J. (1977). Deposition oi'inhaled monadisperse aerosols in small rodena. In inhaled Panicles N, Part I i W. H. WaIIon. Ed.), pp. 3-? 1. Pergamon Press. Oxford. Rajini. P.. and Witschi. H. 11994). Short-'zrm er'eaa of sidestream smoke on respiratory epith<Iium in mice: Cell 'sine:;cs. F.rndam. appl. To.cicol. 22. 105-110. - ~ - Reininghaus. W., and Hackencers. U.(1977). Anlaze zar Lanrzzen-Inhalatmn mit Zigarenen tir kleine Labattiere. }fed. TechnoL 97, 5-6. Renne. R. A.. Wehner. A. P.. Greenspan. B. J.. DeFord. H. S.. Ra_an. H. A.. Westerberg. R. B.. Buschbom. R. L.. Burger. G. T.. Hayes. A. W'.. Suber. R. L.. and l[osbere. A. T. - 199]). '_-Wee< and f i-week innalation studies of aerosolized lvicerol in ra4s. fnrial. ToricoC 4. 95-I11. . Scherer. G.. Conze. C.. von Meperlnck. L.. Sorsa.ll.. and ASlkoier. F. (19907. Importance of exposure to gaseous and particulate phase components of tobacco smoke in ac:ive and oassivt smokers. !ne dreh. Occ:rp. Enrirmr. Health 62. +59--66. Sepp3nen. 0. r199ii. Design -ritzria of venrlauon rar healthv buildings. In Healrhv Buildin;s: 93 Praceedinsr iYl. Maroni. Edi. Ylilano. Stdbec W., and Flachsbarr. H. 1 i969). Size-vepandn2 precipitation oi aerosols in a spinning spiral duct._Enriron. Sci. Tedmoi. 3, 1'_30-L'-96. . Teague, S. V.. Pinkerton. K E.. Goldsmith. YL. GebremshaeL A.. Chang, S.. lenkins. R. A.. and l(one_•nun. J, H. i I99=i. A iidcsream cigareae smoke - _aeneration and exposure system for eavvonmenml tobacco smoke smdies. - lnhaL To.ricul. 6. -9-33, Teredesai. A.. and ?ruhs D. - 199d). Hisaoatholoeical tindin2s '.n the rat and 1V hamster respiratory :racrin a'i0-day inhaiation srudy using r}esi ;ides¢^am 0 O+ . smak< o[ :he standa[d ,-etittr:c< ci~are:re '_Ri. In Tu.crc and Carclno;enic E7ecrs of Solid P:ecfc'es 5t are •4esprrnrnr: Tracr i D. L. DunBwonh. 1. L. Vauderlv, and 0. OberdBrster. Eds.t, pp. 5_9-635. !LSI Monographs. ILSI Press. Washtn'2'on.- W A W N _- Tobacco and Health R<searea institute'1990i.-fte.rearc/e Cl;werte.r. l:nivb7- siry of Kenmeky Pnmmg :erricss. Les;m_ton. L'.S. Envrronmental Prote_:on .>.seaey i :99'_:. Rspvnrnrv HeaL6 Effer.t -rr Pm'sive ]'mukinq: Luo;, Qincer .md Omer Dicurderr. ;CS EPA/6011/6-90/ o--* : 006 F) V,;ivt. J. ',(.. Cuen¢erch. P..lr.u Baron.:. 199i ,. Lacilizwun ,nd;nduc:wn
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N 0 m w m 4~ w ca 0 N
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DRAFT (Hecht and Trushin, 1988; Belinsky et al. 1989; Staretz et al. 1997a), and consequently exhibits less biological activity than NNK (Liu et al., 1990; Castonguay et al., 1983a; Hoffinann et al., 1993). Pyridyl-N-oxidation of both NNK and NNAL to yield 4-(methylnitrosamino)-1-(3- pyridyl-N-oxide)-l-butanone (NNK-N-oxide) and 4-(methylnitrosamino)-1-(3-pyridyl-N-oxide)-1- butanol (NNAL-N-oxide), respectively, are considered to be detoxification pathways of NNK metabolism (Liu et al., 1990; Castonguay et al., 1983a; Hecht, 1994; Staretz et al., 1997a). The determination of hydroxy acid, keto acid and keto alcohol as stable end products of NNK metabolism by a-hydroxylation pathways represents the metabolic activation of the host to produce reactive intermediates with the potential to form adducts with DNA or other cellular macromolecules. Urinary NNK Metabolite Excretion in Laboratory Animals and Man Urinary excretion profiles provide evidence of species-dependent differences in metabolic activation of NNK by a-hydroxylation and detoxification [Table 21. Urinary NNK metabolite profiles in the A/J mouse and F344 rat (Morse et al., 1990) re highly dose dependent [Table 3]. Similar studies have not been performed in other animal w i;o ;J cc . _,._._ o .._ -a 0002234.01 3/16/98 4:14 pm - 6 -
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DRAEr icmol/day, 80-120 µg/day; 72%), occupational exposure (0.15-0.30 µmollday; 25%), cigarette smoking (0.02 /.cmol/day, 3.4 gg/day; 2%), and miscellaneous minor sources, including pharmaceutical products, cosmetics, indoor and outdoor air (0.001 umoUday, 0.1 ug/day; 1%) (Tricker, 1997). Cigarette smoking accounts for only 2% of the estimated total exogenous exposure to N-nitroso compounds; however, tobacco-specific N-nitrosamines (TSNA), as their name implies, are considered to occur only in tobacco and tobacco smoke (Hecht and HofBnann, 1989). To date seven different TSNA derived from the nitrosation of nicotine and other minor tobacco alkaloids have been identified (Amin et aL, 1995). [Figure 1](The following abbreviations will be used in addition to NNK: NNN, N-nitrosonomicotine; NAB, N-nitrosoanabasine; NAT, N-nitrosoanatabine; NNAL, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol; iso-NNAL, 4-(methylnitrosamino)-4-(3- pyridyl)-1-butanol; and iso-NNAC, 4-(methylnitrosamino)-1-(3-pyridyl)butyric acid.) TSNA Present in Mainstream Cigarette Smoke and Indoor Air The U.S. National Academy of Sciences has previously estimated that a smoker of domestic filter cigarettes has a total N-nitrosamine exposure of 16.2 µg/day (6.1 ,ug NNN and 2.9 µg NNK), based on the assumption that the average smoker consumes 20 cigarettes/day (Assembly of Life Sciences, 1981). This exposure estimate was based on unpublished analytical data provided by Hecht and Hof$nann of the American Health Foundation, Valhalla, NY. More recent data for 0002234.01 3/16/98 4:14 pm - 3 -
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DRAFT filter cigarettes [Table 1] yields a lower exposure estimate of 3.4 µg/day (1.5 µg NNN and 1.0 µg NNK) (Tricker, 1997). Since TSNA are also transferred to sidestream cigarette smoke (Adams et al., 1987), and presumably exhaled by smokers, trace levels occur in environmental tobacco smoke (ETS) present in indoor air (Brunnemann et al., 1992; Kius et al., 1992; Tricker et al., 1994). Extensive smoking under poor ventilation conditions results in mean ETS concentrations of 2.8±1.6 (range n.d: 6.0) ng/m' NNN and 4.9±9.6 (range n.d.-13.5) ng/m' NNK (Klus et al., 1992). Similar levels occur in the home; 0.8±1.2 (range n.d.-3.3) ng/m3 NNN and 4.0f4.6 (range n.d.-14.3) ng/m' NNK (Tricker et al., 1994), and other venues (Brunnemann et al., 1992). Putative Metabolism of NNK in Laboratory Animals The major reported pathways of NNK metabolism in experimental animals involve carbonyl reduction, a-hydroxylation of the methylene and methyl groups adjacent to the N-nitroso group, and pyridine-N-oxidation [Figure 2]. Carbonyl reduction of NNK to 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) is probably a detoxification pathway of NNK metabolism since it provides the functional hydroxy moiety necessary for glucuronidation to [4-(methylnitrosamino)-1-(3-pyridyl)but-l-yl]-(3-0- 0002234.01 3/16198 4:14 pm 4
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DRAFT obtained under identical experimental conditions to allow a valid comparison of the metabolism of NNK in laboratory animal and human tissues. • Current data do not support the assumption that NNK, present in tobacco products and cigarette smoke, induces lung and liver tumors in man. This section of our submission briefly reviews published experimental data which formed the basis for our conclusions. To obtain additional scientific data on the metabolism of NNK in different animal species which would be relevant for toxicological assessment of the potential biological activity of this compound to man, Philip Morris is currently funding a research project at the Walther-Straub Institute for Pharmacology and Toxicology, University of Munich, Germany. This research will provide comparative experimental data on the in vitro metabolism of NNK in lung and liver of the A/7 mouse, F344 rat, Syrian golden hamster, and man. Introduction N-Nitroso compounds represent a large diverse group of chemicals of which some, but not all, have been shown to induce a wide range of tumors in experimental animal models (Preussmann and Steward, 1984). According to recent analytical data, total human exposure to exogenous N-nitrosamines is estimated to be 1.10 µmoUday; the major sources are the diet (0.79 0002234D1 3/16l98 4:14 pm - 2 -
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DRAFT D-glucosiduronic acid (NNAL-Gluc) (Maser et al., 1996; Kim and Wells, 1996). a-Hydroxylation of the NNK methyl carbon results in the formation of an unstable intermediate which spontaneously decomposes to yield formaldehyde and 4-(3-pyridyl)-4-oxobutanediazohydroxide, a potential pyridyloxobutylating agent, which can react with water to yield 4-hydroxy-1-(3-pyridyt)-1-butanone (keto alcohol). a-Hydroxylation of the methylene group in NNK produces 4-hydroxy-4- (methylnitrosamino)-1-(3-pyridyl)-1-butanone which spontaneously decomposes to methane diazohydroxide, a potential methylating species, and stable 4-oxo-1-(3-pyridyl)-1-butanone (keto aldehyde), which is further oxidized to 4-oxo-4-(3-pyridyl)butyric acid (keto acid). Glucuronidation of 4-((hydroxymethyl)nitrosamino)-1-(3-pyridyl)-1-butanone, the initial intermediate formed in the "keto alcohol" pathway, may be a detoxification route (Murphy et al., 1995). a-Hydroxylation of the methylene and methyl groups adjacent to the N-nitroso group in NNAL produces unstable metabolic intermediates before ultimately forming stable 4-hydroxy-4- (3-pyridyl)butric acid (hydroxy acid) and 4-hydroxy-l-(3-pyridyl)-1-butanol (diol). a- Hydroxylation of the methyl group and ultimate formation of the diol is a putative detoxification pathway since this pathway has not been reported to result in adduct formation, while a- hydroxylation of the methylene group ("hydroxy acid" pathway) can potentially result in methylation of cellular macromolecules. NNAL is a poor substrate for a-hydroxylation compared to NNK 0002234.01 3/16/98 4:14 pm - 5 -
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DRAFT Background Based on experimental data available prior to 1984, the International Agency for Research on Cancer (IARC) concluded that "There is sujTzcient evidence for the carcinogenicity of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone to experimental animals," but "No data on humans were available" (IARC, 1985). In a footnote it was stated: "In the absence of adequate data on humans, it is reasonable, for practical purposes, to regard chemicals for which there is sufficient evidence of carcinogenicity in animals as if they presented a carcinogenic risk to humans." As far as we are aware, no regulatory agency has further evaluated the carcinogenicity of this compound to humans. Following internal company review of all scientific literature published after the IARC assessment on 4-(methyhiitrosamino)-1-(3-pyridyl)-1-butanone (NNK), it was concluded that: • NNK is a carcinogen in the A/J mouse, F344 rat, and Syrian golden hamster, inducing mainly tumors of the lung, and to a lesser extent, tumors of the liver. • Extensive evidence exists to demonstrate that human metabolism of NNK differs significantly from that observed in laboratory animals. However, relevant data have not been 0002234.01 3/1N98 4: 14 pm
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ROOM-AGED SIDESTREAM SMOKE LtiHPd-ATIOiv dosing of FSS and RASS was based an carbon monoxide (CO), an SS component which has nor been found to change during aging (Eatough er al., 1990: Voncken er aL, 199a). In addition, in experimental studies, the CO concenQation is proportional to and thus representative of the number of cigarettes smoked, a measure frequently applied to determine human ETS expo- sure in experimental studies (Muramatsu et al., 1983; Scherer et al., 1990) and epidemiolo;icai studies (pack-years: e.g., Fontham er al., 1994). Rats were head-only exposed 6 h/day, 7 days/week for 90 days. The concenQadon levels used in the present study are comparable to those used in previous SS inhalation studies ("exueme" to "exaggerated"; Coegins et al., 1993a) to determine lowest- or no-observed effect levels. The major end points in this study were respiratory tract histopathology and the benzo(a)pyrene (B(a)P) metabolism in nasal epithelia and the lungs. '~L4TERG1L5 AND dIETHODS Experimental design. Ra¢ were head-only exposed to FSS and to RASS as well as to rtltered, conditioned Iresh air (sham-exposed aroup) 6 h per day, 7 days per week for 90 days. FSS and RASS were administered a[ three dose levels. The CO concentrations for [he respective leveis were equal for both 5S rypes and were up to 29 ppm. The TPN concentntions were up to 9 u.g/liter. There vas a-t?-day postinhalation period for rats of the connol- and high concentration _roups ro investisnm the delzved accunence. perslstenaeM and/or reversib[lity of findings. The Sisropatholozv of the respira[ary :ract and the &atP metabolism-as a proxy ror polycyclic sromadc hvdrocarbon metabo- lism-In nose and lunrzs 'uere he major ead points studied. The main dose parameter ro compare the bioiovcal acdmty of FSS and R.-1SS was the CO concentration. The results are also discussed on the basis of the respective TPY( concentrations. The study was pedormed in confonnity with Good Laboratory Pracdce [OECD. 1981a: German Gw on Chem[cals. 1990) and the american Assoeu- anan for L•caratory >nimal Sc~enr_ (AALAS) Policy on :he Humane Care and i:se or Laboratory Animals I i99U. Experimenta[ animalr. Male autbred Spraeue-Dawlev rars (C[f:CDBR), bred under spec[ned pa[haeen-2ree conditions. were'obra[ned fram Charks Rive: : Rale[sh. VCi. This stram was chosen because of ehe !arze amount of puolisned dan avaiiabie. in par,:cular because of its Geauent ase-:n .iisarette smoke inhalation studies ~e.g.. Joad er uL. 1993. Cossgins er al.. 199?a.b: Teredesai and Pruhs. !99=). A previous smdv did not show differences between male and remale rats in suscepnbtiity [o SS-ir,duced effects iCorgms er al.. i99'_). Thus. only one sex was used to allow :uidc:ently !crze group sizes snd .roup numbers. Male rars were preferred based on our greater experience with the®. Tne respiratory Jacts of randomiy seieaed :a[s were exam:ned ^israoa[ho- los_ically on arrival: so unusual :iaain2s xe:e ooserwd. Serolo¢ical screening performed on amval. after 90 days of inhalation, and at :he end of [he postinuatanon period did not detecc ann6adies to rai-re!aiec 'rir]ses. such as lympnocvnc choriomeninettis virus, mouse adenovims. munne ooiiovims. paramduenza virus type I. parrov[rus H-L rat parovims, pneumonia virus of mice. rat corona•:ims/sialodacryoadeni[[s virus, and reavvus [ype 3. as +eil as to '.he bacterium t!•:cop[nsmu p[dmonu. The rars were idennded individually asme sabcunneous aanscondea I1M1- 1000• P!e.cx,'cist. Nerhedands: data acquismon by DAS-401. Cno. Zevenuar. Vetherlundsi. Followm2 a 16-.iay oreescamre acc!im5Eiz5nan oenod they were randomly ailuceted to (he six 35 exposcre _roups and :he sham exposure group 143 rats per _roup). Ar :.he ead of :he :nnalaatian eenod _0 and 10 rats per group'were used ior :he hiswpsmological examination .md :he analysis of [0l the B(a)P metabolism, respectively. At dte end of the postinhalauon periad- I_ and 6 rats per sham and td#dose Cups were used for the aforementioned two end points. The age of:lte ra[s a[ the start of the inhalation penod was 17 days. The body weight at that time was 196 - (SD: l3 g). The nes were kept in an animal laboratory unit with controlled hygienic conditions. The laborarory nir (fillered. tresh air) was conditioned. Positive pressure was maintained inside the laboratory unit. Room temperature and relative humidiry were maintained at _ C(SD: 1°C) and 69:0 (SD: 10i). rescectively. The Hghddaric cycle was 12 h! l: h. The rats were housed in transparent polycarbonate cases, two rars per cage. The bedding material was autoclaved sottwdod ;ranulare (SK-:0/550- Bmun & Co.. Barteaber., Germa- ny). A sterilized. foroued pellet diet (.hQ2H EP, EMe[smann. Rinteln• Ger- many) from cage lid ncks and heao-¢ested tap water trom water bottles with autoclaved sipper tubes were suppGed ad !ibirum in each cage. Food and drinking water were not available to the ncs during dte daily exposure periods. Good hygienic condidons within the animal housinrz and exposure rootn were maintained as evidenced among other :ritena• b,v oe,ative results for ;he oacrerioloeical examinauons of the :at diet, drinldn; water, and the laboraton' air and selected surfaces. Generation of FSS and 7d5S. T;.e University of Kentuc.ky reference ci_areae 7Rl was used to eenerate che test atmospheres (,M5 yields per cigareae: a-t.6 ms TPM. '-.-5 mg nicotine, and 25.1 m, CO; Tobacco and Health Research Instimre. 1990). They were conditioned and smoked in basic accordance with the Incemationai Or_wzation for Standardiza[ion (ISO) standards 3402 (1973) and 3308 (I986). cesoec[ivel,v, as generally applied m ,,IS zenerntion. The cisarenes were conditioned at T-°C..604c relative humid- iiy, for at least 3 days. Two automatic JO-port smoking machines were used tor smoke generation (Relninehaus and Hac'senberg, 197 i). Mean puff volumes of 35 ml were generated tal:ing I puf`dmin with a'_-s puff duration usin^_ i four-?iston pump (Baaelle. Geneva. Switzerland) resulting in approximami)' IL puffsicigarette at a mean Sutt !eneth of _3 mm. The MS was exhausted. SS was <ollected using a circular hood made of elass and stainless steel on top of [he smoking machines. The tuee.FSS concentrations were obtained by dilution with filtered. conditioned lresn air. The maximum ase of [he FSS was approx- ima[ely 10 s. Csing a second smoking machine. RASS was generated,.by continuously passng diluted FSS at a rate of 20 m'$ through a i0-m' a.cperimenral a_in3 room wi[h non-inea sur,'aces, resulting in R4SS oi a m2in ase of LSh. Cn lhe asin2 room were materials usually found in res[dences andlor otfices. such as a'alleaper ?ainred with a!a[ax-based white paint ~-9 m-)• window alass-(;],m'). .md a wool carpet (II m°). For experimentsl purposes. the surface areas oi same oi che materials were eaaegemted relawe to the size of nce room, i _.. a 16-m' ool cunain and a booksheif with t surixe area of 7 zr an[r5_[ed ?ine wood with approximately 30 books rr maeaxines vi[h a surface area of ):n=. Tne materials in the aging room '.vere unexposed at [he star, of :`e inhalation and nor replaced throughout he dU days. A ceiling fan was opcr-[ed to facilitate uniform distribution otthe RASS. The room was illuminaeed'ay duorescesr 'dayliehi' lamos LLumilus 11, Osram. Munich. Ge.-nanyt. Two ?ainted'neat exchangers fapprostmateh60 m= sur.'ice irea/ were ased :o keep :h.e room temperature constant finean: '-'_.G'C• SD: !9°C). FSS md RASS were conveved via vlass mbm_ :o the ' exposure ahambers. RASS te^eration was started approximately 3 h'ceiore the start of :he daily exposure to achm.e a steady-state :est atmospnere ror inhalation. During overniu:[. nons-mitin_ perioas. ;he :oom was dushed wuh _ tiltered. eonditmned irsn Sir at _0 m':h _P~ W .. dnalvrica[ characteri-;acon of FSS and 2{SS. A[ designated timt in[er- (\) ' vals. specined analytes :vc:e _e¢nnined :o characterize the rest armospheres.- to evaluate the reproduclbtlity oP the xst atmosphere ,enentiong and :o v" exc!ude cross-con[amination in the sham-exposed ;roup. Samples were mi- _- - lec:ed directly at the accosure chambers. CO .vas continuously monitored usine aondispeaive inlrar:^- phommetry fti!tramat iE. Siemens. BrusseS. Bel¢_iumt a! the eas phase oi :he res[ a[mosphems TPlI was pravimeu:cally tA_COS. Sanonus. GSttin^sn. Germam,y) de:zrmined adeut once per day after aappine particulate matter in nCambrid3qtype _lass nber fif[er(Gelman. Ann Arbor. 1fi). Lie other ana3Ses were da:ermmed at least at weekly inter:als.
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DRAFT Determination of hemoglobin adducts as a possible surrogate for tissue DNA adducts shows less than a 3-fold difference in the mean level of HPB-releasing hemoglobin adducts in smokers (163 fmol HPB/g hemoglobin) compared to nonsmokers (68 finol HPB/g hemoglobin) (Hecht, 1994). A smaller difference was observed in another study (54.7±8.9 vs. 26.7±4.1 finol/g hemoglobin) and self-reported exposure of nonsmokers to environmental tobacco smoke did not increase the background level of HPB-releasing hemoglobin adducts (Richter et al., 1995). Both studies indicated elevated hemoglobin adduct levels were only apparent in about 10% of smokers compared to all subjects. Concluding Remarks The above studies provide intensive evidence that human metabolism of NNK differs significantly from that observed in laboratory animals. These differences are evident from in vitro studies of NNK metabolism in microsomes and tissue samples, excretion profiles of NNK metabolites, and absence of a clear differentiation in NNK/NNN-derived DNA and hemoglobin adducts in smokers and nonsmokers. These data provide little support for the assumption that NNK in tobacco products and cigarette smoke induces similar biological effects in the lung and liver as reported in laboratory animals. 0002234.01 3/IW98 4:14pm - 12 - I
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DRAFT species. The data in Table 3 indicate that a-hydroxylation pathways account for about 50% of NNK metabolism in the A/J mouse and F344 rat, regardless of the administered dose. At the lowest administered dose of NNK (1 µg/kg body weight), excretion of NNAL and NNAL-Gluc does not occur in either species, although NNAL is apparently formed and further metabolized by pyridyl-N- oxidation. NNK-N-oxide excretion increases with decreasing administration of NNK. Humanbiomonitoring studies report the presence ofNNAL, NNAL-Gluc andNNAL- N-oxide in 24-h urine of smokers maintaining constant smoking habits (Hecht et al. 1995; Carmella et al., 1997). NNK-N-oxide is not a urinary metabolite of NNK in smokers (Carmella et al., 1997). Other metabolites of NNK detected in experimental animal excretion studies, such as stable end products of a-hydroxylation , are not specific to NNK metabolism since they are also formed during metabolism of NNN and nicotine. Combined urinary excretion of NNAL, NNAL-Gluc and NNAL- N-oxide in urine of smokers (Hecht et al., 1995; Carmella et al., 1997), when calculated in molar equivalents of NNK, balances well with predicted total NNK exposure [Table 4]. These data provide evidence that NNK metabolism and excretion in man differs significantly from that observed in laboratory animals [Table 21. -P. W c a Total NNAL plus NNAL-Gluc excretion in 5 nonsmokers measured on 2 occasions is reported to be 8.4f11.2 ng/day, and increases after experimental exposure to sidestream smoke used as a surrogate for ETS (Hecht et al., 1993b). Another study reported 17 of 29 nonsmokers to ~ q 0002234.01 3116/98 4:14 pm - 7 -
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DRAFT have a mean excretion of 8.8±9.4 (range 0.8-31) ng/day total NNAL plus NNAL-Gluc compared to 0.68±0.41 (range 0.08-1.68) µg/day in smokers (Meger et al., 1998). Twelve of 29 nonsmokers had no detectable levels of NNAL plus NNAL-Gluc in urine. Metabolism of NNK by Laboratory Animal and Human Microsomes Microsomes from lung and liver have been extensively used to investigate metabolism of NNK under various experimental conditions [Appendix 1]. Despite differences in the individual study protocols (NNK substrate concentration and time of incubation), rodent lung and liver microsomes metabolize NNK to yield significant levels of a-hydroxylation products. Contrary to this, lung and liver microsomes of human origin primarily metabolize NNK by keto reduction to NNAL in the absence of significant a-hydroxylation. Only limited kinetic data are available to document interspecies differences in NNK metabolism by lung and liver microsomes. The available kinetic parameters for NNK metabolism by lung microsomes [Table 5] provide further indication that laboratory animals (A/J mouse and patas monkey) primarily metabolize NNK by a-hydroxylation with no significant formation of NNAL. Human lung microsomes primarily metabolize NNK to NNAL, with no significant formation of a-hydroxylation products. The biological relevance of data for metabolism of NNK by human lung microsomes is 0002234.01 3/16/98 4:14 pm - $ -
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DRAF history of alcohol abuse. Undamaged macroscopically normal tissue is received in the laboratory stored in ice-cold Hanks medium within 30 min of removal. Metabolic studies are performed using precision cut liver slices and lung tissue in dynamic culture (Vickers, 1994). Compared to using microsomes or isolated cells (extensively used in previous studies as summarized in Appendix I), tissue samples maintain structural heterogeneity with intact phase I and phase II metabolism, and cell interaction and communication are preserved to a certain extent, thus resembling the situation in the intact organ. Radiolabeled [5 'H]NNK or [5 'H]NNAL (sp. act. 25-30 Ci/mmol) are incubated over a substrate concentration range of 0.01-100.0 µM with precision cut liver slices and lung tissue for 6 h in Krebs-Henseleit buffer (pH 7.4) under standard laboratory conditions for dynamic culture (Vickers, 1994). The substrate concentration range was selected to include the lowest possible concentration to approach the physiologically relevant concentration in man, and substrate concentrations predicted to occur in animal bioassays. Each incubation is performed in triplicate using lung and liver samples from at least 5 different laboratory animals. Metabolite profiles are determined by reversed-phase HPLC with radioflow detection (Richter and Tricker, 1994). Pharmacokinetic parameters (Km and Vmax) are calculated from Lineweaver-Burk plots with reaction velocities showing linear response to time. Pharmacokinetic parameters will be determined for lung and liver from different human donors. ~ .3 0002234.01 3116/98 4:14pm -14-
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R T partially comprised by the high experimental substrate range compared to actual human exposure to NNK (19-135 ng/filter cigarette; 70-650 pmol [Tricker et al., 1991]). No data are available for microsomes isolated from rat lung. Metabolism of NNK by hepatic microsomes is less well documented [Table 61. Liver microsomes from the patas monkey primarily metabolize NNK via a-hydroxylation with no significant formation of NNAL at low NNK substrate concentrations, while at high substrate concentrations metabolism to NNAL would be predicted to occur. Human liver microsomes primarily metabolize NNK to NNAL, and to a lesser extent NNAL-N-oxide, at low substrate concentrations, with no significant formation of a-hydroxylation products. The formation of NNAL-N-oxide at low substrate concentrations supports the presence of this metabolite in human urine (Carmella et al., 1997). Only kinetic parameters for NNK a-hydroxylation pathways have been reported for A/J mouse and F344 rat liver microsomes. In conclusion, kinetic parameters of NNK metabolism in lung and liver microsomes provide strong evidence that significant differences occur in metabolism between laboratory animals and man. At tow levels of exposure to NNK, human metabolism is characterized by reduction of NNK to NNAL, in the absence of significant a-hydroxylation, while laboratory animals metabolize NNK to yield significant levels of a-hydroxylation products. 0002234.01 3/16/98 4:14pm -9-
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DRAFT (pyridyloxobutyl)deoxyguanosine (HPB) adduct with DNA (Spratt et al., 1989). Linear dose- response relationships are not observed for either NNK-induced methylafion (Belinsky et al., 1990; Murphy et al., 1990a) or NNK-induoed pyridyloxobutylation in the rat lung (Murphy et al., 1990a). An HPB-releasing adduct with hemoglobin has been proposed as a surrogate marker for DNA adduct formation by both NNK and NNN (Carmella and Hecht, 1987; Peterson et al., 1990). However, the exact mechanism of adduct formation still remains to be determined (Murphy and Coletta, 1993). Only two studies have investigated the presence of HPB-releasing DNA adducts in human lung (Foiles et al., 1991; Blomeke et al., 1996). Mean levels of 11t16 and 9.0t2.3 finol HPB/mg DNA have been reported in smokers and nonsmokers, respectively (Foiles et al., 1991). Since the response for the analytical reagent blank was equivalent to 38f16 finol HPB and 1-2 mg DNA were used for analysis, the reported levels are well below the blank response and could easily be due to an analytical artifact. No HPB-releasing DNA adducts were detected in 16 lung tissue samples from current smokers and 16 from nonsmokers (B16meke et al., 1996). The levels of 7- methylguanine in 801ung tissue samples could not be explained by differences in tobacco exposure (measured by serum cotinine), gender, age, or ethnicity (Blomeke et al., 1996). These data suggest that exposure to NNK via smoking does not result in HPB adduction to lung DNA or increase the background level of lung DNA methylation. ., pp02234.01 ~' 3/16/98 4:14 pm - 11 - --
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DRAFT In summary, these results obtained under identical experimental conditions provide further evidence that NNK metabolism in human lung and liver primarily yields NNAL. In contrast to this, NNK metabolism in lung and liver of the A/J mouse and F344 rat results in significant a- hydroxylation to DNA-reactive intermediates thought to be involved in NNK-induced tumorigenesis in these two organs. The research program is predicted to be completed in April 1998. 0002234.01 3/I6/98 4:14pm - 17 - I
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DRAFT considered critical for lung tumorigenesis in A/J mouse lung (Peterson and Hecht, 1991; Belinsky et al., 1992) and F344 rat lung (Staretz et al., 1997b), respectively. Metabolism of NNK by human lung results primarily in the fonnation of NNAL in the absence of formation of significant levels of a-hydroxylation products [Figure 3]. The kinetic data [Table 71 provide support for pyridyl-N- oxidation of NNAL, but not NNK, and are consistent with the reported occurrence of NNAL-N- oxide but not NNK-N-oxide in human urine (Carmella et al., 1997). The kinetic data do not support significant formation of keto alcohol via the pyridyloxobutylation pathway at low NNK substrate concentrations in human lung. These data suggest that HPB-releasing adducts derived from NNK are unlikely to be formed in the human lung and support data for their absence in human lung tissue (Blomeke et al., 1996). Michaefls-Menton kinetics could not be fitted to NNK metabolism at low substrate concentrations in the A/J mouse liver. At high substrate concentrations (1.0-100 /.zM), significant metabolism of NNK by a-hydroxylation pathways occurs in addition to NNAL formation. NNK metabolism in the liver of the F344 rat suggested that hydroxy acid formation is the most favorable pathway for NNK metabolism; however, under conditions used in rat bioassay protocols, significant a-hydroxylation of NNK still occurs. At low physiological NNK substrate concentrations in the human liver, NNK metabolism is predicted to result primarily in the formation of NNAL. 0002234.01 3/16/98 -16- 4:14 pm N © ... m' I
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Table 1. Estimates of Exposure to TSNA in Smokers Exposure estimate (Ecg/day) For a smoker of 20 filter cigarettes TSNA 19811 19912 1994' NNK 3.0 1.0 1.6 NNN 6.2 1.5 1.0 NAB -- -- 0.2 NAT 7.4 -- 1.8 NAB/NAT -- 1.5 -- 1. Academy of Life Sciences (1981). 2. Tricker et al. (1991). 3. Hoffrnann et al. (1994). 0002259.01 3/16/98 4:18 Pm DRAFT I
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DRAFT Research Funded by Philip Morris on the Metabolism of NNK A research project has been fnnded at the Walther-Straub Institute for Pharmacology and Toxicology, University of Munich, Germany, The research has two major objectives: 1. To define the major routes of in vitro NNK and NNAL metabolism under identical experimental conditions in lung and liver of the A/J mouse, F344 rat, Syrian golden hamster, and man. The three animal species chosen represent those most often used for chronic bioassays of NNK. 2. To determine pharmacokinetic constants (Km and Vmax) for each pathway of NNK and NNAL metabolism in both organs of all four species. Experimental Design Lung and liver are removed from laboratory animals killed by decapitation. Human peripheral lung and liver tissue are collected from excess material removed at surgery from patients undergoing routine surgical procedures. Human tissues are rejected from subjects having current treatment with innnuno-suppressants or other drugs known to induce or suppress metabolism, or a 0002234.01 3/16/98 4a4pm - 13 -
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DRAFT Zhang, Z., Huynh, H., Teel, RW., Effects of orally administered capsaicin, the principal component of capsicum fruits, on the in vitro metabolism of the tobacco-specific nitrosamine NNK in hamster lung and liver microsomes, Anticancer Res. 17: 1093-1098 (1997). 0002234.01 3/16/98 4:t4 pm - 26 - I
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References Amin, S., Desai, D., Hecht, S.S., Hoffmann, D., Synthesis of tobacco-specific N-nitrosamincs and their metabolites and results of related bioassays, Crit. Rev. Toxicology 28: 139-147 (1996). Ardries, C.M., Smith, T.J., Kim, S., Yang, C.S., Induction of4-(methylnitrosamino)-1-(3-pyridyl)-1- butanone (NNK) activation in rat lung mincosomes by chronic ethanol consumption and repeated running exercise, Cancer Left. 103: 209-218 (1996). Assembly of Life Sciences, The Health Effects of Nitrate, Nitrite and N-Nitroso Compounds, National Academy Press, Washington, D.C., Chapter 7(1981). Belinsky, S.A., White, C.M., Trushin, N., Hecht, S.S., Cell specificity for the pulmonary metabolism of tobacco-specific nitrosamines in the Fischer rat, Carcinogenesis 10: 2269-2274 (1989). Belinsky, S.A, Foley, J.F., White, C.M., Anderson, M.W., Maronpot, R.R., Dose-response between O6-methylguanine formation in Clara cells and induction of pulmonary neoplasia in the rat by 4- (methylnitrosamino)-1-(3-pyridyl)-1-butanone, Cancer Research 50: 3772-2780 (1990). Belinsky, S.A., Devereux, T.R., Foley, J.F., Maronpot, R.R., Anderson, M.W., Role of the alveolar type II cell in the development and progression of pulmonary tumors induced by 4- (methylnitrosamino)-1-(3-pyridyl)-1-butanone in the A/J mouse, Cancer Research 52: 3164-3173 (1992). Blomeke, B., Greenblatt, M.J., Doan, V.D., Bowman, E.D., Murphy, S.E., Chen, C.C., Kato, S., Shields, P.G., Distribution of 7-alkyl-2'-deoxyguanosine adduct levels in human lung, Carcinogenesis 17: 741-748 (1996). Bouchard, L., Castonguay, A., Inhibitory effects of nonsteroidal anti-inflammatory drugs (NSAIDs) on the metabolism of 4-(methylnitrosamino)-l-(3-pyridyl)-1-butanone (NNK) in mouse lung explants, Drug Met. Dis. 21: 293-298 (1993). Brittebo, E.B., Castonguay, A., Furuya, K., Hecht, S.S., Metabolism of tobacco-specific nitrosamines by cultured rat nasal mucosa, Cancer Res. 43: 4343-4348 (1983). Brunnemann, K.D., Cox, J.E., Hoffmann, D., Analysis of tobacco-specific N-nitrosamines in indoor air, Carcinogenesis 13: 2415-2418 (1992). 0002234.ot 3/16/98 4:14pm - 1$-
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DRAFT Metabolism of NNK by Laboratory Animal and Human Tissues Similar profiles are observed for metabolism of NNK by microsomes and tissue samples [Appendix 1]. The data confirm that significant differences occur between laboratory animal and human metabolism of NNK. The major pathways of NNK metabolism in rodent tissues yield significant levels of a-hydroxylation products while human tissues primarily reduce NNK to NNAL. DNA and Hemoglobin Adduct Formation by NNK Metabolism of NNK by a-hydroxylation is assumed to be a critical event resulting in DNA-reactive intermediates; however, the exact role of methylating and pyridyloxobutylating species in different animal species and organs remains unclear. Metabolism of NNK to the keto acid via a methylating intermediate is thought to be a critical determinant of NNK-induced tumorigenesis in the A/J mouse lung (Peterson and Hecht, 1991; Belinsky et al., 1992) and hamster liver (Liu et al., 1992). Although NNK-induced methylation to yield O6-methylguanine in Clara cells appears to be a suitable indicator of the carcinogenic potency of NNK in the rat lung (Belinsky et al., 1990), pyridyloxobutylation is thought to be the critical event in tumor induction in rat lung (Staretz et al., 1997b) and liver (Liu et al., 1992). Both NNK and NNN can be metabolized to a pyridyoxobutylating species in the rat lung (Hecht et al., 1988) to yield a N- woxx34.oi 3/16/98 4:14pm - 10 -
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Current Results Preliminary results from this research have already been presented at two scientific meetings: AACR Special Conference in Cancer Research "DNA methylation, imprinting, and the epigenetics of cancer," Las Croabas, Puerto Rico, December 12-16, 1997. 2. 37's Annual Meeting of the Society of Toxicology, Seattle, March 1-5, 1998. Further results will be presented at the 89' Annual Meeting of the American Association for Cancer Research, New Orleans, March 28-April 1, 1998. The data currently available from this research project [Table 7, Table 8) demonstrate that NNK metabolism in lung of the A/J mouse and F344 rat results in significant levels of a-hydroxylation products at high tissue substrate concentrations predicted to occur in animal bioassay protocols. The experimental conditions and data support the reported biological activity of NNK in the A/J mouse and F344 rat lung; metabolism of NNK at high substrate concentrations yields the keto acid via a methylation pathway in the A/J mouse lung, while formation of the keto alcohol via the pyridyloxobutylation pathway occurs in the F344 rat lung. These two pathways are 0002234.01 3/16/98 4:14pm - 15 -
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DRAFT Cam7ella, S.G., Hecht, S.S., Formation of hemoglobin adducts upon treatment of F344 rats with the tobacco-specific nitrosamines 4-(methylnitrosamino)- 1-(3-pyridyl)- 1-butanone and N'- nitrosonornicotine, Cancer Res. 47: 2626-2630 (1987). Carmella, S.G., Borukhova, A., Akerkar, S.A., Hecht, S.S., Analysis of human urine for pyridine-N- oxide metabolites of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone, a tobacco-specific lung carcinogen, Cancer Epidemiology Biomarkers Prev. 6: 113-120 (1997) Castonguay, A., Rossignol, G., Modulation of the activation of 4-(methylnitrosamino)-1-(3-pyridyl)- 1-butanone by hamster liver microsomes to protein alkylating species, Toxic. in Vitro 6: 397-404 (1992). Castonguay, A., Lin, D., Stoner, G.D., Radok, P., Furyua, K., Hecht, S.S., Schut, H.A.J., Klaunig, J.E., Comparative carcinogenicity in A/J mice and metabolism by cultured mouse peripheral lung of N'-nitrosonomicotine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone and their analogues, Cancer Res. 43: 1223-1229 (1983 a). Castonguay, A., Stoner, G.D., Schut, H.A.J., Hecht, S.S., Metabolism of tobacco-specific N- nitrosarnines by cultured human tissues, Proc. National Academy of Sciences USA 89: 6694-6697 (1983b). Charest, M. Rossignol, G., Castonguay, A., In vitro and in vivo modulation of the bioactivation of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone in hamster lung tissue, Chem.-Biol. Interactions 71: 265-278 (1989). Desai, D., Numes, M., Chang, L., Lin, J.M., Jiao, D., Amin, S., Ipomeanol analogs as chemopreventive agents: Effect on in vitro metabolism of 4-(methylnitrosamino)-1-(3-pyridyl)-1- butanone (NNK), Cancer Lett. 97: 156-162 (1995). El-Bayoumy, K., Prokopczyk, B., Peterson, L.A., Desai, D., Amin, S., Reddy, B.S., Hoffmann, D., Wynder, E., Effects of dietary fat content on the metabolism of NNK and DNA methylation induced by NNK, Nutr. Cancer 26: 1-10 (1996). Foiles, P.G., Akerkar, S.A., Carmella, S.G., Kagan, M., Stoner, G.D., Resau, J.H., Hecht, S.S., Mass spectrometric analysis of tobacco-specific nitrosamine-DNA adducts in smokers and nonsmokers, Chem. Res. Toxicol. 4: 364-368 (1991). Guo, Z., Smith, T.J., Thomas, P.E., Yang, C.S., Metabolism of 4-(methylnitrosamino)-1-(3-pyridyl)- 1-butanone by inducible and constitutive cytochrome P450 enzymes in rats, Arch. Biochem. Biophys. 298: 279-286 (1992). 0002234.01 3/16/98 4:14 pm -19- I
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DRAFT Table 5. Metabolism of NNK by lung microsomes Metabolite Mouse' Km Vmax Rat Km Vmax Patas monkey2 Km Vmax Human3 Km Vmax Keto alcohol 5.6 56 4.9 19.1 -- -- Keto aldehyde -- -- 10.3 5.3 653 4.6 Keto acid 9.2 4.2 No published -- -- -- -- Hydroxy acid -- -- data -- -- 526 2.9 NNAL 2541 1322 902 479 573 335 NNAL-N-oxide 4.7 54 -- -- -- -- NNK-N-oxide -- -- 5.4 19.1 531 7.7 Substrate range 1-100 µM NNK 1-20 µM NNK 7-200 uM NNK 1. Smith et al. (1990). 2. Smith et al. (1997). 3. Smith et al. (1992). Kinetic parameters: Km (µM), Vmax (pmol/min/mg protein) a 0002259.01 3116/98 4:I 8 pm
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D12A1+ I' Table 4. Biomonitoring of NNK metabolites in human urine Total excretion of NNK Theoretical NNK exnosure and excretion Smoker Cigarettes/day Metabolites (nmol/day)' Exposure (ug/day)z Excretion (ug/day) 1 18.2f2.2 7.32 0.91-1.46 1.52 2 16.7f l.1 6.24 0.85-1.34 1.29 3 16.8t 1.1 4.63 0.84-1.34 0.96 4 15.0f 1.1 4.68 0.75-1.20 0.97 5 15.810.4 4.75 0.79-1.26 0.98 6 9.5±1.2 Data incomplete -- -- 7 14.2f 1.1 2.41 0.71-1.14 0.50 8 13.6t0.7 Data incomplete -- -- 9 19.1t1.7 4.30 0.95-1.53 0.89 10 8.0f 1.4 3.74 0.40-0.64 0.77 11 15.9±1.0 Dataincomplete -- -- Mean 0.78-1.24 0.99 S.D. 0.31 1. Total excretion of NNAL, NNAL-Gluc and NNAL-N-oxide. To convert to /cg NNK multiply by 207. 2. Estimated exposure range based on mainstream cigarette smoke NNK concentrations (Tricker et al., 1991; Hoffman et al., 1994). 0002259.01 3/16/98 4:18 pm w w:. CO ra
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~,--nL-0 Appendix 11. AbstracLg from Philip Morris funded research 1. AACR Special Conference in Cancer Researeh 'ONA methylation, imprinting, and the epigenetiaa of rancef. Las Croabas. Puerto Rico. December 12-16. 1997. 8-32 'ONA Methylation. Imprinting, and the Epiganetics ar Cancer' 'MetaboGsm of 4{methylnitrasunmo}1-(3-pyridy!)-1-butanone to methylating inteimediates in precisiorrait human and rodent Wer and lung tissue slioes_ Elrnar ~ and 'Johannes Er!gl. Vl/a~er. 56?ub Institute for Phannacology and ToodmioSy. Univers~y of Munich. 1}803361Uunich, Germany. and Anthony R Tridcer, FTR. 2003 Neuctr8tel. S1witzartand- 4-(Methylnitrosamino)-1-(3-pyridyl)-1-bufanorie (NNK) present in tobaao and tobacco smoke is melabofaed to at least 14 dBerent produds via several tnetaboric pathways.Including. a-hydrosylallon •to yield ns3etive 6ntermediates capable of both DNA mefhy?ation and pyntlylozobutytation. These metabolic pathv rays have primanly been eluoidaead by ur vifro incubeUon of n-latively high subsirate cartcentrafrons, (YpK2nY 1-10. p.mol NNK, with cED suspensions or _ isolated rniwsames. Compared to these methods, meta6olic studies performed using predsion-cut tissue samples in dynamic culture in which fiver and lung structural heterogeneity w8h functional phase I and phase II metabolism are maintained. while ctli interaction and communicatfon are preserved to a certain extent, n3semble more dosely the in viw situation fii the intact •argan. Using this in vrLo model, atte: spedes differences and kinetic parameters (1Gn and Vmax) for the major me4aboGc pathways of (r3H]NNK (35 Cl/mrnoo metabnl'sm over a subsirate =nea'ntration range of 10.0 nmol to 100,0 pmol in human and rodent (ALJ mouse and F344 rat) hmg and Gverlissue slices have been determined under tdential esperimentai cand-tbons. Correlation studies have been pedorrned for the aetivilies of eight principal P450 drug metabor¢ing enzymes (CYP1A1, 1A1-F'1A2, 2A6, 2B7, 2C19, 2D6, 2E1, 3A4) and 91p-hydroxystereid dehydrogenase (11 P-HSD; EC 1_1.1-146) in the presence and absencr: of en2rme-sefedive chemical inhbtZera. These studies demonstrate eonoarrtration-dependent as we(g as ergarrspecific and inter-species differences In the a-hydroxylaiion of NNK to yield DNA metffyiating species.
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15000 m m 0 HgOniw o NNAL o NNAL-NO * NNK-NO o Keto acid e Diol v HPB ----------~ a -------- ------------- ~- -------------- 20 40 60 NNK (pM) 80 100 I
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DRAFT Spratt, T.E., Trushin, N., Lin, D., Hecht, S.S., Analysis of NZ-(pryidyloxobutyl)deoxyguanosine adducts in DNA of tissues of exposed to tritium-labeled 4-(methylnitrosamino)-1-(3-pyridyl)-1- butanone and N'-nitrosonornicotine, Chem. Res. Toxicol. 2: 169-173 (1989). Staretz, M.E., Hecht, S.S., Effects of phenethyl isothiocyanate on the tissue distribution of 4- (methylnitrosamino)-1-(3-pyridyl)-1-butanone and metabolites in F344 rats, Cancer Res. 55: 5580- 5588 (1995). Staretz, M.E., Murphy, S.E., Patten, C.J., Nunes, M.G., Koehl, W., Amin, S., Koenig, L.A., Guengerich, F.P., Hecht, S.S., Comparative metabolism of the tobacco-related carcinogens benzo[a]pyrene, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone, 4-(methylnitrosamino)-1-(3- pyridyl)-1-butanol, and N'-nitrosonomicotine in human hepatic microsomes, Drug Metab. Dis. 25: 154-162 (1997a). Staretz, M.E., Foiles, P.G., Miglietta, L.M., Hecht, S.S., Evidence for an important role of DNA pyridyloxobutylation in rat lung carcinogenesis by 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone: Effects of dose and phenethyl isothiocyanate, CancerRes. 57: 259-266 (1997b). Tjalve, H., Castonguay, A., Distribution and metabolism in Syrian golden hamsters of 4- (methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), a tobaccl-specific carcinogen. In: Hayes, A.W., Schnell, F.C., Miva, T.S. (Eds.), Developments in the Science and Practice of Toxicology, Elsevier Science Publishers, Amsterdam, pp. 423-427 (1983). Tricker, A.R., N-nitroso compounds and man: Sources of exposure, endogenous formation and occurrence in body fluids, Eur. J. Cancer Prev. 6: 226-268 (1997). Tricker, A.R., Ditrich, C., Preussmann, R., N-Nitroso compounds in cigarette tobacco and their occurrence in mainstream cigarette smoke, Carcinogenesis 12: 257-261 (1991). Tricker, A.R., Klus, H., Begutter, H., Ruppert, T., Scherer, G., Adlkofer, F., Tobacco-specific and volatile N-nitrosamines in indoor air of smoker and nonsmoker homes. In: Weber, L. (Ed.), Indoor Air Pollution, Franz Siegel Buch GmbH, Ulm, pp. 310-316 (1994). Vickers, A.E.M., Use of organ slices to evaluate the biotransformation and drug-induced side-effects of pharmaceuticals, Cell Biol. Toxicol. 10: 407-414 (1994). Yoo, J.S.H., Smith, T.J., Ning, S.M., Lee, M.J., Thomas, P.E., Yang, C.S., Modulation of the levels of cytochromes P450 in rat liver and-lung by dietary lipid, Biochem. Pharmacol. 43: 2535-2542 (1992). 0002234.0f 3/16/98 4:14Pm -25-
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Table 2. Urinary excretion of NNK metabolites in different species % urinary excretion of NNK metabolites Metabolite A/J mice' 0.1 mg/kg i.p. 48-h excretion F344 ratz 0.1 mg/kg i.p. 24-h excretion F344 rat' 0.1 mg/kg i.p. 48-h excretion Patas monkey' 0.1 mg/kg i.v. 24-h excretion Hydroxy acid* 34 16.0 28 41.9-42.9 Keto acid* 19 37.8 21 24.6-26.6 Keto alcohol -- <0.06 -- -- NNAL 1 3.3 7 n.d.-2.0 NNAL-Gluc 3 0.6 2 19.1-19.9 NNK-N-oxide 8 11.7 7 13.6-15.7 NNAL-N-oxide 14 11.4 16 7.7-15.7 NNK -- 0.8 -- n.d.-0.1 6-hydroxy-NNK -- 1.0 -- -- Total a-hydroxylation* 53 54 49 58.1 N O O W ~ 1. Morse et al. (1990). w :O ~ 2. Murphy et al. (1995). 3. Hecht et al. (1993a). aoa22s9.oi 3/16/98 4:18 pm
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Table 6. Metabolism of NNK by liver microsomes Kinetic-parameters: Km (jjM), Vmax (pmoUmin/mg protein) Metabolite Mousel Km Vmax Rat2 Km Vmax Patas monkey3 Km Vmax Human4 Km Vmax Keto alcohol 73.8 239 211 156 474 37.7 1200 500 Keto aldehyde 19.1 173 234 153 8.2 37.4 367 60 NNAL -- -- -- -- 474 3470 56 282 1600 3300 NNAL-N-oxide -- -- -- -- -- -- 53 19 4500 560 NNK-N-oxide Substrate range 1-100 FcM NNK 1-200 µM NNK 1-50 ,uM NNK 5-2000 AM NNK 1. Peterson et al. (1991). 2. Guo et al. (1992). 3. Smith et al. (1997). 4. Patten et al. (1996). oao2259.m 3/76/98 4:18pm
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DRAFT Table 3. Dose response for excretion of NNK metabolites' % 24-h excretion of NNK metabolites at different dose levels (mg/kg i.p.) Metabolite 103.5 10.35 1.035 0.103 0.010 0.001 A/J mouse: Hydroxy acid* 18 32 37 34 27 35 Keto acid* 11 16 26 19 27 23 NNAL 29 11 2 1 -- -- NNAL-Gluc 22 8 4 3 -- -- NNK-N-oxide -- -- 5 8 10 7 NNAL-N-oxide 11 13 8 14 7 6 Total a-hydroxylation* 29 48 63 53 54 58 F344 rat: Hydroxy acid* 26 24 20 28 16 14 Keto acid* 24 39 45 21 38 40 NNAL 25 12 6 7 4 -- NNAL-Gluc 8 3 2 2 2 -- NNK-N-oxide 3 3 6 7 14 14 NNAL-N-oxide 6 6 8 16 13 12 Total a-hydroxylation* 50 63 66 49 54 54 N ; © : ~ -. W k t 1 1. Morse et al. (1990). e W W N 0002259.01 3/16/98 4:18pm
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2063643340
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DRAFT Hamilton, S.M., Teel, R. W., Effects of isothiocyanates on the metabolism of 4-(methylnitrosamino)- 1-(3-pyridyl)-1-butanone (NNK) and benzo[a]pyrene by hamster and rat liver microsomes, Anticancer Res. 14: 1089-1094 (1994). Hecht, S.S., Metabolic activation and detoxification of tobacco-specific nitrosamines -- A model for cancer prevention strategies, Drug. Met. Rev. 26: 373-390 (1994). Hecht, S.S., Recent studies on mechanisms of bioactivation and detoxification of 4- (methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), a tobacco-specific lung carcinogen, Crit. Rev. Toxicol. 26: 163-181 (1996). Hecht, S.S, Hoffinann, D., The relevance of tobacco-specific nitrosamines to human cancer, Cancer Surv. 8: 273-294 (1989). Hecht, S.S., Trushin, N., DNA and hemoglobin allcylation by 4-(methylnitrosamino)-1-(3-pyridyl)-1- butanone and its major metabolite 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol in F344 rats, Carcinogenesis 9: 1665-1668 (1988). Hecht, S.S., Spratt, T.E., Trushin, N., Evidence for 4-(3-pyridyl)-4-oxobutylation of DNA in F344 rats treated with the tobacco-specific nitrosamines 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone and N'-nitrosonornicotine, Carcinogenesis 9: 161-165 (1988). Hecht, S.S., Trushin, N., Reid-Quin, C.A., Burak, E.S., Jones, A.B., Southers, J.L., Gombar, C.T., Carmella, S.G., Anderson, L.M., Rice, J.M., Metabolism of the tobacco-specific nitrosamine 4- (methylnitrosamino)-1-(3-pyridyl)-1-butanone in the patas monkey: Pharmacokinetics and characterization of glucuronide metabolites, Carcinogenesis 14: 229-236 (1993a). Hecht, S.S., Carmella, S.G., Murphy, S.E., Akerkar, S., Brunnemann, K.D., Hbffrnann, D., A tobacco-specific lung carcinogen in the urine of men exposed to cigarette smoke, New England Journal ofMedicine 329: 1543-1546 (1993b). Hecht, S.S., Chung, F.L., Ritchie, J.P., Akerkar, S.A., Borukhova, A., Skowronski, L., Carmella, S.G., Effects of watercress consumption on metabolism of a tobacco-specific lung carcinogen in smokers, Cancer Epidemiol. Biomarkers Prev. 4: 877-884 (1995). Hoffmann, D., Djordjevic, M.V., Rivenson, A., Zhang, E., Desai, D., Amin, S., A study of tobacco carcinogenesis. lI. Relative potencies of tobacco-specific N-nitrosamines as inducers of lung tumours in A/J mice, Cancer Lett. 71: 25-30 (1993). 0002274.01 3/16/98 -20- 4:14pm I
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DRAFT Table 8. Metabolism of NNK in Liver Tissue. Kinetic parameters: Km (FcM), Vmax (pmol/min/mg protein) Metabolite A/J Mouse Km Vmax SG Hamster Km Vmax F344 Rat Km Vmax Human Km Vmax 0.01-1.0 wM NNK No No NNAL -- -- current current 26.0 4640 0.6 254 Keto acid -- -- data data 0.9 18 0.2 9.6 Keto alcohol -- -- 1.9 62 n.d. n.d. Hydroxy alcohol -- -- n.d. n.d. -- -- 0.01-100yM NNK NNAL 174.0 4623 96.0 19220 43.6 6805 Keto acid 677.0 5818 154.0 1159 690.0 8036 Keto alcohol 141.0 2609 260.0 8357 12760 144500 Hydroxy alcohol 199.0 3153 16.2 253 n.d. n.d. Substrate range: 0.01-100.0uM NNK n.d., no detectable formation; --, Michaelis-Menton kinetics could not be fitted. 0002259.01 3/16N8 4:18 pm I
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DRAFC Hoffmann, D., Brunnemann, K.D., Prokopczyk, B., Djordjevic, M.V., Tobacco-specific N- nitrosamines and areca-derived N-nitrosamines: Chemistry, biochemistry, carcinogenicity, and relevance to humans, J. Toxicol. Environ. Health 41: 1-52 (1994). Hong, J.Y., Smith, T.J., Lee, M.J., Li, W., Ma, B.L., Ning, S.M., Brady, J.F., Thomas, P.E., Yang, C.S., Metabolism of carcinogenic nitrosamines by rat nasal mucosa and the effects of dially sulfide, Cancer Res. 51: 1509-1514 (1991). Hong, J.Y., Wang, Z.Y., Smith, T.J., Zhou, S., Shi, S., Pan, J., Yang, C.S., Inhibitory effects of dially sulfide on the metabolism and tumorigenicity of the tobacco-specific carcinogen 4- (methylnitrosamino)-1-(3-pyridyl)-1-butanone, Carcinogenesis 13: 901-904 (1992). International Agency for Research on Cancer, TARC Monographs on the Carcinogenic Risk of Chemicals to Humans, Vol. 37, Tobacco Habits Other than Smoking; Betel-Quid and Areca-Nut Chewing; and Some Related Nitrosamines. International Agency for Reesearch on Cancer, Lyon, (1985). Jorquera, R., Castonguay, A., Schuller, H.M., Effect of tobacco smoke condensate on the metabolism of 4-(methytnitrosamino)-1-(3-pyridyl)-1-butanone by adult and fetal hamster microsomes, Drug. Met. Dis. 21: 318-324 (1993). Kim, P.M., Wells, P.G., Genoprotection by UDP-glucumnosyltransferases in peroxidase-dependent, reactive-oxygen species-mediated micronucleus initiation by the carcinogens 4-(methylnitrosamino)- 1-(3-pyridyl)-1-butanone and benzo[a]pyrene, Cancer Res. 56: 1526-1532 (1996). Klus, H., Begutter, H., Scherer, G., Tricker, A.R., Adlkofer, F., Tobacco-specific and volatile N- nitrosanunes in environmental tobacco smoke of offices, Indoor Environ. 1: 348-350 (1992). Lacroix, D., Desrochers, M., Castonguay, A., Anderson, A., Metabolism of 4-(methylnitrosamino)- I-(3-pyridyl)-1-butanone (NNK) in human kidney epithelial cells transfected with rat CYP2B1 cDNA, Carcinogenesis 14: 1639-1642 (1993). Lin, J.M., Desai, D.H., Morse, M.A., Amin, S., Hecht, S.S., Inhibition of 4-(methylnitrosamino)-1- (3-pyridyl)-1-butanone pulmonary metabolism and tumorigenicity in mice by analogues of the investigational chemotherapeutic drug 4-ipomeanol, Chem. Res. Toxicol. 5: 674-679 (1992). Liu, L., Alaoui-Jamali, M.A., El Alami, N., Castonguay, A., Metabolism and DNA single strand breaks induced by 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone and its analogues in primary culture of rat hepatocytes, CancerRes. 50: 1810-1816 (1990). - 0002234.01 3/16198 4:14 pm - 21 - I
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Table 7. Metabolism of NNK in Lung Tissue. Metabolite A/J Mouse Km Vmax SG Hamster Km Vmax F344 Rat Km Vmax Human Km Vmax 0.01-1.0 µM NNK NNAL 1.7 168 No No 3.6 309 1.0 311 Keto acid 0.3 45 current current 5.2 182 0.4 11 Keto alcohol 0.6 141 data data 2.7 177 1.2 26 Hydroxy alcohol 1.0 56 n.d. -- 0.7 25 NNK-N-oxide 0.7 184 6.8 498 n.d. n.d. NNAL-N-oxide n.d. n.d. n.d. -- 0.6 19 0.01-100,uM NNK NNAL 39.0 4309 -- -- 239 65640 Keto acid 10.1 317 317.0 15620 -- -- Keto alcohol 7.5 556 90.0 8398 -- -- Hydroxy alcohol 160.0 3405 2.4 107 -- -- NNK-N-oxide 25.1 25.1 68.0 7995 41240 413500 NNAL-N-oxide n.d. n.d. 0.9 21 -- -- Substrate range: 0.01-100.0~cM NNK n.d., no detectable formation; --, Michaelis-Menton kinetics could not be fitted. Kinetic parameters: Km (EcM), Vmax (pmoUmin/mg protein) 0002259.01 3/1G/98 4:18 pm
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DRAFT Liu, L., Castonguay, A., Gerson, S.L., Lack of correlation between DNA methylation and hepatogenesis in rats and hamsters treated with 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone, Carcinogenesis 13: 2137-2140 (1992). Liu, Y., Sundqvist, K., Belinksy, S.A., Castonguay, A., Tjalve, H., Grafstrom, R.C., Metabolism and macromolecular interaction of the tobacco-speciific carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)- 1-butanone in cultured explants and epithelial cells of human buccal mucosa, Carcinogenesis 14: 2382-2388 (1993). Maser, E., Richter, E., Friebershauser, J., The identification of 11 P-hydroxysteroid dehydrogenase as carbonyl reductase of the tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1- butanone, Eur. J. Biochem. 238: 484-489 (1996). Merger, M., Meger-Kossien, L, Scherer, G., Metabolites of4-(methylnitrosamino)-1-(3-pyridyl)-1- butanone (NNK) in urine of smokers and nonsmokers, Proc. Am. Assoc. Cancer Res., 40, Abs. 2275 (1998). Miller, C,H., Zhang, Z., Hamilton, S.M., Teel, R.W., Effects of capsaicin on liver microsomal metabolism of the tobacco-specific nitrosamine NNK, Cancer Lett. 75: 45-52 (1993). Miller, C.H., Hamilton, S.M., Teel, R.W., Effects of compounds of plant origin on the mutagenicity and metabolism of the tobacco-specific nitrosamine NNK, Phytotherapy Res. 8: 342-347 (1994). Miller, C.H., Castonguay, A., Teel, R. W., Modulation of the mutagenicity and metabolism of the tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) by phenolic compounds, Mutation Res. 368: 221-233 (1996). Morse, M.A., Ekling, K.I., Toussaint, M., Amin, S.G., Chung, F.L., Characterization of a glucuronide metabolie of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and its dose- dependent excretion in the urine of mice and rats, Carcinogenesis 11: 1819-1823 (1990). Morse, M.A., Kresty, L.A., Toburen, A.L., Inhibition of metabolism of 4-(methylnitrosamino)-1-(3- == pyridyl)-1-butanone by dietary benzaldehyde, Cancer Lett. 97: 255-261 (1995). v y Murphy, S.E., Heiblum, R., Effect of nicotine and tobacco-specific nitrosamines on the metabolism of N'-nitrosonornicotine and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone by rat oral tissue, Carcinogenesis 11: 1663-1665 (1990). 000223401 3/l6/98 4:14 pm - 22 -
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DRAFT Murphy, S.E., Coletta, K.A., Two types of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone hemoglobin adducts, from metabolites which migrate into or are formed in red blood cells, Cancer Res. 53: 777-783 (1993). Murphy, S.E., Palomino, A., Hecht, S.S., Hoffinann, D., Dose-response study of DNA and hemoglobin adduct formation by 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone in F344 rats, Cancer Res. 50: 5446-5452 (1990a). Murphy, S.E., Heiblum, R., Trushin, N., Comparative metabolism of N'-nitrosonomicotine and 4- (methyhiitrosamino)-1-(3-pyridyl)-1-butanone by cultured rat oral tissue and esophagus, CancerRes. 50: 4685-4691 (1990b). Murphy, S.E., Heiblum, R., Kling, P.G., Bowman, D., Davis, W.J., Stoner, G., Effect of phenethyl isothiocyanate on the metabolism of tobacco-specific nitrosamines by cultured rat oral tissue, Carcinogenesis 12: 957-961 (1991). _ Murphy, S.E., Spina, D.A., Nunes, M.G., Puilo, D.A., Glucuronidation of 4-((hydroxymethyl) nitrosamino)-1-(3-pyridyl)-1-butanone, a metabolically activated form of 4-(methylnitrosamino)- butanone, by pherobarbital-treated rats, Chem. Res. Toxicol. 8: 772-779 (1995). Patten, C.J., Smith, T.J., Murphy, S.E., Wang, M.H., Lee., J., Tynes, R.E., Koch, P., Yang, C.S., Kinetic analysis of the activation of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone by heterologously expressed human P450 enzymes and the effects of P450-specific chemical inhibitors on this activation in human liver microsomes, Arch. Biochem. Biophys. 333: 127-138 (1996). Pepin, P., Rossignol, G., Castonguay, A., Inhibition ofNNK-induced lung tumorigenesis in A/J mice by ellagic acid and butylated hydroxyanisole, CancerJ. 3: 266-272 (1990). Pepin, P., Bouchard L., Nicole, P., Castonguay, A., Effects of sulindac and oltipraz on the tumorigenicity of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone in A/J mouse lung, Carcinogenesis 13: 341-348 (1992). Peterson, L.A., Cannella, S.G., Hecht, S.S., Investigations of metabolic precursors to hemoglobin ~, and DNA adducts of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone, Carcinogenesis 11: 1329- _Ch 1333 (1990). ~ ~ Peterson, L.A., Hecht, S.S, 06-methylguanine is a critical determinant of 4-(methylnitrosamino)-1- rw ' (3-pyridyl)-1-butanone tumorigeneiss in A/J mouse lung, Cancer Res. 51: 5557-5564 (1991). a- 0002234.01 J116198 4:14pm - 23 -
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DRAFT Peterson, L.A., Mathew, R., Hecht, S.S., Quantification of microsomal a-hydroxylation of the tobacco-specific nitrosamine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone, Cancer Res. 51: 5495-5500 (1991). Preussmann, R., Steward B.W., N-nitroso carcinogens. In: Searle, C.E. (Ed.), Chemical Carcinogens, 2nd Edition. ACS Monograph Series No. 182, American Chemical Society, Washington, D.C., pp. 643-823 (1984). Richter, E., Tricker, A.R., Nicotine inhibits the metabolic activation of the tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone in rats, Carcinogenesis 15: 1061-1064 (1994). Richter E., Branner, B., Kutzer, C., Donharl, A.M.E., Scherer, G., Tricker, A.R., Heller, W.D., Comparison of biomarkers for exposure to environmental tobacco smoke: Cotinine and hemoglobin adducts from aromatic amines and tobacco-specific nitrosamines in pregnant smoking and nonsmoking women. In: Maroni M. (Ed.), Healthy Buildings '95, Vol. 2, pp. 611-616 (1995). Schulze, J., Schlager, W. Wunch, R., Richter, E., Metabolism of 4-(methylnitrosamino)-1-(3- pyridyl)-1-butanone (NNK) by hamster, mouse and rat intestine: Relevance of species differences, Carcinogenesis 17: 1093-1099 (1996). Smith, T.J., Guo, Z., Thomas, P.E., Chung, F.L., Morse, M.A., Eklind, K., Yang, C.S., Metabolism of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone in mouse lung microsomes and its inhibition by isothiocyanates, Cancer Res. 50: 6817-6822 (1990). Smith, T.J., Guo, Z., Gonzalez, F.J., Guengerich, F.P., Stoner, G.D., Yang, C.S., Metabolism of 4- (methylnitrosamino)-1-(3-pyridyl)-1-butanone in human lung and liver microsomes and cytochrome P-450 expressed in hepatoma cells, Cancer Res. 52: 1757-1763 (1992). Smith, T.J., Guo, Z., Li, C., Ning, S.M., Thomas, P.E., Yang, C.S., Mechanisms of inhibition of 4- (methylnitrosamino)-1-(3-pyridyl)-1-butanone bioactivation in mouse by dietary phenethyl isothiocyanates, Cancer Res. 53: 3276-3282 (1993). Smith, T.J., Stoner, G.D., Yang, C.S., Activation of4-(methylnitrosamino)-1-(3-pyridyl)-I-butanone (NNK) in human lung microsomes by cytochrome P450, lipoxygenase, and hydroperoxides, Cancer Res. 55: 5566-5573 (1995). Smith, T.J., Liao, A.M., Liu, Y., Jones, A.B., Anderson, L.M., Yang, C.S., Enzymes involved in the bioactivation of 4-(methyhlitrosamino)-1-(3-pyridyl)-1-butanone in patas monkey lung and liver microsomes, Carcinogenesis 18: 1577-1584 (1997). a0oz2s4.oi 3n6/9S 4:14 pm -24- I
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4
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DRAFT Lastly, the IARC summary states that: "In a separate exercise, the number of lung cancers occurring in the countries of the European Union that can be attributed to spousal ETS exposure was estimated to be about 800 among women and 300 among men." The reader is likely to assume that the calculation referred to utilized the relative risk of 1.16 obtained from the IARC multi-center study. However, in actuality the authors of the paper used a relative risk for spousal exposure of 1.30, almost two times the uncorrected IARC result (Tredaniel et al. 1997). 0002268.01 7/16/98 3:16Pm -4-
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1 N COOH / N' H CHg Cotinine Acid I i COOH i ~\ ~ NO CH3 N iso-NNAC Enzymatic Oxidation ~Nr Demethylation N' CH3 Nicotine ! Reduction HO I HN N CHpOH ` i ~ N~ NO CHg N NO CH3 iso-NNAL NNAL N H 'N" H Anatabine Anabasine
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Man Oral® 100.g 24.0 98.2 0.8 0.2 - 0.3 2.0 0.2 0.1 2.6 LluNel, 1993 Man Eecphegus(t)239,0 24.0 08.2 1.7 - • 0.1 - 0,1 Caslonguaye/d„1983b Man Kldney ® 2.3 50.0 99.8 0.03 • - 0.05 0.06 0.03 0.11 LsmIe e1 af.,1092 Man Trachea )I) 238.0 24.0 94.2 0.1 - - - 6.7 - 5.7 Castonquey at e/.,1983b 'Abbreviallon in brackels: (tN Ossue; ©, afluced ostl; (h), hapatocyle; (m), mlaosame; -, nal detected, °Calculaled on the basis of total metebofiles detected, c56 a-hydroxyls0on ealalated irom Iha sum at kelo alcohd, kato acld (keto eldehyde) and hydroKy edaf: meleboNc pethways known lo produca reectlve kntennedletea which bind to DNA.. °Keto aldehyde measured Inslead of keoo acid, ve[ue for kela eldehyde preeenled In braskete. +daint determination of kela aldehyde and keio acid. 6tr£$b9E90Z ~
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DRAFT Commentary on the Results of the LARC Multi-Center Study, "Lung Cancer and Exposure to Environmental Tobacco Smoke," Published in the IARC Biennial (1996-1997) Report Results from the International Agency for Research on Cancer (IARC) multi-center epidemiologic study of environmental tobacco smoke (ETS) exposure and lung cancer risk were recently published for the first time in the IARC Biennial Report for 1996-1997 (IARC, 1997; see Appendix A, section 3.7.2). This study, which began in 1988, represents the largest study in Europe and the second largest study ever conducted of its kind. In 1986, IARC made the following comments regarding the epidemiological evidence for the reported association between ETS exposure and lung cancer (IARC, 1986): "Several epidemiological studies have reported an increased risk of lung cancer in nonsmoking spouses of smokers, although some others have not ... The resulting errors could arguably have artifactually depressed or raised estimated risks, and, as a consequence, each is compatible either with an increase or with an absence of risk." It was on the basis of that conclusion that IARC decided to undertake its multi-center study. The results, based on an analysis of 650 cases and 1,542 controls in 12 centers in 7 European countries, were as follows: • RR for spousal exposure -- 1.16 (95% CI, 0.93-1.44) • RR for workplace exposure -- 1.17 (95% CI, 0.94-1.45) • RR for combined exposure -- 1.14 (95°/u CI, 0.88-1.47) 0002268.01 3/16/98 -1- 3:16 pm
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DRAFT interpretation of these results is that they confirm an association between reports of ETS exposure and lung cancer, although the association is extremely weak. It is worth noting that the Introduction to the brief text in the IARC Biennial Report states that ETS exposure is a "likely cause" of lung cancer. This is a far less conclusive statement than made by some other agencies and scientists. There are several interesting points that were made in the discussion of these results in the IARC Biennial Report. The first deals with dose-response trends, where the claim is made that "several quantitative indicators of ETS exposure showed a dose-response relationship with lung cancer risk." It is impossible to analyze this statement from the data provided in the summary; however, the fact that not all such indicators showed a dose-response relationship suggests that such an analysis would be of great importance. Secondly, IARC states that: "A further study of non-smoking women in Moscow, Russian Federation, confirmed the results on ETS of the larger international investigation and suggested a role of environmental air pollution independent of the effect of ETS: " In actuality, the results of this study (Zaridze et al., 1998) "confirm" only the result for spousal exposure. The authors report no increase in lung cancer risk for workplace exposure; moreover, the reported dose- response effects were negative, whether years of exposure or number of cigarettes smoked by the spouse was used as the metameter of spousal exposure. 0002268.01 3/16198 3:IGpm - 3 -
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DRAFT The report also states that there was no evidence of an association between lung cancer risk and ETS exposure during childhood. The two immediate conclusions that can be drawn from these results are that both indices of ETS exposure, spousal and workplace, suggest a small but positive association with lung cancer, but neither association is statistically significant. What cannot be determined from the results as presented is the extent to which they have been corrected for known systematic biases -- in particular, misclassification of smoking habit and confounding by diet (spousal exposure only). The most recent meta-analysis carried out on the possible association of ETS exposure and lung cancer (Hackshaw et al., 1997) arrived at a combined relative risk of 1.24, which the authors estimated should have been reduced by 0.06 for misclassification of smoking habit and by 0.02 for dietary confounding. Although their suggested reduction is almost certainly inadequate, accepting even these values would yield a reduction of 33% for the spousal exposure risk estimate in the IARC study, resulting in a relative risk of 1.10, and a reduction of 25% for the workplace exposure estimate, resulting in a relative risk of 1.13. Although these values are, of course, still greater than 1.0, it is as likely that they suggest no association whatsoever, as it is likely that they suggest an association. However, it should be noted that the results reported by IARC are in line with other studies and, in particular, in line with a number of meta-analyses. Therefore, clearly, a possible 0002268.01 3/t6198 3:16 pm -2-
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A 0 03 W 0' a W W Cti 0
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http://www.iarc.fr/ •:rVr:lai"r,, J\ f....r l' C t~ Wrl(i li\.r`P{1,\ r i() ~?I r C r. Cancer Research far Cancer Contral _~IIrI_)IUlqfq~~~; :>:jlr'.~1~eu~y Ilbl0,
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DRAFT References Hackshaw, A.K., Law, M.R., and Wald, N.J., The accumulated evidence on lung cancer and environmental tobacco smoke, Br. Med. J. 315: 980-988 (1997). Tredaniel, J., Boffetta, P., Saracci, R., and Hirsch, A., Non-smoker lung cancer deaths attributable to exposure to spouse's environmental tobacco smoke, Int. J. Epidemiol. 26: 939-944 (1997). World Health Organization, International Agency for Research on Cancer, IARC Monographs on the Evaluation of the Carcinogenic Risk of Chemicals to Humans, Tobacco Smoking, Vol. 38, Lyon; France (1986). World Health Organization, International Agency for Research on Cancer, Biennial Report: 1996- 1997, Lyon, France (1997). Zaridze, D., Maximovitch, D., Zemlayanaya, G., Aitakov, Z.N., and Boffetta, P., Exposure to environmental tobacco smoke and risk of lung cancer in non-smoking women from Moscow, Russia, Int. J. Cancer 75: 335-338 (1998). 0002268.01 3/16198 3:16 pm - 5 -
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6 N O a., LJ 0+ da GJ W Cr! ~
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sL. -4GC.l~ 9/^_ 'N'CR:_.: ;EALT ORG.wIZ.a.:"C:N INTEXNATIOtiAL AGENCY FOR RESEARCH ON CANCER B IENNIAL REP ORrT 1996-1997 Inte;national Azency `cr 3esearci on Cancer Lvon, =ranc-- :~9''
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Appendix I. !n vitro metabolism of NNK In I(ssues, cultured celta and microsomes hom laboratory eninlels and man Species T1ssue' CondiUona Percentage foanetbn at nralor metabolltese Peroen6age a- Reference NNK Time NNAL NNK-N- NNAL-N- NNAL- Kelo Kelo Hydro>ty 01o1 hydroxylaUon` (µM) (h) oxide odde Gluc alcohol aclda add NJ mouee Lung (t) 4.7 4.0 1.2 49.6 0.6 27.1 19,6 0.6 5.8 47.4 Papln e1 af.,1992 NJ mouse Lim (1) 4.7 0.8 0.6 53.7 1.3 21.8 13.4 1.3 2.1 42.3 Pepln a1 d., 1992 NJ mouse Lung (1) 2.36 2.0 1,6 53.9 0.6 29.7 12,4 0.4 ' 1.4 42.6 Bouchard & Catonguay, 1993 NJ mouse Lung (l) 0.21 24 88.4 7.5 3.7 3.1 10.6 1.6 5,2 15.2 CasWnguay elal,1gg3a NJ mouae Lung (m) 200.0 0.5 78.3 10.4 - 2.6 10.8 2.5 Pateraon al al.,1B91 NJ mouse Lung (m) 10.0 0.5 12.6 43.0 2,0 24,7 (17.1) 41.8 8mllh e/ aL, 1g93 NJ muuse Lung (m) 10.0 0.5 10,3 31.8 - 49.7 2.2 61.9 Sm9h ae aL,1990 NJ mouse Lung (m) 10.0 0.26 13.7 39.1 - 327 14.5 • 47.2 Lln el a1.,1992 NJ mouse Lung (m) 10.0 025 17.6 42.2 - 26,2 (13.8) 40,0 f7sed e1 e1.,1(N5 NJ mouse Lung (m) 10.0 0.18 5.6 46.6 - r 29.0 (19.2) 40.8 Hong of e1.,1992 NJ mousa Lung (m) • 0.8 29.4 21,7 - 48.7 - 48,1 Moraa of a1,19g8 NJ mouse LHer (m) 10.0 0.8 11.7 13.1 - 28.9 (40.3) 69.2 Hang a/ a1.,1902 NJ mouse Liver (m) 10.0 0.25 63.3 - - 16.3 30.4 46,7 lfn of at, 1992 NJ mouse Lirer(m) 10.0 0.25 24.0 - 23.2 (62.6) 47.2 Oesal M a1.,1095 NJ mouse Liver (m) 10,0 0.16 26.6 4.7 3.1 24.4 (41.1) 05.5 SmBh elal., 1993 NJ muse Llver(m) - 0.25 72.0 • - 27,8 49.3 77.1 Morae ef ef, 1995 NJ mouse Intestine (1) 14.0 0.75 87.8 2.9 0.8 2.7 5.4 0.3 0.1 8.4 Pepln of a(, 1090 NJ mcuee Stomech (q 14.0 2.0 07.2 6.0 0.3 5.7 16.2 0.3 0.3 24.2 Pepin el N„ 1900 NMRI (1) IMeedne (I) 1.0 2.0 4.6 28.9 0.1 2.4 44.9 10.8 - 58.1 Schub.e el sL,1g9g NMR) (in) Inteaelne {t) 1.0 2.0 7.1 29.5 4.7 4.2 50.8 3.9 • 58.7 6ehulze 41 eL, 1998 F344 ral Lung (m) 1.0 0.5 22.9 $8.3 • 34.4 4.4 30.8 "ayoumy a1 aL, pgge F344 ret Llver (hl 5.0 2.0 41.7 8.1 0.6 - 8A 35.4 9.0 0.9 50.9 ', Murphy 6 Coletia, 1093 F344ra1 LNer(h{ 1.0 18.0 11.4 2,9 7.9 12.6 3.7 44.7 17.0 - 85.4 Murphy of el„ 1996 F344 ral Llver (m) 1.33 0.5 99.8 0.2 0.004 - 0.2 0.01 0.04 0.21 Ham9ton 6 Teel 1994 F344ra1 LNer(m) 1.0 0.6 98.9 01 - - 0.7 0.3 1.0 EI•Bayoumy of aL,1998 F344 rat Esoph. (l) 40,0 24.0 91.3 4.5 0.1 3.5 4.2 MurphyetaL,lB9ob F344 rat Neaal (t) 23.8 3.0 5.4 • • - 21.3 84,0 5.3 4.0 700 Brigebo e1 W., /9e3 F344 rat Nanel(t) 23.8 24.0 4.9 5.6 09.0 11.2 6.4 06.7 Bd6ebo atel.,1963 F344 rat Oral (t) 1.0 24.0 19.5 51.0 2.0 1e.3 11.2 27.6 Murphy of a/., f990b F344 rel Oral (1) 1.0 24.0 26.2 38.3 0.8 6.9 20:7 0.6 0.5 28.2 Murphy al ab,1990h N ~ z
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DRAFT oxidation metabolites of nicotine were known, they were found only at low concentrations (Turner, 1969). After 1987, the presence of significant concentrations of trans-3-hydroxycotinine (Neurath and Pein, 1987), and glucuronide conjugates of nicotine, cotinine and trans-3-hydroxycotinine (Curvall et al., 1989) in urine were recognized. Although cotinine appears to be the major nicotine metabolite in saliva and serum (Curvall et al., 1990), trans-3-hydroxycotinine (Byrd et al., 1994) or its glucuronide conjugate (Andersson et al., 1997) is the most prevalent in urine. Actually, cotinine constitutes only 10-15% of the nicotine metabolites. At least seventeen (17) metabolites of nicotine have been identified in urine (Kyerematen et al., 1987). Many of these have been quantified (Byrd et al., 1994; Andersson et al., 1997). Cotinine as a Biomarker for Smoker Classification The evolution of cotinine as a biomarker began as a means to discern smokers from nonsmokers. The levels of cotinine in saliva, serum and urine of a smoker of >20 cigarettes per day is relatively easy to distinguish from that of a nonsmoker using a variety of analytical methods including RIA. In large epidemiological studies for which cost, labor, time and other issues are important, saliva samples are the easiest to obtain. It has been reported that analytical determinations of cotinine in saliva provide essentially the same information as similar determinations in serum (Curvall et al., 1990). A mean saliva to serum ratio of cotinine concentration of 1.2 has been reported (Curvall et al., 1990). Although further study is needed, at this time both matrices are 0002191.01 3116/98 3:44pm -3-
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DRAFT nicotine intake, it may not serve as well as a biomarker for ETS exposure. Furthermore, cotinine has a biological half-life of approximately 20 hours and can only represent nicotine intake within an interval of a few half-lives. While a number of alternative biomarkers have been proposed, most of these have been devaluated relative to cotinine (Benowitz, 1996, Table 5). In the search for a biomarker for tobacco use or ETS exposure, by a process of elimination cotinine appears well on the way to becoming the compound of choice for many researchers (Benowitz, 1996). Analytical methods may be described as selective or as specific. Although many analytical methods exist for specific detection and quantification of cotinine, most of these are based on instrumental methods of analysis (Benowitz, 1996, Table 4). Frequently, these methods are considered too expensive, too labor intensive, or to require special skills beyond those frequently available for epidemiological or exposure studies. Radioimmunoassay (RIA) methods for cotinine were developed and widely applied in the scientific community that conducts various epidemiological and exposure investigations (Langone et al., 1973). RIA methods are rapid, economical and require few special analytical skills, but they are selective rather than specific for chemical compounds. Selectivity was likely thought to be an adequate criterion for cotinine. Until approximately 1987, cotinine was considered to be the major, if not the only, known nicotine metabolite of analytically significant concentration in the biological fluids of interest. Although N- 0002191.O1 -- 3/16/98 3:44pm -2-
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DRAFT the degree of ETS exposure in nonsmokers, inaccurate questionnaire-derived data and analytical variance. These levels of uncertainty not only have potential for impact on conclusions drawn from scientific studies, but on decisions regarding individuals such as classification for other purposes. In summary, when an individual is clearly a smoker, or clearly a nonsmoker with moderate ETS exposure, ootinine measurements in saliva, serum or urine may be capable or distinguishing between the two. To determine the classification of occasional smokers or nonsmokers with significant ETS exposure is not an analytical issue, but one of establishing a decision point. Establishing this point is complicated by the lack of a clear definition of a smoker. If the cotinine level used to classify a subject as a smoker is too high, smokers may be erroneously classified as nonsmokers. One study (Saracci and Riboli, 1989) that classified as smokers subjects with urinary levels of cotinine above 50 ng/mg creatinine, later reported that of the 47 subjects that were excluded from the study as smokers, only 20 (1.5% of the total study population) can be considered to be smokers (Riboli et al., 1995). The authors suggested that a cutoff level of 150 ng/mg should be used to avoid exclusion of nonsmokers with high ETS exposure. The position could be taken that a level lower than 50 ng/mg is more realistic to identify light smokers who are deceivers in questionnaire responses. This example clearly demonstrates that a single point value used for all subjects is woefully inadequate for such an important decision. Selection of the point value criteria may be driven inadvertently by the goal of the study. 0002191.01 3/15/98 3:44 pm
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DRAFT As mentioned above, saliva, serum and urine have all been used as matrices for the determination of nicotine and its metabolites. Because urine has been used in a large number of epidemiological studies, and because that matrix appears to contain the largest number of metabolites, it will be considered first. Metabolic Products of Nicotine in Urine At least 17 metabolic products of nicotine have been identified in human urine (Kyerematen et aL, 1987). Of these, cotinine has been the focal component. Analytical methodologies have been developed that may be applied reasonably to those compounds shown in Table 1, along with some of their glucuronide conjugates. The remaining metabolites require more involved analytical methods. Table 2 shows the results of two independent studies that measured in human urine the compounds shown in Table 1. The results are reported as percent of the total found based on nicotine equivalents of each compound, and the cumulative total if the compounds are taken into account sequentially. There is general agreement between these mean values reported in the two studies, although a significant difference was found between the distribution of the glucuronide conjugates, especially the 3HC-G. The variance of the mean of the percentage of each metabolite is somewhat higher in the study by Andersson et al. (1997) than in the one by Byrd et al. (1994). 0002I91.01 • 3/16/98 3:44 pm - 8 "
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Cotinine as a Biomarker for ETS Exposure The analytical chemistry issues for the use of cotinine as a biomarker for classification of smoking status are minor compared to those associated with its use for estimating ETS exposure-dose relationships. The evolution of the use of cotinine levels from a method to make a binary decision (smoker or nonsmoker) to implied quantification of ETS exposure was a slow but relentless process. Cotinine concentrations in the case of regular smokers are normally 2-3 orders of magnitude greater than of the limit of quantification (LOQ) of analytical methods that have been applied. Even the marginal region of 1-5 ng/mL in saliva, serum and urine are adequately measurable using RIA and chromatographic methods. Especially in urine samples, analytical and interpersonal metabolic considerations may become paramount. These factors, coupled with what may be considered a propensity toward analytical expediency in epidemiological ETS exposure studies, may lead to opportunities for contradictory conclusions. Recently, prominent professors of epidemiology expressed the view that contradictory findings in epidemiological research are "common" (statements of Trichopoulos et al. 1997). It is possible that some of these contradictions stem from inadequate experimental design, insufficient number of subjects, or from errors in the analytical data upon which decisions are based. 0002191.01 3/7 G/98 3:+4pm -7-
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DRAFT Introduction There are two reasons that a biomarker for tobacco use or environmental tobacco smoke (ETS) exposure is of considerable interest. The first is to determine whether an individual is a smoker or nonsmoker. The second is to attempt to quantify levels of exposure to ETS for nonsmokers. To answer the first question is relatively straightforward, since in principle the difference between a given biomarker for a smoker and a nonsmoker should be large. The second question, however, requires one to attempt to differentiate between two small and similar values. As a result, the analytical criteria for these two applications are very different, and require consideration of different methodologies. In addition to the analytical concerns, the potential impact of interpersonal variation in metabolic rates and pathways contributes confusion. Add the choice of saliva, serum, or urine as the sample matrix, and it is easy to see why a complex set of measurement and data interpretation uncertainties has evolved. Some of these issues will be addressed in this discussion. Cotinine is a carbon-oxidation metabolite of nicotine. It is found at measurable levels in saliva, serum and urine of subjects who smoke, who use other forms of tobacco, or who have been exposed to ETS at sufficient levels (Benowitz, 1996). Cotinine meets many of the criteria proposed by the National Research Council (NRC) for an ETS biomarker (NRC, 1986), but it must be kept in mind that cotinine is derived from nicotine. Although cotinine may serve as a biomarker for 0002191.01 3/16/98 3:44 pm - 1 -
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F344 ral Oral (1) 1,0 24.0 56,0 251 9.0 8.3 0.4 2.2 17.7 Murphy at d.,1991 F344 ra1 Oral (1) 1,0 24.0 49.9 21.6 6.6 9.1 11.5 0.7 1.8 21.3 Murphy /, Hdblum,1901 m F344 ral Oral (I) 1.0 4.0 35.8 3.9 30.6 14,8 18,6 1.5 - 29.7 a Murphy at aL. 1990b F344 ra1 Oral (1) 1.0 4.0 50.0 20.5 5.1 e.7 14.3 0.6 - 23.8 Murphy at at.,1990b F344 rat Otal (1) 11.0 4.0 81.9 5.5 - 3.1 9.5 • - 12.6 Murphystaf•,1990b SD ral Lung (m) 10.0 0.6 28.6 24.4 0.5 • 48.0 0.5 • 40.5 Yeo at ef., 1902 So rat Lung (m) 0.47 0.33 25.7 40.3 ~ 15.5 12.6 - - 28.0 Ardriert ef al.,1998 SO rat Liver (m) 10•0 0.5 93.6 1.3 0.2 3.9 1.0 - • 4.9 Yooelef„ 1902 SD ra! Masal (m) 10.0 0.5 - 1.0 1.0 - 58.5 38.8 0.2 2.0 96-0 Hong sf eL, 1901 SDral(I) lydesllne(1) 1,0 2.0 0.8 29.4 8.9 • 10.3 4.2 2.7. • 17.2 Schu4eetaL,1998 ~ 50rat(t) Inlestine(11 1.0 2.0 78.2 11.2 L1 - 3.2 6.0 0.4 • 0.6 S+fiulreetat., 199e a 0 SG Hamsler Lung (1) 4.2 3.0 20.4 32.3 1.8 17.9 26.5 0.3 1.e 43.7 Charesl at eL,19e9 SG Hamster Lung (1) 0.86 1.0 37.4 27.4 4,7 12,0 16.6 0.6 1.5 20.0 a T/BtvelCaelon0uay,1983 SG Hamster Lung (I) 0.24 3,0 22.1 33.2 5.5 17.7 20.7 0J • 39.1 Chewsl at af.,1989 SO Hamster Lunp (m) 10.0 0.5 50,5 19.8 1.0 24.3 3,7 • - 28.5 Jorquera ef at„ 1093 SO Hemeter Lung (m) • 0.5 13.0 18.6 7.2 40.6 9.er • 12.9 50,4 Zhang ataL 1997 SO Hamslsr Liver (q 0.68 1.0 t19.0 1.2 3.8 , 2.9 3.1 3.2 8.0 , 7)8Me & Caslonpuay, 1983 SGHamsler Liver(m) 10.0 0,6 e0.2 9.0 0.7 24.1 8.0 0.03 - 30.1 Jorquera el e1.,1993 SG Hamsler Liver(m) 8.0 0.6 44.5 8.7 3.2 35.6 4.0+ • 4•1 60.5 Mlller ef eL, 1094 SO Hamster Liver (m) 5.1 0.5 69.9 12.7 0.9 6..6 1,44 • 1.3 8.2 MIOer et eL,1903 SGHamsler Llver(m) 5.1 0.5 67.7 8.3 4.6 12.0, 6.1+ 0.1 1.2 1e.2 MOleratat„1996 9GHameter Llver(m) 1,33 0.5 $5.4 19.6 2.9 17.5 0.8+ - 3.9 18.3 Hamlltnn 4 Teal, 994 SG Hamsler Llver (m) 1.0 1.0 62.7 2.4 30.6 3,3 0.1 0,9 34,0 Caslonguey i Roselpnoh 1992 SG Hamster Liver (m) • 0.6 31.1 6.1 8.4 24.9 22,1+ O•4 1.0 47.4 Zhanq ef eL,1997 86 Hamater Nasal (1) 0.60 1.0 21.0 1.8 2.0 13.2 61.3 2.2 0.5 06.7 Cherell ef aL,1e8p SO Hamster Tradlea(t) 0.66 1.D 31.1 11.5 16.2 38.0 • 1.2 58.2 Charedalaf., 1999 SO Harneler tntestine p) 1.0 2.0 11,0 25.8 31.1 5,8 14.6 11.7 • 32.1 Schulee at aL, 1998 Man Lung (t) 23e,0 24.0 98.4 2.7 0.9 • 0.9 Caslsnpuay at a1.,1983b Man Lung (m) 10.0 1.0 96,4 1.2 <0.2 <0.2 (1.4) 0.7 - 2.3 . SmOh ef al., 1992 Man Lung (m) 10.0 0-17 96.1 --• (3,0) -• 0.9 - 3.9 Sm6b el a/., 1995 Man Liver4m) 3.0 0.17 e.1,5 1,0 - 7,5 7,0* - 1.0 14.5 Stareta af al., 1997a Man Llver(m) 10.0 1.0 92.4 1.2 07 3.3 (1.6) 0,9 - 5.7 Smllhefa/.,1992 Man Otadder(q 230.0 24.0 99,9 0.1 - 0.1 Caslonguay a/ al., 1993b Man Bronchus (I) 238.0 24.0 95.4 4.5 0.1 • 0.1 Castonguay ef at„ 19B3b Man Oral (1) 239.0 24.0 99.2 0.1 0.1 • 0.1 Castonpuay af a1,1983b Man Oral(1) 6.0 24.0 94.8 0.7 1.6 0.2 0.5 1.8 0.6 2,5 Llue/eL 1993 Man Orat (1) 100.0 24•0 99.5 0.2 0.1 0.1 0.3 0.1 0.1 0.5 , Lfu at eL, 1093 I Man OralQi 6.0 24.0 94.7 1.2 0.4 0.e 2.t 0.4 0.5 J,3 Llu etel., 1993 ZtrEE1T9890~ -;~
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DRAFT used by Zuccaro et al. are more strongly correlated to the [3-HC] than to [COT], although more selective for cotinine. The results of both studies represented in Table 2 indicate that the mean trans-3'- hydroxycotinine is about three times the concentration of cotinine in urine. RIA would yield a result that is a combination of cotinine and cross reactivity with trans-3'-hydroxycotinine. The analytical bias can be as high as a factor of two, and leads to considerable confusion when RIA results are compared to GC or GC/MS results. 3. If enzymatic hydrolysis of the conjugates is included in the analytical step, the resulting nicotine, cotinine and trans-3'-hydroxycotinine concentrations determined by chromatography could account for approximately 85-90% of the total nicotine and its metabolites found in urine samples. This can be concluded from the, cumulative total columns in Table 2. The only reliable approach to relate nicotine dose (and, thereby, uptake) to nicotine metabolites in urine for individuals is to perform a complete suite of metabolite determinations to account for pharmacokinetic differences. 4. The results in Table 2 represent mean values that are reasonably consistent between the two studies. However, the variation of each mean can be up to 100%. Some of this distribution is analytical error; some of it is interpersonal variation. Although correlation between means 0002I9L01 3/l6/98 3:44pm -10-
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DRAFT It is clear from Table 2 that free (i.e., not conjugated) cotinine accounts for only about 10% of the total nicotine metabolites analytically detectable in human urine. This observation requires a number of considerations: 1. Whether a selective (i.e., RIA) or specific (i.e., GC/MS) analytical method is used for cotinine determination, only about 10% of the nicotine equivalent as cotinine will be measured unless an enzyme-catalyzed hydrolysis is conducted to free the conjugated cotinine. Even then only a portion of the nicotine metabolites is measured. 2. In the case of RIA, a cross reactivity of approximately 34% for trans-3'-hydroxycotinine (3- HC) in urine samples has been reported (Zuccaro et al., 1997). Multiple regression of data presented by Zuccaro et al. (1997) yields an expression for cotinine concentration determined by RIA ([COT]pjA) versus cotinine concentration ([COT]) and trans-3'-hydroxycotinine ([3- HC]) determined by HPLC as follows (concentrations in ,ug/L): [COT]wA = 0.97±(0.63) [COT] + 0.29(f0.14)[3-HC] + 557(±371) Interestingly, both the coefficient of variation and the "p" value for the coefficient for 3-HC ~ 0 are smaller than that for COT. This suggests that the results from RIA determinations as ow 00o21B1.al 3/16/98 3:44pm -9-
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DRAFT in different matrices has been established (Benowitz, 1996), no controlled experiments are known to determine the variance between individual values either on an interpersonal or intrapersonal level. In summary, with frequently used analytical approaches for determination of cotinine in urine, only about 10% of the total nicotine is accounted for in the analysis. When RIA is used, the representation as a cotinine determination may be in error by as much as a factor of two. Approximately 25-35% of the metabolites is present as conjugates not detected unless enzymatic hydrolysis is conducted. Interpersonal variation is an important issue dealing not only with nicotine exposure, but also with metabolic rates and distribution among the various metabolites and their conjugates. When only 10% of the metabolite products of nicotine are used, it is not unexpected that large variations in results will be obtained. Finally, urine volume is a variable associated with many factors including liquid consumption. It is possible that careful management of 24-hour urine sampling can be used successfully (Benowitz et al., 1997). Unfortunately, many studies related to ETS exposure make determinations using only one sample. Use of the cotinine-creatinine ratio (CCR) to adjust for urine output has limitations because of the variables that influence creatinine output. 0002191.01 3/16/98 3:44pm ' ll-
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DRAFT pharmacokinetic and lifestyle factors must be given more consideration when attempting to use a point value for decision making. Salivary Cotinine Concentration Related to Air Quality Measurements Benowitz (1996) has discussed relationships between cotinine measurements and nicotine intake. The relationships proposed have been described earlier and provide some opportunity to assess nicotine intake on a daily average. Three ETS exposure studies involving subjects from 16 cities in the United States (Jenkins et al., 1996), Stockholm, which has exceptionally low ETS (nicotine) levels (Phillips et al., 1996), and Barcelona, which tends to have relatively high levels of ETS both in the workplace and at home (Phillips et al., 1997), provide an opportunity to consider such relationships. In all three studies, salivary cotinine measurements were made using the RIA method both prior to and after the subjects' breathing zones were sampled using personal monitors. In the case of working subjects, dual personal monitors were used for workplace and away from workplace environments. The data obtained from the monitor samples were used to calculate 24-hour time weighted average (TWA) nicotine concentrations. Figure i shows a plot of the median salivary cotinine concentration versus the 24-hr TWA nicotine in air concentration for different subject categories for all three studies. The median was used because at very low levels near analytical detection limits it is generally a more 0002191.01 3116198 3:44 7m - 16 -
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DRAFT representative value than the mean. In most cases, at higher concentrations the mean and median were relatively close in value. One clear outlier point is observed in the results shown in Figure 1. This point represents non-working housewives or househusbands living in a nonsmoking home environment in Stockholm (Phillips et al., 1996). That datum was not included in the regression. No explanation was provided for this result, which represents by far the highest nicotine and cotinine levels found in the Stockholm study, and is almost 10-fold higher than that found for homes of workers with smoking environments. Only nine (9) subjects were in this group, and it is possible that the values obtained were not representative. There could also have been unknown extraneous sources of nicotine. After removing the datum discussed above, the remaining data shown in Figure 1 yield the following linear regression. Salivary Cotinine (ng/mL) = 0.76(±0.07)*Nicotine ().cg/m3) + 0.38(±0.07) For both the coefficient and intercept, p<0.001. However, again it should be emphasized that this correlation is for median values of groups of subjects ranging from as few as 9 to over 100. Individual variation is expected to be large, but the data were not made available in the publications. Furthermore, geographic location appears to play a significant role (Jenkins et al., 0002191.01 3/16/96 3:ae pm - 17 -
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DRAFT nicotine, and nonsmokers exposed to ETS 0.08 mg. These compare favorably with similar calculations using the data of the study by Jenkins et al. (1996). For the cell with nonsmoking home and nonsmoking workplace, the calculated daily intake is 0.013 mg (vs. 0.024), whereas, for the subjects in the cell with smoking away from work but not at work, an estimated daily intake of 0.07 mg (vs. 0.08) is calculated. In the most extreme case of ETS exposure both at work and away from work, a daily nicotine intake of 0.16 mg is estimated. These results appear to be consistent findings, although the Jenkins data were less so when salivary cotinine levels were below 2 ng/mL. All comparisons are based on mean or median values and may not be valid for individuals. Although salivary cotinine measurements have been used as a biomarker for nicotine exposure for means (or medians) of large numbers of subjects from within given exposure groups, a wide range of levels of salivary cotinine is found for individual smokers and nonsmokers alike that does not correlate well to their nicotine exposure levels (Saracci and Riboli, 1989). Because of the consistent relationships between cotinine in saliva, serum and urine, it is concluded that none of these matrices can be reliably applied to individual subjects using a single measurement. As stated above, analytically valid measurements of cotinine in any of these matrices can distinguish a smoker of 20 cigarettes per day from a nonsmoker who is not exposed to ETS. Distinction between a light smoker and a nonsmoker exposed to high ETS levels is very problematic. Kemmeren et al. (1994) have used a statistical equation shown below, and based on expressions for "t" values, to predict the 0002191.01 3l]6/98 xa4 Pm - 14 -
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DRAFl' no reported ETS exposure, reported ETS exposure and reported smokers is 0.024, 0.08, and 40 mg, respectively. Using a similar approach to a different set of data, a group of 91 smokers with mean saliva cotinine concentrations of 261.1(tl 16.2) ng/mL would convert to a daily intake of 20.9 (±3.8) mg of nicotine, which may be compared to the experimentally determined 19.8 (f6.3) (Andersson et al., 1997). Other examples reinforce the evidence that serum or saliva cotinine levels can be used to estimate the mean values of nicotine intake, but the variance about those estimates for individuals may be significant, as described below. Furthermore, most of the available data are based on single samples from each individual, and very little is know about intra-individual variation. Use of Cotinine as a Nicotine Biomarker in Exposure Studies In a large study by Jenkins et al. (1996), saliva samples for cotinine measurements were taken for nonsmoker subjects in 16 cities in the United States the day before and the day affer personal monitors were used to measure ETS parameters, including nicotine, in workplace and non- workplace environments. Four cell types were defined depending on smoking taking place away from work or in the workplace. A strong correlation (Rz = 0.991) was found between the median 24-hr time weighted average (TWA) nicotine exposure and the median average salivary cotinine level on a cell-by-cell basis. Salivary cotinine levels were not well correlated with individual 24-hr TWA nicotine levels (RZ=0.105). Based on an earlier study discussed above (Saracci and Riboli, 1989), nonsmokers not exposed to ETS had an apparent estimated daily intake of 0.024 mg of 0002191,01 3/16/98 3:44Pm -13-
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DRAFT However, one does not know what the values may be if they are below the LLD or LOQ. The analytical method used by Pirkle et al. (Pirkle et al., 1996; Bemert et al., 1997) has a reported detection limit of 50 ng/L (0.05 ng/ml). A significant percentage of the data for nonsmokers is found in the region below 0.5 ng/ml, further bringing into question the data treatment used in exposure studies. Other Factors that Affect Cotinine Concentration in Biological,Fluids From the data discussed so far, it appears that salivary and serum cotinine concentrations may be correlated to exposure to nicotine in ETS for nonsmokers. Almost all exposure studies have been conducted with mature adults >17 years of age. However, a number of studies have suggested that cotinine levels for a given exposure are higher in children and blacks (Zuccaro et al., 1997; Wagenknecht et al., 1993; Pattishall et al., 1985). In one of the few studies to attempt to use multivariate methods of analysis of exposure data, Pirkle et al. (1996) performed multiple regression of log serum cotinine concentration versus such parameters as age, ethnic background, number of smokers in the house, size of household, number of rooms in the house, etc. Dietary intake was considered in the form of consumption of bell peppers and found to be of marginal statistical significance (0.001<p<0.05) in children 4-11 years of age, but not significant in adults. The regression coefficients that were obtained had a wide range of statistical reliability. 0002191.01 3116/98 J:44 pm - - 19 -
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DRAFT individual subject. Ranges of exposure that result from daily lifestyle variations are not considered. This variability may well be much greater than the analytical variability. As a result, uncertainty exists over the cotinine concentration that unambiguously differentiates a smoker from a nonsmoker. At the present time, somewhat empirical decisions are made. These decisions may be influenced by the goal of the investigation. It has been suggested that cotinine concentrations of 10-50 ng/mL in saliva or serum, or 50 nanogram cotinine per milligram creatinine in urine (cotinine-creatinine ratio: CCR) are levels below which a subject is considered to be a nonsmoker and above which is classified as a smoker. Conflicts with questionnaire data arise and are occasionally resolved by dismissing the subject from the study. The latter decision is prudent for experimental purity, but could eliminate an important segment of the total population. The complex distribution of cotinine species in urine would recommend the use of serum or saliva for smoking status classification. Pirkle et al. (1996) have determined serum cotinine levels for over 4,000 subj ects spanning wide age groups, smoking status and ETS exposure. Their results show a finite percentage of the population with serum cotinine levels below 10 ng/mL that report tobacco use, and above 10 ng/mL that report no tobacco use or ETS exposure at home or at work. Three regions of classification should be considered: smokers, nonsmokers and indeterminate. The overlap is a combined result of interpersonal metabolic variances, variation in m 0002191.01 3/l6/98 3:44 pm ' S '
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76 CARCINOGENESIS BY ORGAN SITE 3.7 Cancer of the lung Lung cancer is the most frequent malig- nant neoplasm worldwide: tobacco smoking is responsible for most cases, and the control of smoking represents the most important approach to prevent lung cancer (see Section 2.4). Among the important research ques- dons still to be answered are the contribu- tions of other risk factors (occupation, diet, environmental pollution) in both smokers and non-smokers and the role of genetic predisposition: these questions are being addressed in a series of studies conducted in areas of high and low risk for lung cancer. 3.7.1 Case-control study of lung cancer in northern Thailand D.M. Pactia and P. Pisani; in collaboration with P. Srivatanalrul, Bangkok Thailand; N. Martin, Chiang Mai, Thaitand; V. Saensiogkaew, Bangkok Thailand; and T. Bishop, [xds, UK This study is investigating the reasons for the relatively high incidence of lung cancer, particularly in women, in northern Thailand. Age-standardized incidence rates in Lampang province are 41.8 per 10 000 in men and 20.1 per 10 000 in women. A case- control study comparing 196 cases of lung cancer with two groups of controls (217 hospital controls and 156 community con- trols drawn at random from the population of this province) was carried out from 1993 to 1995 and data analysis began in 1996. Because one hypothesis under investi- gation is the role of air pollution from numerous coal-faed electricity generating plants, place of residence is an important variable of interest, linked to corresponding environmental measurements of arsenic and cadmium. Other factors investigated include tobacco habits, exposure to domestic smoke, and cooking practices. Blood samples from all subjects have been stored for analysis of heavy metals and of metabolites and adducts of components of tobacco smoke. DNA is being extracted from white blood cells of cases and controls to study metabolic poly- morphism at the GSTM l and CYP1A1 loci. 3.7.2 Lung cancer and exposure to environmental tobacco smoke P. Boffetta, P. Bremun. S. Lea and G. Fenn; in collaboration with W. Aluens, Beemeu. Germxny; E' Benhamou and S. Bentutnou- vittejuif, France; S.C. Dar6y, Oxford UK: F. Forasuere and C. Fortes, Rame, Italy; C.A. Gotzfflez and A. Agudo, Barcelona, Spain; J. Trddaniet, Paris. France; S.K. Jindal, Chandigarh, India; K. H. Jackel. Bssen. Germauy: A Mendes, tistan, Portugal; F. Mecktti, Tusin. Italy; G. Peeahagen and F. Nyberg, Stockholm, Sweden: R Saracci. Pisa, Ifaly; L. Simonato, Padua Italy; H. Wichmann, Munich, Germany: C. Winck, Pono, Portugal: and D. Zandze, Moscow, Russian Federation Environmental tobacco smoke (ETS) is a likely cause of lung cancer [27, 37], while evidence of an association with other neoplasms is inconclusive. However, the quantitative aspects of the association between ETS exposure and lung cancer risk are not yet well established, nor is the interaction between exposure to ETS and exposure to othercarninogens. . An IARC-coordinated international collaborative case-control study was aimed at investigating the relationship between exposure to ETS and to other environmental and occupational risk factors and the risk of lung cancer in subjects who have never smoked tobacco. A total of 650 cases and 1542 controls have been enrolled in 12 centres in seven European countries. Information on exposure to occupational carcinogens, urban air pollution, background radiation and dietary habits, as well as lifelong exposure to ETS, has been collected by personal interview of cases and controls. Self-reported (non-)smoking status was cor reh 1.1 ET wo 1.4 exF ET rel: hig sm, (Fil ber dur con Chu con tica furt Mo rest invt env the exe: ring exp- ~ • amc u Figui of a frotn
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yRar T Metabolic Products of Nicotine in Serum and Saliva Little has been reported in the literature concerning nicotine metabolites other than cotinine in blood. From the point of view of analytical significance, cotinine appears to be the most important nicotine metabolite in serum. The conversion of nicotine to cotinine in blood by the liver of smokers has been related to daily nicotine intake as 0.08 [(mg/24 hr)/ng/mL], with a coefficient of variation of 21.9% (Benner et al., 1989) Thus, a serum cotinine level of 250 ng/mL corresponds to a daily intake of20t4 mg of nicotine. It was suggested that this factor also applies to nonsmokers (Benowitz, 1996). Benowitz (1996) has described a calculation that suggests a urine to blood ratio of 6, but cautions that the interpersonal variability described previously would contribute to variability in this ratio. There are insufficient data to estimate the interpersonal variability. The ratio predicted by Benowitz has had some experimental verification with a urine to blood cotinine ratio of 5 reported (Jarvis et al., 1984). Benowitz (1996) also reports that saliva to blood ratios are 1.1-1.4, leading to an essentially interchangeable use of saliva and serum cotinine data. These conversions have been summarized in Table 3. Using the serum to daily nicotine intake conversion given above, and data from a large study (Pirkle et al., 1996, Figure 2), the approximate daily intake for nonsmoking subjects with 0002191.01 3/Lfi/98 3:4aPm - 12-
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DRAFT 1996; Phillips et al., 1996; Phillips et al., 1997). Thus, there is no evidence that salivary cotinine concentration can be used to estimate ETS exposure for an individual using such a correlation. The intercept in the regression of the data shown in Figure 1 is different from zero with statistical significance. The implication of this intercept is that the mean salivary nicotine concentration in all groups has a base level that is not derived from nicotine in ETS. Using the factor shown in Table 3, a daily intake of 30 mg/24 hr from an additional source is estimated. It is tempting to suggest that there are sources of cotinine in saliva of nonsmokers other than that from ETS exposure. Dietary sources have been suggested (Castro and Monji, 1986; Sheen, 1988; Davis et al.,1991; Domino et al., 1993; Domino, 1995). There are still open questions concerning potential cross-reactivity and other issues related to the RIA method of analysis, so that the potential for an artifact cannot be ruled out. A factor that contributes to the intercept described above is the data analysis policy used. In the indoor air studies mentioned above (Jenkins et al., 1996; Phillips et al., 1996; Phillips et al., 1997), if the concentrations of salivary cotinine were found to be below the limit of quantification (LOQ) or limit of detection (LLD), the values were empirically set equal to one-half of the LLD or LOQ. Depending on a number of factors and the criteria used to establish the LLD or LOQ, this means that in some cases up 60% of the data was set to 0.5 ng/ml for salivary cotinine. With so few data points, a linear regression with an intercept near 0.5 ng/ml appears reasonable. 0002191.01 3/16198 3:44pm 18
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DRAFT As an illustrative example, Figure 2 shows a plot of serum cotinine concentrations calculated using regression coefficients from Pirkle versus age for white and black children living in a 5-room house, with 3 members in the household, with 1 smoker, and after consuming 20 g of bell pepper. There are two observations from Figure 2: all other factors being equal, young children show cotinine levels higher than adolescents, and black children have higher cotinine levels than whites. The statistical significance of many of the regression coefficients was marginal, but a trend is suggested. In a 1984 report, Greenberg et al. determined urinary and salivary cotinine levels in infants and reported urinary cotinine to be the most reliable indicator of exposure. Median levels of cotinine in urine were 351 ng and 4 ng of cotinine/mg creatinine for ETS exposed and not exposed, respectively. From these data, Van Vunakis et al. (1987) have estimated that on a creatinine basis, urinary cotinine levels in infants are approximately 60 times greater than those found in adult males. Based on urine volume, the concentration of cotinine in the urine of infants is 8 and 1.6 times as much as the ETS-exposed and highly exposed groups, respectively. Weaver et al. (1996) determined the urinary cotinine levels of 79 inner-city children and found a mean of 54.7 ± 45.6 ng/ml with a range of 1-244 ng/ml. Clearly, the age and ethnic background of young children must be taken into account when the cotinine levels of this group are used to infer levels of exposure to ETS. If this factor is ca J 00 0002191.O1 3/16/98 3:44 pm - 20-
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nxnrT thought to be relatively free of other analytically significant metabolites of nicotine. The principal reason that nicotine is not used as a biomarker for its uptake is that the half-life of nicotine in biological fluids is only approximately 2 hours (Benowitz, 1996). Measurement of nicotine would not give a representative estimate of nicotine intake during the previous few days. In epidemiological or exposure studies, classification of an individual as a smoker or nonsmoker is critical to the results, especially in the interpretation of data at low relative risk (RR) levels such as those reported with regard to ETS exposure (Jenkins et al., 1996). Wide ranges of smoking status misclassification have been suggested based on questionnaire data (Riboli et al., 1995), and misclassification of 2 -5% smokers claiming to be nonsmokers is not uncommon (Jenkins et al., 1996; Phillips et al., 1996). RR values below approximately 2.0 are considered weak associations (Greenberg). In the area of ETS epidemiology, low RR values, as for example 1.2, could be significantly influenced by smoking status misclassification of less than 5%. Utilization of cotinine as a biomarker for smoker classification is less of an analytical issue than a policy issue. Interpersonal variability in metabolic processes, lifestyle, and other factors creates variations in cotinine levels that make it very difficult to distinguish an occasional smoker from a nonsmoker with recent exposure to high levels of ETS (Phillips et al., 1996). An additional issue that is rarely addressed in the literature is "intra-personal" variability. For example, researchers who conduct exposure studies infrequently collect more than one, possibly two, samples from an 0002191.01 3116/98 3:44pm -4-
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DRAFT A number of these issues are being addressed directly or indirectly by Philip Morris- supported programs. 1. Which biological fluid is overall the most appropriate as the measurement medium? Each of the principal biological fluids used for biomarker measurements, serum, saliva and urine, has advantages and disadvantages as described above. The analytical methods that may be applicable also have a range of strong and weak points. In 1986, a group of experts met to discuss these issues in detail (Watts et al., 1990). This group concluded that "estimation based on urinary cotinine excretion would be less reliable than estimation based on plasma or salivary levels." They also found that "good correlations were reported between saliva and blood for results from the same subject," as found by others (Curvall et al., 1989). The number of nicotine metabolites in urine, the interpersonal variation, and the issues of using the CCR method that result from creatinine concentration variation, all counter the relative convenience of collecting urine samples. Following the recommendations given above, Philip Morris is participating in a program to carefully investigate the use of saliva as the sample medium of choice. Although urine has considerable interest from a pharmacological perspective, as a sample medium used strictly to estimate nicotine exposure, either serum or saliva appears to have the advantage. Work 0002191.01 3/16/98 3:aa pm - Z4 -
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DRAFT number of replicate measurements needed to estimate the "habitual" serum cotinine level of a subject within a selected percentage of the "true value." The expression used is ko,95 = (1.96 x CV/D)z in which "k" is the number of measurements needed to estimate the habitual cotinine level within a certain percentage "D" of the intra-personal variation "CV" at a 95% confidence level. The intra- personal coefficient of variation (CV) was estimated to be 16.1%, although there is little supporting evidence for this value. Using this value, one calculates that a single sample can estimate the "habitual" cotinine concentration to within approximately ±32%. Ten (10) replicate samplings would be required to obtain a measure within ±10% of the "habitual" value. The issue of the time period over which sampling and measurements were or should be made was discussed briefly by Kemmeren et al. (1994), who observed that samples should be taken over a longer period of time than just a few days. In summary, it does not seem appropriate to debate a point value for a cutoff to distinguish smokers from nonsmokers based on a single measurement of salivary cotinine. In this case, the intra-personal variation in the concentration will contribute significantly to the cotinine level, and less is known about the interpersonal variation with a fixed uptake of nicotine. These 0002191.01 3116/98 a:4a pm - 15 -
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What Is Philip Morris Doing About Cotinine Until an even more appropriate biomarker for nicotine intake is found, cotinine is currently the one of choice. The issue is not cotinine per se, but the limitations and misinterpretations associated with measurements and data. Several factors need to be considered. Some of these are: 1. Which biological fluid is overall the most appropriate as the measurement medium? 2. What are the recommended procedures and protocols for the acquisition, transportation, preservation and storage of these samples? 3. Which nicotine metabolites should one measure to obtain improved and appropriate accuracy in the estimation of nicotine intake? m to o+ . ~ w w 4. What analytical method is best suited to the task? 03 .-• 5. How should data be analyzed? 6. How can the proliferation of expedient but questionable analytical practices be contained? 0002191.01 3/16/98 3:a4 pm - 23 -
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DRAFT not taken into consideration, cotinine levels found in exposure studies can be misinterpreted as excessive exposure. Conclusions Cotinine is not a perfect biomarker for nicotine exposure. Issues such as interpersonal variation in metabolism, non-tobacco sources of nicotine, and how well nicotine represents ETS exposure plague all biomarkers. For the present, cotinine appears to be the most reliable biomarker for estimating day-to-day exposure to tobacco smoke provided that it is applied only to mean (or median) values of large numbers of subjects. There is much room to improve upon the use of nicotine metabolites as biomarkers for nicotine intake. Cotinine in serum and saliva appear to be equally advantageous over the use of urine samples; principally because of the limited number of analytically significant metabolites in these media. This is especially true if RIA methods are used, because of the potential for cross- reactivity of metabolites other than cotinine. Further, because cotinine represents 10-15% of the total nicotine metabolite in urine, variation in the larger concentrations of the other metabolites could have significant impact on the use of cotinine only in relating the result to nicotine intake. 0002191.01 3/!6/98 3:44pm ' -21-
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DRAFT number and variation in the nicotine metabolites. Philip Morris is conducting a statistical analysis of existing data to address this issue. The outcome of this analysis will be an opportunity to define the analytical needs to minimize variations resulting from the complexities of a mixture of metabolites. 4. What analytical method is best suited to the task? Philip Morris is participating in the development of an analytical method for cotinine and possibly trans-3-hydroxycotinine in saliva. For cotinine at least, the lower limit of quantification (LOQ) is expected to be approximately 0.05 ng/mL using conservative statistical policies. This may be compared with the current approximately 0.5-1.0 ng/mL using RIA methods. The method will have relatively high initial capital costs, but is capable of a throughput of over 100 samples per day. Under consideration is the participation in the development of an alternative method for urine that will involve enzymatic hydrolysis of the glucuronide conjugates of nicotine, cotinine and trans-3-hydroxycotinine with the subsequent determination of these three metabolites. 5. How should data be analyzed? There are two issues with data analysis relative to cotinine that are present in the literature. 0002191.01 3/16/98 3:44 pm - 2,6 -
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DRAFT Table 1. Nicotine and Metabolites that Have Been Determined Analytically Compound Abbreviation Nicotine MC Nicotine glucuronide conjugate NIC-G Cotinine COT Cotinine glucuronide conjugate COT-G trans-3'-Hydroxycotinine 3HC trans-3'-hydroxycotinine glucuronide conjugate 3HC-G Nicotine-N'-oxide NNO Cotinine-N-oxide CNO Demethynicotine DMC i 0002201 01 31198 10 13 am
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DRAFT determinations that may be used for various purposes, such as establishment of smoking status for insurance applications. The complexities described above all but preclude successful use of such simple, nonselective "dip-stick" tests. One such device is known as NicCheckT"t on the market and is FDA approved for physician use for detection of smokers. Philip Morris is participating in an investigation of this device to ascertain its reliability, especially against potential false positive results. 0002191.01 3/]6N8 3-44 pm - 28 -
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DRAFT a.) In exposure studies, cotinine levels below the limit of quantification (LOQ) of the analytical method used are empirically set equal to 50% of the LOQ. This has the negative impact that regardless of how much ETS exposure is reduced, a finite cotinine level is guaranteed. This can result in a misuse of values below an LOQ. As ETS exposure is reduced, new analytical methods are required to demonstrate reduction in nicotine intake. b.) Smoker-nonsmoker discrimination is based on the use of a point value "cut-off' approach. This assumes that anyone below a set value of cotinine level is a nonsmoker, and anyone above that value is a smoker. This is not consistent with biological diversity. New methods of distributive data analysis that permit incorporation of a variety of factors to predict the probability of a single result are needed. Philip Morris is participating in the evaluation of such methods of data analysis. 6. How can the proliferation of expedient but questionable analytical practices be contained? For a variety of reasons, often commercial, new quick and easy methods for the detection and alleged quantification of cotinine or other biomarker for nicotine appear in the literature, in patents or in the marketplace. These devices offer quick, simple and near-patient 0002191.01 3/1W98 3:44 pm - 27 -
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DRAFT is underway to establish appropriate sampling protocols and analytical methodologies for saliva. An excellent method for cotinine determinations in serum was recently published (Bernert et al., 1997). 2. What are the recommended procedures and protocols for the acquisition, transportation, preservation and storage of these samples? A number of recommendations for sample acquisition and handling for saliva have been reported. The subject is too detailed to be reviewed here. Philip Morris is participating in a study to evaluate a number of factors in the subject preparation, sample collection, sample preservation, sample transportation and storage for saliva. A method and procedures document is planned. 3. Which nicotine metabolites should one measure to obtain improved and appropriate accuracy in the estimation of nicotine intake? For a variety of reasons, the recommendations reported above (Watts et al., 1990) have not been adopted by many researchers who conduct epidemiological investigations, particularly with respect to the use of urine as the sample medium. If nicotine metabolites are to be used in urine, improved analytical methods are required for acceptable results because of the 0002191,01 3/16/98 3:44 pm -25-
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DRAFT A viable analytical method that has a detection limit much below that of RIA and gas chromatography is needed. The method must be rugged and capable of processing large numbers of samples economically. Such a method has been developed for cotinine in serum based on liquid chromatography combined with atmospheric pressure ionization tandem mass spectrometry (LC/APUMS/MS) (Wagenknecht et al., 1993). Work is underway to apply the method to saliva samples. Initial capital outlay is high, but the ruggedness and throughput capabilities of the method are excellent. However, saliva and serum have their own limitation as a sample matrix. Only intensive or concentration measurements may be made. To obtain data on the equivalent of nicotine eliminated over a period of time, total nicotine metabolite excretion by collecting urine samples has a number of advantages. Table 2 shows that approximately 90% of the total nicotine metabolite in urine could potentially be estimated by that approach. Therefore, a method using enzymatic hydrolysis of the glucuronide conjugates of nicotine and its metabolites in urine, with the subsequent determination of nicotine, cotinine and trans-3-hydroxycotinine is required. This method should have a detection limit to permit quantitative data to be obtained for subjects with the lowest level of nicotine exposure. It is likely that modification of existing procedures coupled with LC/API/MS/MS could meet these needs. oaaxi9i.oi 3nvros 3:44pm -22-
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Table 3. Conversion Factors for Cotinine TO CONVERT MULTIPLY BY Cotinine in serum to cotinine in urine 5-6 ngfmL - ng/mL Cotinine in serum to cotinine in saliva l.1-1.4 ng/mL -+ ng/mL Cotinine in serum to daily (24 hr) nicotine intake 0.08±0.18 ng/mL -+ mg/day i 0002201 01 3 11,98 10'. 13 am
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and urban air pollution will be integrated into the common database. 3.7.4 Multicentric case-control study of lung cancer in central and eastern Europe P. Brennao and P. Boffetta: in collaboration with E. Fabianova, Banska Bystrica. Slovakia; J. Fevotte. Lyon. France; A. Fletcher. London, UK: D. Mates, Bucharest, Romania: P. Rudnai, Budapest. Hungary; J. Siemia- rydd. Monneal, Canada; N. Szeszenia-Dabrowska, t.odz, Poland; D.G. Zaridze, Moscow, Russian Federation: and W. Zatonski, Warsaw. Poland Countries of central and eastern Europe have the highest incidence and mortality of lung cancer ever recorded. Air pollution is often blamed as the main contributor to this excess, but evidence for its role is limited. A study has been initiated in six areas of Hungary, Poland, Romania, the Russian Fede- ration and Slovakia, to assess the relative contributions of tobacco smoking, occupa- tional exposures and outdoor air pollution in lung carcinogenesis. Enrolment of a total of 3000 cases and a comparable number of controls has started. Special efforts are being made to assess past occupational exposures using detailed employment histories evalu- ated by panels of local experts. Blood samples will also be collected, to investigate polymorphisms of metabolic enzymes. 3.7.5 Case-control studies of lung cancer in Brazil, Uruguay and Argentina P. Boffeea: in crollaboration with E. de Srefani, Monnevidao, Uruguay; & Ma[os, Buenos Aires, Argentina; and V. W unsch, Sao Paulo, Brazil The urban areas of Brazil, Uruguay and Argentina have among the highest death rates in the Americas for cancer of all sites and of the lung in particular. Three similar studies have been designed to identify asso- ciations between environmental and occupa- tional exposures and risk of lung cancer in Sao Paulo, Brazil, in Uruguay and in Buenos Aires. Argentina, and to examine the synergistic -effect of selected occupational exposures and tobacco smoking. The study in Uruguay confirmed the important role of known carcinogens, such as tobacco smoking and asbestos, and suggested an increased risk among workers of the meat industry and workers exposed to pesticides [86]; it is also addressing the risks for other cancer sites. The study in S5o Paulo suggested a smaller role than expected for occupational exposures, with increased risks in only a few categories, such as machinery and pottery workers. Data collection for the study in Argentina was completed in 1997 and analysis will be carried out in 1998. 3.7.6 Multicentric case-control study of lung cancer in India P. Boffena and R. Sankarsnarayanan: in mllaboration with M. K. Nafr, Tnvandrum, India; D.N. Rao, Bombay, India; and V. Shanta. Madru, India Although the industrial population in India is very large and many hazardous industries are present, virtually no informa- tion exists on occupational risk factors for cancer- The presence of a network of well organized cancer registries is a favourable condition for conducting multicentric case- control studies, and therefore such a study has been started in Bombay, Trivandrum and Madras, to investigate occupational and environmental factors for lung cancer. A series of cases of lymphatic and haemato- poietic neoplasms has also been included. Data collection was completed in 1997 and the analysis will be completed in 1998. 3.7.7 Case-control study of environmental tobacco smoke and genetic susceptibility to lung cancer P. Boffetta, M. Lang, N. Malats. M. Friesen, S. Atawodi, S. Lea and J. Hall; in collaboration wilh W. Ahrens. Bremen. Gernwry; S. Bentumou, Villejuif, France; L BNske-Hohlfrld and H. Wichmann, Munich, Gemuny: V Forastiere a Poman. Pal Finland: A t naly; G. Pen L. Simonuu Russian Fed. Amonr have beet levels of , susceptibi. of lung ca In ten many, Ita Federatior being coB, lung canct Cance an impon showing i of the wol tobacco s; these can papilloma- important -_ tional exp "'. head and t C N _: developin; o+ -plasm, ma ~ ' in which b m .A W >- 3.8.1 Multicen laryngea and Uruy P. Boffetts, I with E. d Kogevinas, fane'vo, Bra: Menezas, P, Canada:and
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~ 16 City ~ Barcelona i . Stockholm 1 2 3 24-Hr. TWA Nicotine in Air (uglm3) Figure 1. Plot of Salivary Cotinine Concentration versus 24-hr. TWA Nicotine Concentration Exposure in Nonsmokers 1
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DRAFT Phillips, K., Bentley, M.C., Howard, D.A., Alvan, G., and Huici, A., Assessment of air quality in Barcelona by personal monitoring of nonsmokers for respirable suspended particles and environmental tobacco smoke. Environ Inter 1997:23:173-196. Pirkle, J.L., Flegal, K.M., Bernert, J.T., et al., Exposure of the US population to environmental tobacco smoke: The Third National Health and Nutrition Examination Survey. JAMA 1996:275:1233-1240. Riboli, E., Haley, N.J., Tredaniel, J., Saracci, R., Preston-Martin, S., Trichopoulos, D., Misclassification of smoking status among women in relation to exposure to environmental tobacco smoke. Eur Respir J 1995: 8:285-290. Saracci, R., Riboli, E., Passive smoking and lung cancer. current evidence and on-going studies at the International Agency for Research on Cancer, Mutat Res. 1989: 222:117-127. Sheen, S.J., Detection of nicotine in foods and plant materials. J. Food Sd. 1988: 53:1572-1573. Statements attributed to D. Trichopoulos, E. Kakalamani and A: Linou in Greek Newspaper "Ta Nea," Apri130, 1997. Tumer, D.M., Biochem J 1969: 115: 889. Van Vunakis, H., Gjika, H.B., Langone, J.J., In: Environmental Carcinogens: Methods ofAnalysis and Exposure Measurement, V. 9 -- Passive Smoking, O'Niel, I.K., Brunneman, K.D., Dodet, B., Hoffmann, D. (Eds.) International Agency for Research on Cancer, Lyon, 1987, p.322. Wagenknecht, L.D., Manolio, T.A., Sidney, S., et al., Environmental tobacco smoke exposure as determined by cotinine in black and white young adults: The CARDIA Study. Environ Res. 1993:63:39-46. Watts, R.R., Langone, J.J., Knight, G.J., Lewtas, J., Cotinine analytical workshop report: consideration of analytical methods for determining cotinine in human body fluids as a measure of passive exposure to tobacco smoke. Environ Health Perspect 1990: 84: 173-182. Weaver, V.M., Davoli, C.T., Murphy, S.E., Sunyer, et al., Environmental Tobacco Smoke Exposure in Inner-City Children. Can Epidemiol Biomarkers & Prev. 1996:5:135-137. Zuccaro, P., Pichini, S., Altiere, L, Rosa, M., Pellegrini, M., Pacifici, R., Clin Chem 1997:43:180- 181. 0002 t91.Ot 3/16/98 3:44pm -31-
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Cancer >i he.unfl ;)!sa while other the iation r risk ; the and donal imed veen ental ;k of .ever • and :r 12 thes. :ona1 aund 11 as xreti aols. '+vas contirme:i Sy :nterviews oi reianves. TSe xiauve risk RR; of ;ung cancer nsk was • _6 95% l.; 0.93-i.1a1 for exposure ;o =1'S iom the spouse, t.:'. ,09a--i.4 _1 'oP workplace ETS exposure and i.11 ;0.33- 11",l ior zombfned spousal and workplace ,tp,ure. Several quantitative ;ndicators oi - ~ y exposure showed a 3ose-response telauonship with lung .:aneer risk: ZAs vere Signer :or squamous cell carcinoma ana imail::eil :arcinoma han tor adencarcinoma ~Fgure =01. 'Laere was no associarion between :ung cancer cisk and 5TS exposure during :!riidhood. Additional analyses are co-timmng on tisk'actors nher ttan ETS. A parailcl study was conducted in CSandigarh. India. where = =xposure .omes mainly from bidi smoking. The ;tatis- scal analysis will be completed ln 1998. A $rther study of non-smoking women ln Yloscow. Russian °ederation• confirmed 'he resuits on 9TS of the larger kntetnational .~ esdgadon and suggested a role of environmental air ?ofludon independent of the affect of ETS [517]. trt a separate exercise• the aumcer of :ung cancers occur- ring in 'he _ountnes of :he European Union hat can ~e attributed to spousal E-TS exposure was estimated to be about 3C0 .tmong women attd JCO among :nets ;36 i'. Vwwwranwne a . lMw at+'IEe %ty9J1 a1.et F;gure 20. 9elative risk of'ung :ancer bv'tears of exoosure to environmental tooacco smoke 'trom spouse or rrorkplace 3nd ay histological 3.'J Cambined analvsis of ,;ase-eantroi studies )f lung cancer in •vesrern Europe F. 3offetro. ?. 3rennan mu '•. .]aouneau. n :una- iomtwn 'vnh 4. Utrens mu :i. 'nnlacem. 3remen. ~emm~v: 3. 3ennamuu mu i, 3ennamoa. 'rillami. ?rance: S.C. 7arbq Jxcord, _K, ? Forasnere una :. Fones. 3ome. :tatr. CA. Gonzxlez Ad x. Aguuo. 3arcelonn, iown; L 3. lhcxe,. =ssen. Germmr. ? Pderiem. Tunn, !LVV; G?ersnagen mo ? Vvperg, $mckhoim, 3wene¢ 3. iuLattL 1i55. Italv: .. SienuatvN.u• Montreti. _.maaa: ~. Simonaro.-?aeu~: ;talv: md H. -h'ichn:mn. ltmucn. jennzny :n Jarallel .0 :be itUdV )n ion-5mokers descdoed :n ?ecqon i."._. :as•es of :ung cancer Ind .:ontrois :iave ~een snroited !n i series of studies in '0 .;entres 's .vestem Europe, uresoective of :heir ;mok:ng iabits. Comparable in[otmat)on an :ooacco smoking, ~xoosure to iccaoauonai ;arcino- Tens and irban air ooiluucn :-tas been .:oilec:ed from aoout A000 aaes ind 10 OC0 controis. T:re lnatysis :ocases )n ietaiied aspects or tobacco :arcinogenesis that can- not Se addressed !n smaller >tudies. such as he ~ffect )f +er• :ignt ;tnoicng, bng-<erm quitting and smok'ng ot iroducts other han cigarenes iFi;ure ~';. ?Sese maivses wiil be :omnietea :n 1998. in neailei, lnfcrma- :ion On JXnosure A JCellpational larcinoP,.e1s 2 "gure 2.. ?etative risk >r ung cancer by 9ne ;ategories )f :igarette ~onsumption and duration of smoking. Aererenca cateqory: tever smokers.
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Predicted Serum Cotinine Concentration versus Age _  White I 1.00 o Non-HP is nic Blacks m tn yC ~ o 0 °a' e 0.50 ~ ~  ~ a 9 c ~~ 0.00 ~ °- - - ---J I 0 5 10 15 , N , O . Age (Years) w L --------- .p ... W ~ Figure 2. Use of Multivariate Analysis of NHANES III° Data to Illustrated the Effect of Age and Ethnicity (NHANES III = Third National Health and Nutrition Examination survey)
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DRAFT References Andersson, G., Vala, E.D., Curvall, M., The influence of cigarette consumption and smoking machine yields of tar an nicotine on the nicotine uptake and oral mucosal lesions in smokers. JOral PatholMed 1997:26:117-123. Benner, C.L., Bayona, J.M., Caka, F.M., et al., Chemical composition of environmental tobacco smoke. 2. Particulate-phase compounds. Environ Sci Technol 1989:23:688-699. Benowitz, N.L., Cotinine as a biomarker of environmental tobacco smoke exposure, Epidemiol. Rev. 1996:18:188-204. Benowitz, N.L., Zevin, S., Jacob, P., Br. J. Clin Pharmacol 1997:43:259-267. Bernert, J.T., Wazman, E.T., Pirkle, J.L., Sosnoff, C.S., Akins, J.R., et al., Development and validation of sensitive method for determination of serum cotinine in smokers and nonsmokers by liquid chromatography/atmospheric pressure ionization tandem mass spectrometry. Clin Chem 1997:43(12):2281-2291. Byrd, G.D., Robinson, J.H., Caldwell, W.S., deBethizy, J.D., Nicotine uptake and metabolism in smokers. Paper presented at the 48th Tobacco Chemists' Research Conference, Greensboro, NC, September 25-28, 1994. Castro, A., Monji, N., Dietary nicotine and its significance in studies on tobacco smoking. Biochem Arch 1986: 2: 91-97. Curvall, M., Elwin, C.E., Kazemi-Vala, E., et al., The pharmacokinetics of cotinine in plasma and o saliva from non-smoking healthy volunteers. Eur J Clin Pharmacol 1990:38:281-287. o+ s.o o, ', Curvall, M.,Vala, E.D., Englund, G., Enzell, C.R., Urinary excretion of nicotine and its major metabolites. Paper presented at the 43rd Tobacco Chemists' Research Conference, Richmond, VA, September 29-October 3, 1989. Davis, R.A., Stiles, M.F., deBethizy, J.D., Reynolds, J.H., Dietary nicotine: a source of urinary cotinine. Fd Chem Toxic 1991: 29: 821-827. Domino, E.F., Nontobacco sources of cotinine in the urine of nonsmokers. Clin Pharmacol Ther -t' CO ,a w m -J 1995: 57: 479. 0002191.01 7/l6/98 3:44 pm - 29 -
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C.mcar x he ung the onal tndv e of acco I art neat :ides tther .'auio I :or asks nerr the :99' ;ennanv: ', J~nstanunescu. 9ucaarest. 'Lomama: F ?nmsuere ma --. =onei, Rome. ivv: 3. iabnel. ?nman. 7mand: :C. ausgaivel-Tnsr,unen. iesmw. ?,mand: , k. bleneus, ?eaotas. 3mzr1: .' Mer 1eni. Tunn. 71yv: Q?ersnugen ma ? Vvoerg. itocWmm. Sweaen: L. Simonato. ?aoua. :mlv: md D.G. andze. ~loscow. 3usslan P^-uemnon Among ung .attce: :ases. aon-smokers tave been axposed on average :o lower ,evels oC aarcinogens han ;mokers; Zenetic susceptibility may ?lay a ;reater -ole :n ask of :ung :ancer ln the iormer , oup ot .ases. in :en ,entres from 3razil. ?rance. Cror- nanv. :taiv, ?oiand. Romania. the 2usstan Federation and Sweden, Slood samples aave being ~oilected irom zbout '0 aon-smoiting :un, _,ancer aases, l:o smoking '.ung .ancer 0 :ases and _CO ton-;moking :antroi >ubjects, in traer to 3etermtne ~ i1 ;enetic Dolvmor- ihtsm ar 'lutarhione S-cransferase •LIi ana .'. ii; :he ;eveis of he '_FA reoair .nzvme :°-methviguanine-DNA methyitransterase. : iiii :he :ormanon .)( Sae:Rogiobin adduc:s with 1 ~tydrox•i-i-.;-~yriayil-;-butanoqe ~a ;netaooiite of ;obacco-specitic aitrosamines 1. :md iv, ;eneac ilterations in the p-5? ;ene .tnu :C-ras mutations in iung aeopiastic ;issue of eases. Cases ma controls Save been interie•.ved ai:out heir smoiting habits and ~ xposure :o anvironmenrai tobacco smoke. =nroiment of padeats and laboratory .maivses :iave )een comoleted and statistical anaivsts viil take :iace in 1908. 3.3 Head and necz cancer Ikuon Zw, I in '.ous na- :or well able rse- ady md and A ato- ,kd' attd .al C.tncers o[ :he 'aead and aeck ,:omonse an imponant nouo n[ 'teooiasms that are showing Lncreasing ;ncidence n manv parts ir he vorid. k.though ,ilconoi ,ir.aicing and tooacco smoic:ng se estabiished :auses of these cancers, infection with he human papillomavirus :nav represent an additional :mportant risic factor, as do somee xcuna- aonal axposures. In iddition, patients w«h iead and necs cancer are at increued isk oi. developing a ;econd :obacco-reiated neo- ?iasm, making hem an :mnorclnt ?ooulation :n .vhich :o axpiore 3enetic suscatibtiitv. 3.3.1 Multicentric case control study of lar+ngeal cancer in Brazii, A: ;entina and Uruguay .'. 3alitra ?. 3rennan ana 2. :ierero: :n :oilaboranon vith =. Ae iteiani. Ytomewdeo. ;mguay: A :Cogevinas 3arcetona. Soain: 3. :tmimun. 2io ae :aneuv. 3nviL '_. Matos. 3uenos . Vres. vgendns A. Neneves. ?etous. 3mzul: J. iiemiaryckt. Nonaea. Canaaa: md `s,'r/unsch. $ao 2suio. 3rav1 rW, I Argentina, -:ruguay md southern Brazil 't"'f' Save :vgh incidence -rates of larvngeai vch :ancer, hat io zot seem ]o Se =xplained only Sv _-xposure :o : -town .arcinogens such as tobacco smoking and alcohol drinking. Following a series of studies oi lung :ancer • see Section 3.".5i, a muittcenttic studv of 'aryngeai ~ancer has been initiated in three areas oi 3razrl Cio de Janeiro. Sjo Paulo and ?eiotas and Porto .a;egrel, in Buenos kires and in Montevideo. "he study nms to identitv occupational ;isk factors of this disease: additional aims are ate assessment af the -ole of FIP`I !ntec:ion, quantification of ine _ontribudon of tobacco smoking and aicanoi drinking, and clariflcation of he role o[ other possible atestyle asic .actors. such as die: and mate drinking. Collection of interiiew data vtd biological samples started in 199- and will ~x compieted in 1999. .n some ot the zentres• the stud,v is 3eing .:onducted in Darailel with an investt;ation of he role of Sumen papiilomavirus '.nie:::ion in oram ~ancer see Section 3.3-.1?. 3.3.2 Combined analysis of case-controi studies of sinonasal cancer '. 3offetta, S. Merter md D. CaGn: in coilaoomtion .vith R.3. yaves, md L.A. Bnnron. Bahesd. S4D.
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DRAFT Table 2. Distribution of Nicotine and Metabolites in Two Studies (Each compound was converted to an equivalent amount of nicotine) NIC + METABOLITES % OF TOTAL FOUND' CUMULATIVE % TOTAL % OF TOTAL FOUNDZ CUMULATIVE %TOTAL 3HC 35 35 36 36 COT-G 17 52 14 50 COT 13 65 9 59 NIC 10 75 9 68 3HC-G 9 84 23 91 NIC-G 3 87 5 96 NNO 7 94 3 99 CNO 4 98 1 100 DMC 2 100 Not Determined (Smokers) N = 11 91 PERCENT N-OXIDE I 1 4 Byrd et al. (1994) (SD values are 3-8%, taken from Table I, RSD values calculated to be 21- 75%). 2. Andersson et al. (1997) (SD values are 0.9-10.6 taken from Table 4, RSD values calculated to be 28-100%). uao22o i m 3 11 98 10 Uam
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DRAFT Domino, E.F., Horbach, E., Demana, T., Relevance of nicotine content of common vegetables to the identification of passive tobacco smokers. Med. Sci. Res. 1993: 21: 371-572. Greenberg, R.A., Haley, N.J., Etzel, R.A., Loda, F.A., Measuring the exposure of infants to tobacco smoke. Nicotine and cotinine in urine and saliva. New Engl JMed 1984:310:1075-1078. Greenberg, R.S., "Prospective studies" In: Encyclopedia ofStatistical Sciences, V. 7, pp 315-219, S. Koty and N.L. Johnson, Eds., John Wiley & Sons, NY. Jarvis, M., Tunstall-Pedoe, H., Feyerabend, C., et al., Biochemical markers of smoke absorption and self-reported exposure to passive smiling. JEpidemiol Community Health 1984:38:335-339. Jenkins, R.A., Palausky, A., Counts, R.W., et al., Exposure to environmental tobacco smoke in sixteen cities in the United States as determined by personal breathing zone air sampling. JExpo Anal Environ Epidemiol. 1996:6:473-502. Kemmeren, J.M., van Poppel, G., Verhoef, P., Jarvis, M.J., Plasma cotinine: stability in smokers and validation of self-reported smoke exposure in nonsmokers. Environ. Res. 1994:66:235-243. Kyerematen, G.A., Taylor, L.H., deBethizy, J.D., Vessell, E.S., Radiometric-high performance liquid chromatography assay for nicotine and twelve of its metabolites. J Chrom Biomed Appl 1987:419:191-203. Langone, J.J., Gjika, H.B., Van Vunakis H., Nicotine and its metabolites. Radioimmunoassays for nicotine and cotinine. Biochemistry 1973: 12: 5025-5030 - National Research Council, Committee on Passive Smoking, Board of Environmental Studies and Toxicology, Environmental tobacco smoke: measuring exposures and assessing health effects. Washington D.C.: National Academy Press, 1986. Neurath, G.B., Pein, F.G., Gas chromatographic determination of trans-3'-hydroxycotinine, a major metabolite of nicotine in smokers. J Chrom Biomed Appl 1987:15:400-406. Pattishall, E.N., Strope, G.L., Etzel, R.A., Helms, R.E., Haley, N.J., Denny, F.W., Serum cotinine as a measure of tobacco smoke exposure in children. AJDC 1985:139:1101-1104. Phillips, K., Bentley, M.C., Howard, D.A., Alvan, G., Assessment of air quality in Stockholm by personal monitoring of nonsmokers for respirable suspended particles and environmental tobacco smoke. Scan J Work Environ Health 1996: 22 (Suppl. 1)1:1-24. 0002191.01 3/16198 3:44 pm - 30 -
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G. Schepers et al., S-Nicotine metabolism in rats H . SHOC COT •.•••`NJ NIC CHOC DHMNIC J /\ /\ k . ~ N O CH3 i H3C CH3 NMNN'0 PASAc POBAc TNN'0 CH3 NN'DMNIC PBAc Allahydroxy- demethyicotinine CNN'O ~ 0 DMCNO NN1C PHOBAc PTHF Demethyicotinine- A4.'S-enamine NCOT N'MN(C ~ COOH PAAc PMABAc H q-(3-Py(dyI)-4oxc- 6utyramide I COOH NH I CH3 H 189 Fig. 1: Savctural fomwlae of nicoflne metaboliics and refexence compounds.
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Nicrotine phase 2 metabolites in human urine-structure of metabolically formed eranr-3'-hydroxycotinine glucuronide in our urine sample the major nicotine phase 2 metabolite was lrans-3'-hydroxycotinine-0-f~-D-glucuronide: a" OH 1 61 CH3 This is the first time that a metabolically formed nicotine phase 2 metabolite has been identified. Using MS and NMR spectrometry, this metabolite was found to be the O-{3-D- glucuronide of THOC. In addition, evidence is given of 865 further urinary nicotine phase 2 metabolites, i.e., sulphuric acid conjugates of COT and THOC. The differences between our results and those reported in literature, but also between the published data itself concerning the quantitative results, can be explained by inter-individual variation in both nicotine phase ] and phase 2 metabolism. I. Curvall, M., Kazemi-Vala, E., Englund. G. and Enzall, C. 1959. Absr. J3rd Tobacco Chemhn' Research Confrrelrte; Richmond. VA. Octoher 1989, Papcr No. 54 . . 2. K,cmmutcn. O.A., Morgan. 6LL.. ChauopadhYa,. B. rt al. 149t1. Clin. Ytrnrruucnl. Tfter., da, fiJIfi51 .. Cunall. M.. Kazemi-Vala. E. and Englund. G. 1991. In: Adlkofer. F. and Thurnu, K. (cds). Effecu of A4corlne on Rinfq;iral Systcros, pp. 54-75. Birkhauur Vcrlag. Bascl 4. Byrd, G.D., Chang, K: M.. Grecne. L6l- et n/. 1992. Dm_. Metab. Dispos.. 20. 192-197 - 5. Caldwell. W.S., Grcene, J.M.. Byrd. G.D. a al. 1992. Clrem. Res. Tosicol.. 5- 28(l-235 6. Rustemeier. K.. Demetriou. D.- $chepars, G. and Voncken, P. 199? AbsL IIJTOX Satellitc Meeting an Absorption. Disnbution, Melabolism and Excretion of Nicotine and ReWted Alkaloids, Salsomaggiore Termc, lun. 23-26. P4:1 CBromatograph. (submittcd) . Reprint requests tn: Dr Gcorg Schepers, INBIFO Institut fdr bialogishe Forschung- Fuggerstr. 3, D-5000 Kola 90. Germany_ Paper reteived: 10th September, 1992.
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G. Schepers el aL, S-Nicarine metabolism in rats 193 ary metabolitcs via COT led us to the assumption that The values for the shoRest excretion half-time, cal_ mefabolite A might be also a secondary metabolite via culated from the steepest slope in the sigma-minus COT. plots, a:e shown'in Table L The convex sigma minus 0 6 12 16 24 OAIJ f a 7 12 1--7 18 24 i4dE Or} i3vE Dd b--I 0.1-I ( 0 7 6 T 6 -9 : Poanc ---0 : PCIvMPm -~:an - • 3Hoc ...... Q : A b lF ---- 1 12 18 24 0 6 12 18 24 ladE 01 7YvE Oi) Fig 4: Sigma-minus plots of the urinary exaeeflon kinetics of S-nicotine and metabolites in the male mt a nonindueed (M t SE, n- 5): b: A:oclor-induccd (M t S$ n- 5). -
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DRAFT to be less than 0.007% of the total estimated emissions in the U.S. Thus, SS is certainly not a major contributor to BaP in the atmosphere, compared to other sources. Occurrence of BaP in Air The concentration of BaP in ambient air is dependent on a number of factors: 1) Season -- generally highest in winter and lowest in summer; 2) Source of emission -- industrial and transportation; 3) Meteorological factors; 4) Urban vs. rural settings; and 5) Geographic location (Europe vs. U.S.). Table 3 and Table 4 (after Pucknat, 1981) illustrates some of these factors; they are cited in the literature as being used for various calculations. The U.S. average for urban sites for the 5-year period 1966-1970 is about 2.0 ng/m3 (Pucknat 1981, p. 85). The BaP concentration range in urban air of U.S. cities as determined by various authors in recent years (published during the period 1971-1977) is 0.13 to 3.2 ng/m3 (Pucknat, 1981, p. 169). As can be seen in Table 5, BaP levels in European countries have historically been much higher than those reported in the U.S.; there is also a wide variation from winter to summer. 0002235.01 3/16/98 4:00pm -3-
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Arch Toxicol (1933) 62: 395-397 NUWCS°t Toxlcology m Springer-Verlae 1933 Short Communication Cotinine determination by immnnoassays may be influenced by other nicotine metabolites Georg Schepers and Rudiger-Alexander Walk 1NB1FO Institut fiir biolo,°,ische Forsehung, Fuggerstrasse 3, D-S000 [<oln 90, Federal Rcpublik of Germany Abstract. Polyclonai rabbit anticotinine antiserum, which can be used for biomonitoring nicotine uptake by the de- termination of cotinine in body fluids, was checked by a competitive ELISA for its cross-reactivity with nine nico- tine metabolites. The highest percentage of relative cross- reactivity (about 30%) was observed with transJ'-hydroxy- cotinine, a metabolite which is known to be excreted in 3-fold higher amounts than cotinine in the urine of human smokers. Therefore, it is possible that cotinine determina- tions performed by immunochemical methods - especially in urine - may yield overestimated cotinine concentra- tions. Key words: Cotinine - 3'-Hydroxycotinine - Cross reac- tion - Enzyme-linked immunosorbent assay - Biomoni- toring Introduction Several parameters have been proposed for biomonitoring the exposure of individuals to tobacco smoke. They have been reviewed recently by the International Aeency for Research on Cancer (IARC monographs 1986), and with respect to environmental tobacco smoke by the National Research Council (NRC [986) and Jarvis (1987). Among those parameters, the determination of nicotine uptake is the one most recommended, since nicotine is rather speci- fic for tobacco smoke. Nicotine itself has a short biologicalhalf-life (about 1-2 h in human plasma: Benowitz et al. 1982; Kyerematen et al. 1982, Luck and Nau 1984). Since cotinine, a nicotine metabolite, has the advantage of a longer plasma half-life of 16-20 h (Benowitz et al. 1983), it has been widely used as a marker for nicotine uptake. Sensitive methods for the determination of cotinine in biological fluids are available. Some of these are based on immunochemical techniques using specific antibodies. The antibodies used, however, should not cross-react with other compounds possibly present in the test fluid. There- fore, the mono- or potyclonal anticotinine antibodies have been tested for cross-reactivity with nicotine and nicotine metabolites (Langone et al. 1973; Langone and van Vuna- kis 1982; l3jerke et al. 1986, 1987; Hansel et al. 1986; van Vunakis et al. 1987). Offprint requests to: R-A. Walk However, in none of these studies were the used anti- cotinine antisera tested for cross-reactivity with trans-3'-. hydroxycotinine (THOC), a nicotine metabolite which is excreted in amounts 3 times higher than cotinine in the urine of human smokers (Neurath et af 1987). The objec- tive of this study was to investigate in a competitive coti- nine ELISA the cross-reactivity of an anticotinine antise- rum which was raised in rabbits according to the method described by Langone et al. (1973). Special emphasis was placed on cross-reactivity with the nicotine metabolite THOC. Material and methods Materials. The anticotinine antiserum was raised in rabbits by immunization with a thyroglobutin-cotinine conjugate. The conjugates of cotinine with thyroglobulin or human sei•um albumin (HSA) were prepared by reaction with trans-4'-carboxycotinine as described by Langone et al. (1973). Alkaline phosphatase-conjugated antirabbit IeG (goat) was purchased from Medac (Hamburg, FRG). p-Nitrophenvlphosphate and the diethanolamine buffer were supplied by Boehrinzer (Mannheim, FRG), bovine serum albumin (BSA, fraction V) by Baker (Gross-Gerau, FRG), HSA by Sigma (Deisenhofen, FRG)]and thyro- globulin and (-)-nicotine by Serva (Heidelberg, FRG). (-)-Cotinine was purchased from Roth (Karlsruhe, FRG), (-)-nicotine-I'-N-oxide, trans-4'-carboxycotinine, (_)-cis- and (_)-trans-3'-hydroxycotinine, (±)-demethyl- cotinine, and (-)-cotinine-I-N-oxide from Dr Georg Neurath (Hamburg, FRG). All other chemicals were ob- tained from commercial sources and were of analvtical reagent grade. ' ~ Buffers. PBS: phosphate buffered saline, 0.002 mof/I phos- phate, 0.15 mol/1 NaCI, pH 7.4; PBS-T: PBS supplement- ed with 0.0>",0 (v/v) Tween 20; PBS-BSA-T: PBS-T supple- mented with 0.1% (w/v) BSA. Methods. The competitive ELISA for cotinine determina- tian was performed in 96-welt polystyrene microtiter plates (Immunoplate 1, Nunc, Wiesbaden, FRG). The plates were coated with 50 µl cotinine-HSA conjugate di- luted in 0.1 molll carbonate-bicarbonate buffer, pH 9.6, by incubation over night at 4° C. The wells were washed three times with PBS-T. Competitor solutions (I00 ia) of various concentrations (concentration range see Table 1) were preincubated with 150 µl anticotinine antiserum I
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DRABT Mainstream smoke concentrations of 9.2 ng BaP/cigarette have been reported for the Kentucky Reference Cigarette IR4F (R.J. Reynolds, 1988). The value for a filtered cigarette could be rounded to 10 ng BaP/cigarette. Thus, an average (1 pack/day) smoker of filtered cigarettes would be exposed to an additional 0.2 kg/day of BaP, a level which is approximately 4 times the estimated daily intake of BaP by inhalation. [See Table 8] However; one should keep in mind that more than 90% of the daily intake of BaP is derived from other sources, primarily food. This can be examined from the perspective of the daily lung burden as described by Chen and Thilly (1996). Instead of using the value of 10 m'/day for breathing, the more widely used value of 20 m'/day will be chosen. The value of 2.5 ng BaP/m3 as the "urban air concentration" will be used, which, as shown in Table 9, is probably an over-estimation of the urban air BaP concentration in the 1990s, if the downward trend has continued. Using the formula (breathing rate/day X urban air BaP level) = ng BaP by inhalation/day, for a breathing rate of 20 m3/day X 2.5 ng BaP/m3 = 50 ng BaP/day or 0.05 ,ug/day by inhalation/day. The level of 0.05 ug/day by inhalation corresponds to what Hattemer-Frey and Travis reported [See Table 7]. OOO2235.01 3/16/98 4:00 pm ' ~ '
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7
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DRarr tracks with RSP, then 0.13 /cg/m3 would correspond to 0.0013 µg/m3. From the perspective of human exposure, this level of exposure would be equivalent to 0.0013 µg/m3 X 20 m3/day = 0.026 µg/ or 26 ng/day, which is about half the daily level by inhalation estimated by Hattemer-Frey and Travis. 0002235.0I 3/16/98 4-00 pm
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864 NMR spectrometer. tH-NMR-and H, H-COSY-90 spectra of the samples (approx. 250 µg in 0.5 mL CD3OD) were recorded. Results and discussion: In the washed urine, the peak repre- senting nicotine phase 2 metabolites, designated C for conjugates in Table 1, completely disappeared after treat- ment with p-glucuronidase and with sulphatase but only partially decreased after acidic and alkaline hydrolysis (Table 1). The differences found between alkaline and acidic hydrolysis might indicate that COT is conjugated by different substituents and/or at different sites than THOC and NIC. In the TSP-MS of the partially purified fraction 1(Figurc 1a), [M + H)+ ions corresponding to sulphuric acid conjugates of COT (mlz 257) and THOC (mh 273) were found. The assumption that these ions represent nicotine phase 2 metabolites is supported by the occurrence of fragments with mlz 177 (COT) and m/z 193 (THOC). Several other ions in this spectrum might represent amino acids still present in this fraction (e.g., m/z 134: asparagine, m/z 150: methionine). In the TSP-MS of fraction 2 (Figure 1b), [M + H1' ions corresponding to a gtucuronide of THOC (mJz 369) and of its 0. Schcpers, D. Demetriou, K. Rustemeier, P. Voncken and B. Diehl }_6 IIl:S' ' I II 9 8 7 6 5iM 4 3- 2 1 0 Figure 2: One-dimensional rH-NMR spectrum of partially purified nicotine phase 2 merabolites, fraction 2. 3 5' t- z [b <'b ppm 2.0 50 2.6 3-C' 3.2 3.8 b v't zp zw nn aaa z . ... Figure 1: TPM-MS of partial/y purified nicotine phase 2 metabolites. a: fraction 1. b: fraction 2. Table 2: Chemical shifts of pyridine, pyrrolidine and glucuronic acid ring protons. H-No. Chemical shift (ppm) THOC Chloramphenicol- Fraction 2 (1-D-glucuronide 2 8.4 - 8.4 4 7.7 - 7.7 5 7.5 - 7.5 6 8.5 - 8.5 3' 4.5 - 4.8 4'a,b 2.4, 2.3 - 2.6, 2.4 5' 4.8 - 4.8 fi' 2.7 - 2.7 1" - 4.4 4.4 - 2" - 3 3 3.3 3- - 3.4 3.4 I dl i I le n 4.4 3.8pPM 3 2.6 4.4 5.0 2.0 Figure 3: Two-dimensiana! H, H-COSY 90 spectrum of partially purified nicotine phase 2 metabolites, fraction 2. aglycon THOC (m/z 193) were found. The fragment at m/z 351 can be explained by the loss of water (18 Daltons). In the one-dimensional 'H-NMR spectrum (Figure 2) and in the H,H,-COSY 90 spectrum (Figure 3) of fraction 2, the signals of a substituted THOC were observed. A comparison of the chemical shifts with those of THOC (Table 2) shows that the substitution has to be at the oxygen of the hydroxyl- group at the 3'-position in the pyrrolidine ring. The tH-NMR- signals of the sugar moiety show complex unresolved multiplets. - Therefore, a straightforward analysis of this part of the spectrum was not possible. The structure of the sugar moiety, however, could be established by comparison with the spectrum of chloramphenicol-(3-D-glucuronide, which showed similar chemical shifts for the glucuronic acid ring protons (Table 2). Spin simulation analysis based on the coupling constants obtained from the first order evaluation of this spectrum predicted fine structures for the multiplets of
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tine metabolites in the urine of smokers is not yet known (Neurath and Pein 1937). ThereFore, one should be aware that biomonitoring the nicotine uptake by the determina- tion of cotinine using immunochemical methods may pro- duce questionable results concerning the cotinine concen- trations. Acknowledgemersrs. The authors wish to thank Ms Gabriele Fleger and Heidi BGlles for helpful technical assistance. References Abraham GE (1969) Solid-phase radioimmunoassay of cstradiol- 17 ber.r. J Clin Endocrinol Metab 29: S66-S70 13enotvitz NL, Jacob P, Jones RT, Rosenberg 1(1952) Inter-indi- vidual variability in the metabolism and cardiovascular cl=fects of nicotine in man. 1 Pharrnacol Exp Tlter 221: 365-372 Benowitz NL, Kuyt F. Jacob P, Jones RT, Osman A-L (1983) Co- tininc disposition and effens. Clin Pharmacol Ther 34: 604-611 Biber A, Scherer G, Hoepfner I, Adlkofer F, Heller N-D, Had- dow JE, Knieht GJ (1937) Determination of nicotine and co- tinine in human serum and urine: an interlaboratory study. Toxicol Lett 35: 45-52 Bjerckc RJ, Cook G, Rychlik N, Gjika HB, van Vunakis H,.Lan- gone JJ (1936) Stereospecifre monoclonal antibodies-to nico- tine and cotinine and their use in enzyme-linked immunosor- bent assays. J Immunol Meth 90: 203-213 Bjercke RJ, Cook G, Langone 1J (1987) Comparison of mono- clonal and polyclonal antibodies to cotininc in nonisotopic and isotopic immunoassays. J Immunol Meth 96: 239-246 Dagne E, Castagnoli N (1972) Structure of hydroxycotinine, a nicotine metabolite. J Med Chem 15: 356-360 Hansel MC, Rowetl FJ, Landon J, Sidki AM (1936) Single-re- agent potarisatidn fluoroimmunoassay for cotinine (a nicotine metabolite) in urine. Ann Clin Biochem 23: 596-602 . IARC monographs on the evaluation of the carcinoAenic risk of chemicals to humans (1986) Tobacco smoking, vo13S. Inter- national Agency for Research on Cancer, Lyon. pp 163-170 Jarvis JM (1957) Uptake of environmental tobaceo smoke. In: O'Neill IK, Brunnemann KD, Dodet B, Hoffmann D (eds) Environmental carcinogens - methods of analysis and_ ex- 397 posure measurement vol9 - passive smoking. International Agency for Research on Cancer, Lyon, pp 43-53 Johnson LC. Letzel H, Kleinschmidt 1(1955) Passive smoking un- der controlkd conditions. Int Arch Occup Environ Health 56: 99-1I0 Ryeremte.^. GA, Damiano MD, Dvorchik BH, Vesell ES (195?) Smokiaa-induced chanees in nicotine disposition: Applica- tion of a ncw HPLC assay for nicotine and its metabolites. Clin Pharmacol 32: 769-780 Langone JL. Gjika H73- %an Vunakis H (1973) Nicotine and its metabolices. Radioimmunoassays for nicotine and cotinine. Bioche:nistry 12: 5025-5030 .- L:mcone JL van Vunakis H(1932) Radioimmunoassavs for nico- tine, cctinine, and v-(3-pyridd).y-oxo-N-methylbutyramide. Meth Enzemol 5-:6]5-640 Letzel H. rscher-Brandes A. Johnson LC, Uberla IC, Bibcr A (1957) ldnsuring problems in e.timatine tite exposure to pns- sive smal:inc using the excration of catinine. To<icol Lett 35: 35-4- Luck W, Nau H(1934) Nirotine and cotinine concentracions in serum and milk of nursing smokers. Br J CIinPharmacol IS9 Ic NRC (1996) EEnvironmental tobacco smoke: measuring exposures and assessing health effects. P.,ional Research Council, Na- tionai Academy Press-Washinston, pp 133-159 Neurath GB, Dunger M, Orth D, Pein-FG (1987) Trans-3'-hy- droxvcotinine as a main membolite in urine of smokers. Int Arch,Occup Environ Health 59: 199-201 Neurath GB, Pein FG (1957) Gas chromatographic determination of trans-3"-hydroxycotinine, a major metabolite of picotine in smokrs. 1 Chromatosr 415: 400-406 _ van Vunakis H, Gijka HB, Lan_one 11 (I9S7) Method 16 - Ra- dioimmunoassav for nicotine and cotinine. In: O-Neitt IK, Brunnemann KD, Dodet B, Hoffmann D (eds) Environ- mental carcino8ens-methods of analysis and exposure mea- surement vol 9 - passive smoking. International Agency for Researci on Cancer, Lyon, pp 317-3±0 Received S(a-: 31. I98SiReceived in revised form August 21. 1983/Acccpted Aucust 22. 19SS N '. O . O1
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Table 1. Percentage by Source of Estimated BaP Emission in the United States. EMISSION SOURCE PERCENTAGE Automotive exhaust 1.7 Heating 38 Refuse burning 45 Industrial plants 16 uuo_±is.ul 3.1196 10_'1 am
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DRAF" Jenkins, R.A., et al., Exposure to tobacco smoke in sixteen cities in the United States as determined by personal breathing zone air sampling. JExpos Analysis Environ Epidemiol 6: 473-502 (1996). National Academy of Sciences (NAS), Petroleum in the Marine Environment, Washington, D.C., National Academy of Sciences (1975). Osborne, M.R., and Crosby, N.T., Occurrence of benzopyrenes in the environment. In: Benzopyrenes. Cambridge University Press, Cambridge, pp. 301-316 (1987). Pucknat, A.W., Health Impact of Polynuclear Aromatic Hydrocarbons. Noyes Data Corp., Park Ridge (1981). R.J. Reynolds Tobacco Company, Chemical and Biological Studies on New Cigarette Prototypes that Heat Instead of Burn Tobacco, R.J. Reynolds Tobacco Company, Winston-Salem, North Carolina, pp.119-180 (1988). Suess, M.J., The environmental load and cycle of polycylic aromatic hydrocarbons. Scf. Total. Environ. 6: 239-250 (1976). Tobacco Merchants Association of the United States, Inc., Vol. 1, Tobacco USA (Table la), Princeton, N.J. (Revised October 1997). U.S. Environmental Protection Agency, Respiratory Health Effects of Passive Smoking: Lung Cancer and Other Disorders. US EPA/600/6-90/006 F (1992). 0002235.0I 3/76/98 4:00pm -11-
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DRAFT As one can see, the levels vary significantly depending upon the sampling location and the type of water, but in general they are rather low. This is not unexpected since PAH compounds in solution are readily adsorbed on to the surface of dust, soil or other insoluble particles. These particles will fall slowly to the bottom, and thus PAH compounds are removed from solution. The levels of BaP reported in sediments on the other hand can be rather high in the order of gg/kg or even mg/kg of dry sample. Soil: Sources and Occurrences The majority of investigations of PAHs in soils have been carried out by Soviet investigators between 1967-1977; these papers only reported the BaP content (Osbome and Crosby, 1987). The concentrations of BaP measured in the U.S.S.R. ranged from 0.0008 mg/kg to 200 mg/kg, with the maximum value found in the vicinity of an oil refinery. Similarly high concentrations (650 mg/kg) were measured in the area of a carbon black factory. In samples of sandy and forest soil collected in West Germany, considerably lower concentrations of BaP, ranging from 0.001 to 0.0004 mg/kg, were found. The contamination of soil can be attributed almost exclusively to emissions from combustion processes. In the majority of surface soil samples taken in Iceland, where hardly any fossil fuels are burnt, the most commonly found PAHs were not detected (detection limit for BaP, e.g., 0.02 ,ug/kg soil). Soil samples taken at the Reykjavik, Iceland airport, however, were extremely contaminated, with BaP concentrations reaching 0.785 mg/kg. 0002235.01 3/16198 4:O0pm -5-
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DRAFT "Human Exposure to Benzo(a)pyrene" Hattemer-Frey and Travis (1991) used a multimedia transport model to evaluate environmental partitioning of BaP. Measured and predicted environmental concentrations were used to estimate the accumulation of BaP in the food chain and the subsequent extent of human exposure from inhalation and ingestion. Their results showed that the food chain is the dominant pathway of human exposure, accounting for about 97% of the total daily intake of BaP. See Table 7. This value of approximately 2.2 µg/day average daily intake of BaP is in agreement with other values reported in the literature (e.g., Suess, 1976). Hattemer-Frey and Travis (1991) then went on to discuss human exposure to BaP from smoking and indoor air pollution, referencing a paper by Butler and Crossley (1979) that reportedly estimated that one cigarette delivers approximately 39 ng of BaP. Further, Hattemer-Frey and Travis used in their calculations an estimate that the average smoker smokes 20 cigarettes per day. Based on these calculations, they suggested that the smoker receives an additional 780 ng/day (0.78 µg/day) BaP from smoking. Additionally, they again referenced Butler and Crossley (1979), who reported that concentrations of BaP measured indoors (2.2 ng/m3) were comparable to outdoor air concentrations (2.5 ng/m3); thus, indoor activities would not substantially increase the BaP intake, since inhalation is not a major pathway of human exposure to BaP. 0002235.01 3/16/98 4:00pm ' '6'
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DRAFT Examining BaP Exposure from ETS or Room-Aged Sidestream Smoke (RASS) Attributing levels of PAH (BaP) in the air of restaurants, public rooms, etc., to ETS is difficult since other sources may be present, and other factors, such as ventilation rates, number of smokers, etc., may confound the issue. However, Grimmer (1983), under controlled conditions, reported 22 ng/m3 BaP where cigarettes were being smoked and less than 3 ng/m ~vhere no cigarettes were being smoked. He calculated that ETS contributed about 7 times the background BaP level. Grimmer states that "the measured concentrations of 22 ng BaP per cubic meter has to be considered as a maximum BaP concentration attainable by smoking. In practice nobody would tolerate this concentration" of smoke due to eye irritation, etc. A 12-month inhalation study in rats using room-aged sidestream smoke (RASS) (INBIFO, data enclosed) reports the following concentrations of BaP in RASS from 1R4F cigarettes: 0.13 u.g/m3 (upper limit) for the whole-body 12-month exposure group, and 0.121 ,t.cg/m ~upper limit) for the head-only 12-month exposure group (Haussmann et al., 1998). Again, if we consider urban ambient air to contain an average of 2.5 ng/m3 BaP, one can see that the rats in this study were exposed to approximately 50 times the level of BaP found in ambient air. The RASS concentrations in this study were approximately 100-fold higher than the maximum of the average concentrations of respiratory suspended particles (RSP) reportedly attributable to ETS (Guerin et al., 1992; U.S. EPA, 1992; Jenkins et al., 1996). Thus, if one assumes that BaP, which is in the particulate phase, 0002235.01 3/16/98 4:00pm -8-
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DRAFT Table 3. Average BaP Concentrations (ng/m') in U.S. Urban and Rural Areas (after Pucknat (1981), Table 5.14, p. 168). 1966 1970 1976 Urban 3.2 2.1 0.5 Rural 0.4 0.2 0.1 0uozzaa.oi
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DRAFT Pucknat (1981) cites a paper within a paper which reports a "safe" lifetime BaP dose for human lungs as 4.3 mg. On the basis of this value, he then states that the concentration of atmospheric BaP should not exceed 120 ng/m3. A standard BaP concentration for industrial workers was determined to be 200 ng/m3. (OSHA Workplace Exposure Limit (PEL) for coal tar pitch volatiles of 0.2 mg/m3 averaged over an 8-hour workshift (Final Rule, January 1989); NIOSH- recommended airborne exposure limit for coal tar pitch volatiles of 0.1 mg/m3 over a 10-hour workshift; ACGIH recommendation that worker exposures, by all routes, be controlled to levels as low as can be reasonably achieved; New Jersey Hazardous Substance Fact Sheet -- Benzo(a)Pyrene -- Micromedex, Inc., 1974-1998.) Water: Sources and Occurrences According to a National Academy of Sciences (NAS) report (Petroleum in the Marine Environment, NAS 1975), about 6 million tons of petroleum hydrocarbons enter the oceans annually; the major contributors are marine transportation and runoff (urban and river). Other sources of PAHs in the oceans are coastal refineries, industrial and domestic waste, natural seeps, and atmospheric fallout. BaP levels found in water are shown in Table 6 (after Osborne and Crosby, Table 17.5, p. 307). 0002235.01 3116199 4:00 pm - 4 -
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396 Table I. Rclative cross-reactivity of aniicotininc antibodies with nicotine mciabolites Compound Concentration in assay (moi/I) Steepness of i i i i Relative cross-reactivity (°o) _ Min. %lax. - At 50N inhibition nh b t on curve• (absorbance This stud9" Published data ( x 10-°) ( x 10-') ( x 10-°) units/loa Langone Bjercl:e (mol/I)) etal.(193?)" etal.(1936)~ (-)-cotinine 57 0.057 3.5 -0.40 100 t00 t00 I-N-methylnicotinium iodide 33 ln -- - <0.037 - I'-N-methylnicotiniumiodide 33 I0 13100 -0.53 0.077± 0.006•' - nicotine-1'-N-oxide 56 I' 6 660 -0.S- 0.053-_ 0.006° 0.01 0.0"'S (-)-eotinine-I-N-oxide 52 i6 13J0 -0S± 0.19 =0.01< 0.1 - (-)-nieotine 1'-0 - 10 - 1360 -0_S .0'6 =0.0.e 0.05 0.025 -. (=)-demethylcotinine G_ s;.5 -0._9- . .__ti fi --,1.0^ 0.3 [_9 ( r)-cis-3'-hydrosycmininc ' 5'_ In . 30.7 . -O.;S - . ._ _ , li: =1.1, (+)-trans-3 -)tydroxyootininc (+)-nicotine 51 - I6 - I1.8 - - - - - - - <OAI 0-01' (+)-cotinine - - - - - <0.0t (-)-nornicotine - - - - - - - -- - - 0.01 0.30 (-)-cotinine-N'-oxide - - ?.3 Onl y published data regarding those metabolites which have an intact pyridine and pyrroiidine rin__ structure were salectcd • At 50% inhibition ° Polyclonal antibodies raised in rabbits ° Monoclonal antibodies - ' Mean±standard deviation, the coefficients of variation range between 3.7% (for trans-3'-hydrosycotinine) and 22.2% (for I'-N- methylnieotinium iodide) (1:4000 diluted with PBS-BSA-T) for 30 min at room tem- perature (RT). After pipettingg 50 µl of the preincubation mixtures into the wells of the coated microtiter plates, these were incubated for fh at RT and washed three times with PBS-T. Finally, 50 ul alkaline phosphatase conjugat- ed antirabbit I-G (1:2000 diluted with PBS-BSA-T) aas added to each well and the microtiter plate was incubated for I h at RT. After the plates were washed three times with PBS-T and one additional time with bidistilled water. 50 µl of freshly prepared substrate solution (10 mmol/t in diethanolamine buffer, pH 9.5, containing 0.5 mmol/I magnesium chloride) was added to each well. The enzyme reaction was performed for 30 min at RT in the dark and was stopped by adding 50 ul of 20% (w/v) N1aOH solution. Absorbance was read at 405 nm. For optimal comparison each individual nicotine met- abolite wus assayed together with cotinine on one microti- ter plate. Duplicate or triplicate assays were performed on different davs. The inhibition curves for cotinine and the other nicotine metabolites were calculated bv a least square fit to the logistic function y = A/(t+(B/x)c) in which B represents the logarithm of competitor concentra- tion at 50% inhibition. The logarithm of the competitor concentration and the value of the absorbance of the sam- ple are given by x and y. A corresponds to the maximum absorbance (i. e, that at zero competitor concentration) and C determines the curvation of the concentration-response curve. The relative cross-reactivity was calculated from the molar competitor concentrations at 50% inhibition (Abra- ham 1969). Results and Discussion The nicotine metabolites demethylcotinine and cis- and trans-3'-hydroxycotinine showed moderate to marked cross-reactivity to the anticotinine antiserum (Table 1). It is of special interest that the highest cross-reactivitv of about 30% was observed with THOC. High cross-reactivity would lead to an overestimation of cotinine in the pres- ence of THOC and thus would explain the variations be- tween the results which were obtained by determining the cotinine concentrations in urine by immunochetriicalver- sus gas chromatographic methods (Johnson et al. 1955; Biber et al. 1957; Letzel et al. 1957). Benowitz et al. (1963) has already discussed the possibility that the discrepanec between the cotinine half-life determined by gas chroma- - tosraphy (t'ri = 16-20 h) and that determined by radioim- munoassay (t'/: = 30-40 h, Langone et al. 1973) might be due to the different methods. In addition, they expected that "metabolites cross-reacting with cotinine antibodies mieht give a false lono estimate of t'/when measured by radioimmunoassav". The monoclonal anticotinine aTIti- bodies which have been produced and described (Bjercke et al. 1936) had not been tested for the cross-reactivity with THOC. Funhermore, these monoclonal antibodies do not seem to be more sDeciflc than the polyclonals raised in rabbits (Table I). Additionally, anticotfnine antibodics mieht have a hi-h stereoselective specificity, as has been shown by Lansone et al. (1982) for (a-)- and (-)-cotinine (Table I). Such a stereoselective specificity of anticotininc antibodies cannot be excluded for the cross-reactivit_v ~% ith (+)- and (-)-THOC. Since in the present study the cross- reactivity of THOC was assayed with its racemic form (=)-THOC, and since the naturally occurring metabolite (-)THOC (trans-I-methyl-3-(R)-hydroxy-5-(S)-3-pyridyl- ?-pyrrolidinone, Dagne and Castagnoli 1972) is only part of the racemic (±)-THOC, the relative cross-reactivity of the anticotinine antiserum with the (-)-THOC enantio- mer might be even hieher than 30%. Cross-reactivity of anticotinine antibodies with furthcr nicotine metabolites in the biological test fluids cannot b; excluded, especially since, e. s. the whole spectrum of ni'.r-
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Table 6. BaP Levels in Water. SAMPLE COUNTRY BaP (ng/L) Tap water FRG 0.25 - 9 Tap water USA 0.2 - 1.6 Groundwater FRG 1 - 10 Groundwater USA 0.2 Rainwater FRG 4 - 80 Reservoirs UK 0.7 - 3.8 Well water UK 0.2 - 0.6 Well water FRG 2- 15 Lake Erie USA 0.3 River Rhine at Mainz FRG 50 - 110 River Rhine at Koblenz FRG 10 - 60 River Thames UK 170 - 280 River Thames UK 4.2 - 430 River Trent UK 5.3 - 504 River Severn UK 1.5 - 48 Ohio River USA 5.6 Delaware River USA 41.1 Motorway run-off UK 570 Domestic effluent FRG 38 Human urine 1300 Sewage sludge FRG 1.7 (mg/kg) 002248.01 3.11.98 10.21 am
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DRAFT Table 4. Summer-Winter Average of Ambient BaP Concentrations (ng/m') in the Air of Selected Cities (after Pucknat (1981), p. 169). CITY BaP (ng/m') Atlanta 4.5 Birmingham 15.7 Detroit 18.5 Los Angeles 2.9 Nashville 13.2 New Orleans 3.1 San Francisco 1.3 000224801 31198 10 ? l am
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Med. Sci. Ra., 1992; 20, 863-865 Nicotine phase 2 metabolites in human urine - structure of metabolically formed trans-3'-hydroxycotinine glucuronide 863 G. Schepers, D. Demetriou, K. Rustemeier, P. Voncken and B. Diehlt INBIFO Institut ftir biologische Forschung, Fuggerstr. 3, D-5000 Kofn 90, Germany and 'Spectral Service, Vogelsanger Sir. 30, D-5000 Koln 30, Germany Keywords: Urine,,nicotinc, cotinine, rrans-3'-hydroxycotinine, glucuronic acid conjugates, sulphuric acid conjugatcs, TSP-MS, NMR. Introduction: Evidence for phase 2 metabolites as major urinary nicotine metabolites in humans has recently been presented by several authors [e.g., 1-4]: conjugates of nicotine, cotinine, and 3'-hydroxycotinine were quantified in urine samples indirectly by measuring the increase in the amounts of nicotine, cotinine, and 3'-hydroxycotinine after enzymatic hydrolysis using (3-glucuronidase. This led to the suggestion that nicotine phase 2 metabolites are glucuronides of nicotine and its respective phase I metabolites. Caldwell et al. [51 recently tanied out thermospray liquid chromatography/mass spectrometry (MS) of a smoker's urine and electrospray MS of a high performance liquid chromatography (HPLC) fraction of it. The retention time and mass spectra were compared with those obtained for the fully synthesised and spectrometrically characterised reference substance. They claimed that N-(i-D-glucopyra- ncuronosyl-(S)-(-)-cotininium inner salt is a major urinary nicotine phase 2 metabolite. In a very recent study (6], we analysed a smoker's urine by HPLC after derivatisation with 1,3-diethyl-2-thiobarbituric acid (DETBA), which is selective for compounds containing unsubstituted pyridine nitrogen: in addition to large peaks derived from trans-3'-hydroxycotinine (THOC), nicotine (NIC), and cotinine (COT), a large peak representing nicotine phase 2 metabolites was found. This peak was not detectable in the DETBA assay when the urine was treated with an enzyme preparation containing p-glucuronidase and sulphatase. The products of the enzyme cleavage were mainly THOC (67%), COT (31%) and a small amount of NIC (2%). The objective of the present study was to determine which substituents are conjugated to the nicotine phase 1 metabolitcs and at which position. Materials and methods: CHzCIz was from Merck (Darmstadt, Germany), DETBA from Aldrich (Steinheim, Germany), S- nicotine, chloramine-T, chloramphenicol ]3-D-glucuronide, (3-glucuronidase (Helix pomaria type HP-2) and arylsulphatasc (Hel& pornaria, type H-2) from the Sigma Chemical Co. . (Deisenhofen, Germany), COT from Roth (Karlsruhc, Germany), and THOC from Dr G. Neurath (Institut ftir Biopharmazeutische Mikroanalytik, Hamburg, Germany). The pooled human urine sample was randomly collected (male Caucasian smoker, approximate daily consumption 50 cigarettes, nicotine delivery 1 mg per cigarette). In order to remove most of the nicotine phase I metabolites, aliquots of the urine were alkalised by NH,OH to pH 10.8 and exten- sively washed with CHzCIz (4 times, twice the sample volume). Hydrolysis of the CHzCIz-washed urine sample was performed enzymatically and by acid and alkaline treatment. The conditions are given in Table 1. The nicotine metabolites before and after hydrolysis were analysed using the DETBA HPLC assay [6]. Partial purification of nicotine phase 2 metabolites for MS and NMR analysis was performed starting with 500 mL of the CHzCIz-washed urine- The purification procedures consisted of size-exclusion chromatography on Sephadex G10 (Pharmacia, Freiburg, Germany), collection of the fractions representing the first DETBA-positive peak, chromatography of these fractions by cation-exchange HPLC on a Partispherc SCX cartridge (Whatman, Maidstone, UK), repetitive chromatography of the DETBA-positive fractions by ion-pair HPLC on Radial Pak Cl8 cartridge connected in series with a Nova-Pak C18 column (Waters, Fschborn- Germany). and repetitive chromatography of the DETBA-postirve fractions by reverse phase HPLC on the above-mentioned C18 columns. Two DETBA-positive fractions were obtained. For - NMR analysis, fraction 2, representing the major DETBA- positive peak, was further purified by hydrophilic interaction liquid chromatography on a polyhydroxyethyl-aspartamide tv column (ICT, Frankfurt, Germany). Thermospray mass spectrometry (TSP-MS) was performed on a mass spectrometer coupled to a TSP interface (QUATTRO, Fisons Instruments, Mainz, Germany). The solvent was a mixture of methanol and 0.1 mol L-t ammonium acetate in water (50/50, v/v). The sample was directly applied by loop injection. NMR spectrometry was performed on a Bruker AC-P 300 Table l: Hydrolysis of CNzC(i-washed smoker's urine. Treatmcnt Hydrolysis condition Metabolite dctected' Activity (units mC') pH Incubation time (h) Temperature (°C) C NIC COT THOC (i-glucuronidase 6,000 4.7 18 37 0 + + + Arylsulphatase 1,000 7.0 18 37 0 + + + Aikaii - 14.U 16 70 +(1) a + ll Acid - I.0 16 70 +(j) + 0 +
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G. Schepers et aJ., S-Ntcotine metobolIsm in rtffs T.bakfaneh., 8, 339-349. 38. by P. (1973) : Piaynvdc orid.dan of vimfice m nirotine 72 bnmiumn ion. J. Bict Cbem.248, 2796-2800. 39. NBuyeo T: L. (humke i-D. CS.agooli N. (1979) : Metabolic oziduiou of niwtine to ebemidiy re.eeve ;wermea:.res. J. Med. Ch., 22, 259-263. 40. 1<mcine A. Wd]im. D.E, Qe.rel T. Lemu 7.-P. (1991) : Hammil RBul.tim of miac.om.l iLviaawinmg mooooz7~ •~' Tisae-0epmdtntexpRssionmd.ubsnnfe peei5eity. MaL Phmm.eol., 40, 211 217. 41. T1egle DM. (1980). Min®md Hario-wntaming monooxy8wuc Oxygeu.tion of uuoleophil'c vina8a ®d .ultur cwmpomdi. ls Jacoby W.B., ed. Esymstic Basis of Derezintion. Vol. 1. New Yorr Academic Press, pp. 201-227. 197 42 D.mmi L.A. Pool W.P. Cmoks PA., KaderLt R%., Teglet DM (1988) : Slaeoeeleai.ity in the N'ocidntion of nicotine itaneis by fltrIDbcatanring monoacy8eaue. MaL Ph.onawL.33, 70L705. 43. Booth J, Boy4nd F'i (1970) : The meabolism of nimeme into two op[ic.IIy-.ctire daeoiaomaa of nicatmo-i'oxide by ®a1 timun m.iau and by cigmene ®oFas. Blacben. Phso,aco1,19, 733-742 44. JemaP. Goncd J.W. BsYea AH. (1971) : Campu.rive C- .nd Nax~on of (+). and (-}uimdm by vuicu..peeiez. Xmobiotics, 1,497-498. 45. Vesken P, Rusremcia R:., Schrym (3. (1990) : IdrndSntion of ds-3'-hydmxymCaix au a urinrty aiwdne metebolira. Xcaobiotica,20,1353-1356.
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Table 7. Pathways of Human Exposure to B(a)P (after Hattemer-Frey and Travis, 1991). SOURCE DAILY INTAKE (/-cg/da}•) % OF TOTAL DAILY INTAKE Food (total) 2.1 97 Inhalation 0.05 2 Water 0.01 1 TOTAL 2.16 100 0,- 000'_?08.01 71198 10 11 am
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Table 8. Estimated Exposure of a Smoker to BaP (ng/day). Cigarettes/day 10 20 40 I R4F filtered cigarette 100 200 400 OW2238.01 3111 98 10QI am
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DRAFT Table 5. Atmospheric Benzo[a)pyrene Concentrations (ng/m') for Various Locations Around the World in Summer and Winter (after Osborne and Crosby (1987), Table 17.1, page 302). LOCATION WINTER SUMMER YEAR Sydney 8 0.8 1962-63 Liege, Belgium 110 15 1958-62 Ontario, Canada 15-20 1.2-18.5 1961-62 Prague 122 19 1964 Copenhagen 17 5 1956 Helsinki 5 22 1962-63 Paris 300-500 1958 Budapest 1000 32 1968 Teheran 6 0.6 1971 Belfast 51 9 1961-62 Milan 610 3 1958-60 Amsterdam 22 2 1968 18 2 1969 5 1970 8 1971 Oslo 15 1 1956 Poland 130 30 1966-67 Madrid 120 0 1969-70 Stockholm 10 1 1960 1 000224801 31198 I0'_I am
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DRAFT References Butler, J.D., and Crossley, P., An appraisal of the relative airborne sub-urban concentrations of polycyclic aromatic hydrocarbons monitored indoors and outdoors, The Science of the Total Environment 11: 53-58 (1979). Chen, J., and Thilly, W.G., Mutational spectra vary with exposure conditions: benzo[a]pyrene in human cells. Mutation Research 357: 209-217 (1996). Grimmer, G., Chapter 3 -- Sources and occurrence of polycyclic aromatic hydrocarbons. In: IARC Environmental Carcinogens, Selected Methods ofAnalysis, Vol. 3--Analysis ofPolycyclic Aromatic Hydrocarbons in Environmental Samples. H. Egan (Ed.). IARC, Lyon, pp. 31-54 (1979). Grimmer, G., and Pott, F. Occurrence of PAH. In: Environmental Carcinogens: Polycyclic Aromatic Hydrocarbons. G. Grimmer (Ed.). CRC Press, Inc., Boca Raton, FL, p. 61-128 (1983). Guerin, M.R., Jenkins, R.A., and Tomkins, R.A. The Chemistry of Environmental Tobacco Smoke: Composition and Measurement. Center for Indoor Air Research, Lewis Publishers, Chelsea, MI (1992). Hattemer-Frey, H.A., and Travis, C.C., Benzo-a-Pyrene: Environmental Partitioning and Human Exposure, Toxicology and Industrial Health 7(2): 141-157 (1991). Haussmann, H.J, et al., Comparison of fresh and room-aged cigarette sidestream smoke in a subchronic inhalation study on rats. Toxicological Sciences 41: 100-116 (1998). International Agency for Research on Cancer (IARC), IARC Monographs on the Evaluation of the Carcinogenic Risk of Chemicals to Humans, Polynuclear Aromatic Hydrocarbon Compounds, Part 1, Chemical, Environmental and Experimental Data. IARC, Lyon, Vol. 32, pp. 211-224, pp. 36-37 (1983). International Agency for Research on Cancer (IARC), IARC Monographs on the Evaluation of the Carcinogenic Risk of Chemicals to Humans, Biological Data Relevant in the Evaluation of Carcinogenic Risk to Humans. Tobacco Smoking. IARC, Lyon, Vol. 38, pp. 389-394 (1986a). International Agency for Research on Cancer (IARC), IARC Monographs on the Evaluation of the Carcinogenic Risk of Chemicals to Humans, Biological Data Relevant in the Evaluation of Carcinogenic Risk to Humans. Tobacco Smoking. IARC, Lyon, Vo138., pp. 101-102 (1986b). 0002235.01 3116198 4:00 pm - t0-
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DRAFT Table 2. Estimated B(a)P Emissions in the United States, after Grimmer (1979). SOURCE TONS/YEAR TOTAL Vehicle exhaust Gas-powered cars 10 Gas-powered trucks 12 Diesel fuel-powered trucks and buses 0.4 22.4 Heating Coal Hand-stoked residential furnaces 420 Intermediate units 10 Coal-fired steam power plants 1 Oil Low-pressure air atomizer and others 2 Gas 2 Wood 40 475 Refuse burning Commercial, residential, institutional and apartments 33 Open burning Forest and agricultural 140 Vehicle disposal 50 Coal refuse fires - 340 563 Industrial plants Petroleum cracking 6 'i ' Asphalt air-blowing <I Coke production 192 200 Total (all sources) 1260 Wo2lba,o
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8
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DRAFT Submission by Philip Morris U.S.A. to The National Toxicology Program Chapter 8: Section on Carcinogenesis from Philip Morris U.S.A. Comments on Health Effects of Exposure to Environmental Tobacco Smoke Final Draft for Scientific, Public and SRP Review Office of Environmental Health Hazard Assessment California Environmental Protection Agency May 5, 1997 March 19,1998 0002193.01 3/1G/98 4:33 pm
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DRAFT Table 9. Average BaP Concentrations (ng/m') in U.S. Urban and Rural Areas During 1966-1977 (after Pucknat 1981, Table 5.14, p. 168). Location Year 1966 1970 1976 Urban 3.2 2.1 0.5 Rural 0.4 0.2 0.1 0001248,01 71I,98 10 ._'1 am
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F-,9 ure 1. ESTIMATED BaP EMITTED INTO ATMOSPHERE FROM SIDESTREAM SMOKE 0.12 o.i 0.02 4 4 4 - 1 1 1 ~ W D m O n n m w w rn N m w C m o ~D m ~ T W m m O m m N v m rn m a [a m m
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COMMENTS ON CHAPTER 7-- CARCINOGENIC EFFECTS Summary Overview Based on Chapter 7, "Carcinogenic Effects," OEHHA contends that (1) the data support a causal relationship between ETS exposure and lung cancer, (2) the data support a causal relationship between ETS exposure and cancer of the nasal sinus and (3) the data are "suggestive" of a causal relationship between ETS exposure and cervical cancer. Philip Morris herein provides comments on those three conclusions. Given the limited time made available for public comments, Philip Morris has not been able to address in detail the other cancer endpoints discussed by OEHFIA; this does not mean that the Company endorses OEHHA's conclusions or the reasoning described therein, or that Philip Morris believes that OEIIIIA's review is complete, impartial, or appropriate. For instance, Philip Morris notes that OEHFIA has apparently not considered a number of potentially relevant references that could contribute to its discussion of non-lung cancers; thus, the review in Chapter 7 is incomplete on this basis alone. A list of those references is provided as Attachment A to this comment.
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COMMENTS ON: Health Effects of Exposure to Environmental Tobacco Smoke Final Draft for Scientific, Public and SRP Review Office of Environmental Health Hazard Assessment California Environmental Protection Agency PHILIP MORRIS U.S.A. May 5,1997 ff, --
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194 Erv. J. Drug Merab. Phmraacakiner., 1993, No. 2 Tablel: Shortest exetetion half-time t[n, of S-nicotine and metabolites in male rats. MaraboQre rra (h) Non-indtteed Aroclor indrrced NIC 1.6 0.9 CNN'O 2.1 13 NNIC 2.7 1S COT 3.1 1.2 POBAc 43 1.9 POMBAm 4.8 13 CNO 4.0 iS 3HOC 4.9 2.0 A 5.0 2.6 C.ucul.ted fmm the areepe.t slope in the sigzne-mimu plois plota can be explained in that the formation rate is more rapid than the elimination rate within the first 3 h. For CNN'O, this period might be missed due to the very fast metabolism of NIC to this primary metabo- lite and the fast excretion kinetics of CNN'O. Comparison of the metabolite profiles of male and female rats In general, NIC metabolism and its inducibility were mmi3ar for male and female rats. All urinary »etabo- lites found in the male rat were also found in the fe- male rat For the female as for the male rat, unidenti- fmd metabolites accounted for approx. 20% of the uri- nary excreted radioactivity in the noninduced and for 35% in the Aroclor-induced rat Statistically signifi- cant differences in the excreted amount of NIC and its metabolites were found between the noninduced and induced female rats (Fig. 5): Aroclor induction leads to a reduction in the formation of CNN'O and to an enhanced formation of secondary metabolites via COT, e.g. POBA,c, POMBAm, CNO, and 3HOC, as it was also found for the male rat. For some metabolites, however, different urinary excretion was observed between male and female rats. The ratios for the excretion over 24 h and the results of the statistical evaluation of these differences are given in Table IL Major differences were seen be- tween the noninduced male and female rats. The uri- nary excretion of CNN'0, which for both sexes is the major NIC metabolite, was lower and the excretion of COT was higher in the female rat The lower N'-oxi- dation of nicotine observed in the female rats is in ac- cordance with the recently reported sex differences in flavin-containing monooxygenase activity (40). Fur- 6-1 6-I a fll rh fTl aao rI. t1z1si.i6teiiTeFQ-rA a C o n~ a ecT b + LJ~ rZ, Iil ~ rt*7 GLJLJLJC.I~OCILJC~o 7 Z 3 1 6 9 7 a G A a C D D A E F II G H Fig. 5: Profile of minary nicotine metsbolitrs excreted over 24 h after iv. S-nicotine wdm+clstmdon to fcmalc zats. a: noninducod (M ± SB, n - 5): b: Aroclor-in- duced (M t SE, n- 4). 1: CNN'O; 2: NIC; 3: POBAc; 4: POMBAm; 5: CNO; G. COT; 7: NNIC; 8: 3HOC, 9: TNN'O; 10: PHOBAc; 11: NCOT; 12: PAAc A to H: unknovm (F: phase 2 metsbolites?). Statistically significant differences (P < 0.05) bo- twccn noninduccd and Amclor-induccd rats aro in- dicated by stars. thermore, noninduced and Aroclor-induced female rats excrete significantly higher amounts of the unknown metabolites F, which are assumed to represent phase 2 metabolites of NIC, than the male rat Stereoselectivity of S-nicotine metabolism During the formation of nicotine-N'-oxide WO) and 3HOC, two of the major NIC metabolites in the rat, a second siereogettic center in their molecular structure is formed. Therefore, it can be expected that the me-
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issues relevant to OEHHA's claims. OEHHA should review this literature and incorporate it into the ETS assessment. 3
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Section 7.3.1 -- Nasal Sinus Cancer OEHHA Claim: Nasal sinus cancer is listed in Table ES. 1 (p. ES-2) under "effects causally associated with ETS exposure." [emphasis added] Response: • Nowhere does OEHIIA indicate how it reached a conclusion of "causality." In fact, OEHHA's statements in the text of the document do not even mention a causal relationship. • The reader of the OEHHA document is led to assume that the conclusion of "causality" is scientifically justified -- because it appears in the Executive Summary of the document -- when, in actuality, OEffi-IA provides no rationale at all for such a conclusion, and when the Executive Summary states a conclusion different from the conclusions stated in the text of the document. • In the text, OEHHA states: "Of the studies examining the effect of ETS exposure on nasal sinus cancers, all three show consistent associations, presenting strong evidence that ETS exposure increases the risk of nasal sinus cancers in nonsmoking adults" (p. ES-7) and "Existing studies consistently show a significant positive association between exposure to ETS and nasal sinus cancer in nonsmokers. The results have been observed in studies conducted in eastern and western countries, in males and females, in cohort and case-control study designs, and with some adjustment for possible confounders." (p. 7-28) -4-
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Section 7.2 -- ETS and Lung Cancer Appendix A of the Final Draft purports to be a "Summary of Public Comments and Responses" received on previously released draft chapters of the document. Pages A-2 and A-3 are described as a "List of Those Commenting"; however, this list is incomplete. In particular, the Comment of Philip Morris U.S.A. on the January 1996 External Review Draft, Carcinogenic Effects of Exposure to Environmental Tobacco Smoke -- Excerpt: ETS and Lung Cancer, is not included on the list. In so omitting the Comment of Philip Morris, OEHHA has failed to respond to the many issues raised therein, as it is required to do. Moreover, the essence of the discussion on lung cancer in the Final Draft is unchanged from the January 1996 External Review Draft. Therefore, the Comment already submitted by Philip Morris on the lung cancer claims made by OEHHA is still relevant. That Comment is resubmitted as Attachment B to this submission. In the Comment on lung cancer already submitted by Philip Morris, a number of articles overlooked by OEHHA in its previous draft were referenced; however, OEHHA has failed to incorporate most of those references into the Final Draft document. Attachment C to this Section of this Comment lists those articles. It is incumbent upon OEHHA to address the important issues raised by these articles. In the year since Philip Morris submitted the Comment that OEHHA ignored, additional relevant literature has accumulated in the area of ETS exposure and lung cancer. Attachment D to this Comment lists new lung cancer literature, which raises a number of important 0080035.01 - 2 -
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fi[BiOPFAN 70URNA1. OF DRUG METABOLLSM AND P&ARMACOffiNEIICS, 1993, VoL 18, No. 2, pp. 187-197 Metabolism of S-nicotine in noninduced and Aroclor-induced rats G. SCHEPERS, K. RUSTEMEIER, R: A. WALK, and U. HACKENBERG INBIFO Institut fzir biologische Forschung, Cologne, Germany Received for publication : July 17, 1992 Keywordr : Nicotine, urinary metabolites, excretion kinetics, Arocior induction, sex differences, stereoselectivity SUMIviARY The urinary excretion of nicotine and its metabolites in noninduced and Aroclor-induced male and female rats has been determined following intravenous <rm+nima*:on of 2'-[i4C]-labeled S-nicotine at a dase of 4.6 I.tmol/kg. Complete xecovcry of the administeted tadioectivity was achicved: 95% in urine and 4% in feces over 96 h and 1% mmaining in the body. More dtan 40 nicotine metabolites were found by xadio-HPLC; 19 we:e identified including the eLdtraastliaatercomczs of nicotino-N'-oxide and 3'-hydroxycotinine. 'ihe urinary metabolite profile and excretion kinetics of nicotine and its metabolites wcae significandy diffetent between nonindueed and Axoclor-induced rats. The major urinary nicotine metabolite in the noninduced rat was eis-nicotine-N'- oxide. In the Aroclor-induced rat, cotininc metabolites were the major metabolitcs found. Sex differences wexa found for the urinary nicotine metabolite profile, mainly expressed in the excretion of cu aicofinoN'-oxide, 29% in the male and 17% in the female noninduced tat, and the excretion of cotininc, 5% in the male and 12% in the female noninduced rat 11•igh staeoselectivity was found for the formation of the crs/trans-diastencamcts of nicotine-N'-oxide ss well as of 3'-hydroxycotinine, the stexeoselectivity being mo® pronounced in male rats. INTRODUCTION Nicotine mefabolism has been extensively studied, and more than 20 nicotine metabolites resulting from 5 different metabolic pathways have been described [for review, see (1-5)]. The metabolism of the naturally occurring S-enantiomer and of the synthetic R-enanti- omer of nicotine were shown to be different in vitro (6) as well as in vivo (7), and one enantiomer was found to interfere with the metabolism of the other (8). In the aforementioned in vivo metabolism study in the rat using enantiotnericaliy pure nicotine tritiated at the Please send reprint requestr to : Dr Georg Schepers, INBIFO Institut fdr biologische Fotschung, Fuggetstr. 3, D-51149 K61n, Germany methyl group (7), only a limited nuslt~r of inetabo- lites could be detected. Tlus is because the methyl group is lost at early steps in the metabolic pathways. TLerefore, the present study was performed using the enantiomerically pure S-nicotine, [14C]-labeled at the 2'-position in the pyrrolidine ring. It is known that the metabolism and biokinetics of nicotine are influenced by genetic (e.g. stain, sex) and exogenous factors (e.g. drug treatment, diet) [reviewed in (5)], which can affect the induction status of the metabolizing enzymes. The influence of selective in- ducers of various cytochrome P-450 isozymes, e.g. phenobarbital, 5,6-benzoflavone, ~-naphthoflavone, 3- methyicholanthtene, or ethanol on nicotine metabolism bas been shown in vitm on subcellular fractions or isolated organs [e.g. (9-15)]. In vivo, thg influence of 0 r1+ -
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190 Elr. J. Drug Metab. phormocokinet., 1993, No. 2 Administration of S-nicotine The 14C-NIC stock solution was diluted with physio- logical saline to a final conceutration of 0.9 µmollmL The rats (230-300 g body weight) received an i.v. dose of 4.6 Nmol 14C-NIC/kg (0.75 mg nicotine basdkg) at a rate of 13.3 pll(kg• s) by a motor-driven Hamilton syringe via the implanted vascular access port Following taC-NIC administration, the rats were housed in metabolism cages for up to 96 h. Collection of urine and feces Urine was collected in 50 ml ice-cooled polypropylene tubes pieloaded with 200 µl of L-tartaric acfd (5 mol/l). Feces were collected in ice-cooled glass containers. The surfaces of the metabolism cages were rinsed with water and the wash water collected- The urine samples were centcifuged and stored at -20 'C until chromatograpbic analysis. Determination of radioactivity Radioactivity was detennined by liquid scintillation counting (LSC) using a TRI-CARB 2550 TR liquid scintillation analyzer (Canbesa-Paclcazd, Frankfurt, Ge>many)- Urine and wash water samples were pre- pared directly in Aquasafe 300 (Znsser Analytic, Frankfurt, Germany). Feces and tissue samples were homogenized and the radioactivity was determined following oxidative combustion in an OX500 sample 260 240 200 y160 i tu m m ~ c01120 .. 60 401 E a m 0 n x 6 u a g oxidizer (Harvey Instruments, via Zinsser Analytic, Frankfurt, Germany), by trapping the COz in Oxysolve C-400 (Zinsser Analytic, Fianldurt, Germany). Radiochromatography HPLC of the urine samples was perfomted using a Hewlett Packard 1090 L Equid chromatograph (Wald- bronn, Getmany) equipped with a temary solvent de- livery system and a diode array detector (10 lil cell) connected in series to a RAMONA 90 radioactivity detector (150 }d calcium fluoride solid scinhllator flow cell, Raytest. Straubenhardt, Germany). The analog signals of the UV detector (254 nm) and of the radioactivity detector were evaluated by calculating peak areas using a chromatography data system (Multichrom, VG Inrn,i,,,rnts, VoTiesbaden, Germany). Peak identification was perfommed by comparison with the retention times of the reference compounds Radio-HPLC was performed by ion-pair clnomato- graphy on two analytical Nova-Pak ClTcolumas con- nected in series. A nonlinear temary solvent program was applied. The mobile phase consisting of a potas- sium phosphate buffer (pH 3.6), coniaining pentane-, heptane-, and octane-sulfonic acid, and acetonitrile was pumped at a flow rate of 0.6 mllmin. A typical radiocLromatogram is shown in Figure 2 To verify the results obtained by ion-pair chromatography, a cat- ion-exchange HPLC method based on the method of Cundy and Crooks (21) was used following modifica- tions. Full delai3s of both HPLC methods are de- scribed by Demetriou et al. (22). 0 i u vlow .k.....J 0 i r 10 20 30 40 50 60 70 Tine (minutnsl Fig. 2: Ion-pair r4dio-HPLC of a 24 h urine ®ample from a noninduced female rat dosed with 2'-[14C]-labeled S-nicetine.
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G. ScHepas et aL, S-Nicotine metobalLmt in rats Evaluation of the urinary excretion kinetics To evaluate the urinary eurrtion kinetics of MC and its major metabolites, sigma minus plot analysis was used (23): the differeace between the total amount of a metabolite excreted (CTb) and the amount excreted up to a given time point (UJ relative to the administered NIC was plotted logarithmically versus time. RESULTS AND DISCUSSION Total excretion and recovery of the administered radioactivity Rats dosed with 14C-MC e.xaeled 955'0 of the admin- istered radioactivity in utine within 96 h. The excre- tion in feces was 4% and the radioactivity determined in the body 1%. The exhaled radioactivity was deter- mined to be less than 0.1%. Hence, complete te- covery of the administered radioactivity was achieved. The results were the same for both noninduced and Aroclor-induced rats. Profile of urinary metabolites More than 40 peaks were found in the radiochromato- grams of the urine samples; 18 of them coincided with chromatographic properties of authentic synthetic compounds. For the diastaeomets of nicotine-N'- oxide, CNN'O and TNN'O, baseline separation was achieved with both ion-pair and ion-exchange HPLC. However, due to insufficient separations THOC and CHOC were determined in sum and designated as 3'- bydroxycotiaine (3HOC). Furthermore, PMABAc and its lactam COT as well as 5FfOC and its ring-opened tautomer POMBAm were not separated. They were designated as COT and POMBAm, respectively. When considered according to the amounts ex- creted in the urine of nonmduced male rats within 24 h following the administration of NIC, the first 20 me- tabolites accounted for more than 90% of the excreted radioactivity (Fig. 3). Of these 20 metabolites, 12 were identified. Unidentified MC melabolites repre- sented 20% of the radioactivity excreted in the urine of noninduced and 35% of the radioactivity excreted in the urine of the Aroclor-induced rats. The major urinary MC metabolite in the nonin- duced male rat is CNN'O (29%), followed by MC (11 %) and by POBAc (10%) (Fg. 3a). COT ac- di counted for 5% of the administered radioactivity. The MC found may have been elir++lnn+M either unn~_ta- bolized or after muabolic formation followed by the reduction of nicotine-N'-oude (6, 24-27). In the me- tabolite profile of the Aroclor-indnced male rats, the formation of secondary metaboGtes via COT predomi- nated, thus indicating a shift from the N- to the C-oxi- dation pathway. The major metabolites in the induced rat were CNO (14%), POBAc (12%), POMBAm (9%), and 3HOC (7%), whereas CNN'O decreased to 5% (Fig. 3b). COT accounted for less than 2% of the x 101 5-I GJ i°l p-i 5-1 0-' di 191 a LILI0000000 oooQ.m I 113 4 6 0 7 0 9 A B C 0 1101111 E F 4 G H *~* F~II11 + rh + + rn + 0 11 b 111213141510171819 A B C 0 O 7f E F II G H Fig. 3: Profile of urinary nicotine metabolites excreted over 24 h ancr i.v. S-nicotine •dm+n+straHon to male rats. a: noninduced (M ± S$ n- 5); b: Aroclor-induced (M ± SB, a-5). 1: CNN'O; 2: NIC; 3: POBAc, 4: POMBAm; 5: CNO; 6: COT; 7: NHIC; 8: 3HOC; 9: TNN'O; 10: PHOBAG 11: NCOT; 12: PAAc; A to H: unlaown (F: pbase 2 metabolites?). Statisticslly significant differences (P < 0.05) be- tweea noninduced and Aroclor-induced rats aze in- dicared by staxs.
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overlap 1.0 and are quite wide (as the authors themselves acknowledge), particularly the one for maxillary sinus cancers. • Sample Size: OEHHA's conclusion of causality is based on three studies reporting data based on only 91 cases in total. Neither the number of studies nor the number of cases is sufficient support for such a conclusion. • OEHHA even notes that the Hirayama (1983) study results are based on only 28 cases, but fails to point out that such a small sample size compromises the interpretation of the reported results. The low number of subjects results in very unstable risk estimates. • Fukuda and Shibata (1990) acknowledge that their data set was of "relatively small size." However, OEHHA's discussion of this study obscures the fact that there are only 35 nonsmoking cases. OEHHA focuses on the risk estimates by exposure category and the purportedly statistically significant "trend," while failing to acknowledge the fragility of those risk estimates, based as they are on 15 and nine cases, respectively. • While the Zheng, et al., study is fairly large (particularly given the rarity of this tumor), the risk estimates reported for spousal smoking are based on only 28 cases (those who were nonsmokers). Again, sample size is a distinct limitation of this study, a point which OEHHA fails to clearly state in their summary of the paper. -7-
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• OEHHA should formally indicate how it reached the determination of causality that warrattted the inclusion of nasal sinus cancer in Table ES.1. OEHHA Claim: "Active smoking is firmly established as a causal factor for cancers of the. . . nasal sinus cavity." (p. 7-26) Response: • No references are provided for this contention. However, the 1982 and 1989 Surgeon General's reports and the 19861ARC Monograph (No. 38), cited elsewhere in the chapter by OEHHA, do not even mention nasal sinus cancer in their discussions of cancers purportedly associated with active smoking. OEHHA Claim: "There are some data on the role of ETS for other cancer sites, including cancers of nasal sinus cavity. ..." (p. 7-26) • The 1982 and 1989 Surgeon General's Reports, cited by OEHHA, do not even mention nasal sinus cancer in their discussions of ETS. OEHIL4 Claim: "Existing studies consistently show a significant positive association between exposure to ETS and nasal sinus cancer in nonsmokers." (p. 7-28) -5-
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Response: • Lack of Statistical Significance: Only one of the risk estimates cited by OEHHA in its discussion of the studies has a confidence interval that excludes 1.0. The single risk estimate that is "statistically significant" has an extremely wide confidence interval, based as it is on only nine cases. • In Table 7.8 (p. 7-65), OEHHA lists the reported risk estimates from the three nasal sinus cancer studies. The table clearly shows that only one of the reported risk estimates has a confidence interval that excludes 1.0. • None of the three spousal smoking risk estimates cited by OEHHA from the Hirayama study is statistically significant -- all the confidence intervals include 1.0. Thus, those risk estimates are consistent with neither an increase or a decrease in risk. • Fukuda and Shibata do not even discuss the statistical significance of the individual risk estimates in their paper, but OEHHA has calculated confidence intervals. The great width (1.7 to 19.4) of the CI on the risk estimate for more than one smoker in the home illustrates the uncertainty associated with that risk estimate. When coupled with the fact that it is based on only nine cases, this makes any interpretation based on that number highly suspect. • The authors of the Zheng, et al., study provide only limited information on the spousal smoking analysis. Moreover, the confidence intervals for both risk estimates presented -6-
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188 Ecv. J. Drug Metab. Phmmacokinet.,1993, No. 2 selective cytocbrome P-450 isozyme induction on ni- cotine metabolism in the rat was shown using racemic nicotine (16, 17). To our knowledge, no in vivo study with nicotine has been performed using a nonselective inducer for various enzyme systems, e.g. Aroclor 1254, a mixture of polycblorinated biphenyls commonly used for the preparation of the postmitochondrial (S9) fraction for metabolizing promutagens in in vitro assays (18). The influence of such a nonselective inducer on nicotine metabolism, however, is of special interest since nico- tine metabolism includes not only P-450-related but also other enzymatic pathways. In the present study, the in vivo metabolism and biokinetics of S-(pyrrolldine-2'-14C]-nicotine as well as the influence of enzyme induction by Aroclor 1254 are presented in detail for male and female rat In ad- ditio, a comprehensive analysis of major and minor urinary nicotine metabolites, based on the completely recovered radioactivity, is given. MATERIALS AND METHODS Test substance S-(Pyrrolidine-Z 14C]-nicotinedia,-tartrate salt (14C- NIC) was synthesized by Chemsyn Science Labora- tories (Lenexa, Kans, USA): specific radioactivity: 58.8 mCilmmol, chemical purity: > 973'0, radiochemi- cal purity: > 989'0, optical purity: > 989'a. The optical purity was confiimed by determining the enantiomeric excess by HPLC (19) and GC on chiral columns (pub- lication in preparation). The 14C-NIC was dissolved in phosphate-buffered saline (phosphate 10 mmolll, pH 7.2, NaCI 140 mmolll) to a final activity of 2.5 mCi/ml, and aliquots of this stock solution were stored at -80°C. Reference compounds S-nicotine (NIC) was from Sigma (Deiseahofen, Ger- many): cotinine (COT) from Roth (IfarLcmhe, Ger- many); 3-pyridylacetic acid (PAAc) from Aldrich (Steinheim, Germany). Dihydrometanicotine (DHM- NIC), cis-3'-hydroxycotinine (CHOC), trans-3'-hy- droxycotinine ('I'HOC), cot.inine-N-oxide (CNO), nor- cotinine (NCOT), nornicotine (NNIC), 4{3-pyridyl)-4- oxo-N-methyl-butyramide (POMBAm), 4(3-pyridyl)- 4oxobutyric acid (POBAc), N,N'-dimethylnicotinium diiodide (NN'DMN), 4-(3-pyrldyl)-4-methyl-amino- butyric acid (PMABAc), 4-(3-pyridyl)4-aminobutyric acid (PABAc), 4-(3-pyridyl)-butycic acid (PBAc), 4- (3-pyudyq-4-hydroxybutycic acid (PHOBAc), and 5- (3-pyridyl)-tetrahydmfuranone (P1IIIF) were from Dr G. Neurath (Institut hir Biopharmazeutische M>3cmanalytik, Hamburg, Germany). 5'-Hydmxyco- tinine (SHOC), deme8rylcotinine-N-oxide (DMCNO), and the methyl ester of POBAc were from Philip Mor- ris (Richmond, Va, USA); 1'(R)-2'(S)-cir- and 1'(S)- 2'(S)-trans-oicotine-N'-oxide (CNN'O and TNN'O) were from Prof. 7. Gosod (Kings College, London, UK); N-methylnicotininm iodide (NMNIC), N'- methylnicotinium iodide (N'MI9IC), N-methyicotinin- ium iodide (NMCOT), and N-methyl-N'-oxonicotin- ium iodide (NMNN'O) were from Prof. P. Crooks (University of Kentucky, Lexington, USA). For the structural formulae of nicotine metabolites and refer- ence compounds, see Figure 1. Other chemicals Aroclor 1254 was from Analabs (via Antechnica, Eflingen, Germany) and was dissolved in corn oil (200 gl1). All other chemicals were from commercial sources and of the highest purity available. ' Animals Male and female Sprague Dawley CrL:CD(SD)BR outbced albino rats (Charles River Wiga, Sulzfeld, Germany) were used. 14 days befon administration of t4C-NIC, a vascular access port (Implantofix-R, Braun Melsungen, Melsungen, Geimany) was subcuta- neously implanted with access to the vena jugularis (20). The rats were individually housed in polycarbo- nate cages with bedding material. During the whole study, water and diet (iEtn atJ MRH-HALTUNG, Eggersmann, Rintein, Germany) were provided ad libitum. The room temperature was maintained at 22 ± 2`C. The light/dadc cycle was 12:12 h, the light cycle starting at 06.00 am. Administration of Aroclor Those rats used to study the influence of enzyme in- duction on the metabolism and biokinetics of NIC re- ceived a single i~. dose of Aroclor 1254 (OS g/kg) 5 days prior to t C-NIC adm+nistration. The protocol was the same as that used for the preparation of the postmitochondrial fraction for metabolizing promuta- gens in in vitro assays (18).
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G. Schepers er aL, S-Nicotine metabolism in rats 195 Toble II : Ratio of urinary excretion of S-nicotine and metebolites over 24 h betweea male and female rats. Metabolite Rado Non-indaced Aroclor induced CNN'O t.7>(a) 1.4 NIC 1.2 2.6* POBAc 1.0 0.9 POMBAm 0.8• 0.7 CNO 1.3' 1.6 COT 0.4R 0.4~ NNIC 0.8 12 3HOC 1.1 12 3NN'O 13 1.0 A 1S• 1.8• B 03* 0.6" C 0.7 1.2 D 43" 23' PHOBAc 12 0.9 NCOT 1.5• 1.1 E 1.8• 1.5 F 0.1* 0.1* PAAc 1.9 23• G 2.8• 10.0• H 1.4 1.7 (a) Statistically significzm diffemms (P < 0.05) in the urinary exaetion of nicvuoe meuboli= between ooovnduced male aad female cats as well as betwcm Arocxor-iaduccd male and female mts are indimted by as(msks ta6olism of NIC may lead to the predominant forma- tion of one of the diastereomets of these metabolites. We found that the in vivo N'-oxidation of NIC in both male and female rats leads predominantly to the formation of fhe cis-diastereomer of NN'O, CNN'O (see Table IlI). This steteoselectivity was statisrically significantly more pronounced in the noninduced com- pared to the Aroclor-induced rats. This highly stere- oselective N-oxidation pathway is catalyzed by flavin- containing monooxygenases (41) and has been pre- viously reported for several in vitro studies (6, 42-44). As already menfioned, separation of the diastereomers of 3HOC was not achieved by the radiochromato- graphic methods. However, we achieved their separ- ation by HPLC after diethylthiobarbinuic add derivat- ization (34), and/we were able to show that in both . male and female rats the trans-diaste *nmPr of 3HOC. THOC, is predominantly formed (see Table III). This confimu recently reported results (45). The steteose- lectivity of the 3'-hydroxylation of COT was statisti- cally significantly higher in the Aroclor-induced com- pamd to the non-induced rats. The sfereoselective for- mation of diastereomers of NN'O as well as of 3HOC was mon: pronounced in male than in female rats. The diffetences in stereoselectivity found for the for- mation of NN'O as well as of 3HOC in the nonin- duced and Aroclor-induced rats lead to the assumption that different isozymes of the respective enzymes are involved and that these isozymes as induced and/or repressed to a different extent CONCLUSION The results of this study demonsfrate that there are considerably more S-nicotine met•ebolites present in the urine of rats than previously described. Important differences exist in the metabolism and excretion kine- tics between nonindticed and Aroclor-induced rats and in the urinary excretion of some S-nicotine metabolites between male and female rats. The forniation of the cis/rranr-tliastereomers of nicotine-N'-oxide and 3'-hy- droxycotinine is highly steteoseiective. The results underline the importance of exogenous facto>:s and genetic composition on the complex metabolism and biokinetics of S-nicotine. ACKNOWLEDGEMENTS We thank Prof. I. Gorrod and Prof. P. Crooks for the gift of mfetencc compounds, Dr P. Voncken for helpful discussions and for providing the OC-MS analysis. Mr D. Haaselmann for eritical reading of the manusecip[, and Mrs G. Fieger. Mrs 0. Kunz, Mr J. Doll as well as Mr D. Demetriou for their excellent technical assistanoe. Table f77 : Ratio of the urinary 24 h excietion of the ds~trans-diastnwmcxs of nicotinc-N'-oxide and 3'-hydroxycotinine in the rat (M t SE). MetaboJite ratio Male Femak Noninduced (n=S) Aroelor-induced (n=5) Noninduced (n=4) Aroclor-induced (n=5) CNN'O/I'NN'O 9.4 t 03 6.0 t 0.5 7.2t0.4 4.4t0.5 THOCCHOC 7.7 --0.4 13.6 t 1.3 4.6t0.3 9.0t1.3
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OEHHA Claim: "The relationship between ETS exposure and cervical cancer was investigated in one cohort and three case-control studies." (p. 7-29) Response: • OEI IIIA's review of the literature on ETS exposure and cervical cancer is incomplete. When additional studies are taken into account, however, no clear picture of "risk" emerges. What seems most likely, based upon the data from these and other studies, is that ETS exposure is a marker for a number of interrelated factors, and that the reportedly elevated risk estimates represent the residual effect of insufficient control for these variables. • Munoz, et al. (1996), report cervical cancer risk estimates for husband's smoking status that are not statistically significant when adjusted for a number of potential confounding factors. OEHHA did not reference this article. • Bosch, et al. (1996), report statistically significant cervical cancer risk estimates for husband's smoking, but conclude that the "two most likely reasons" for their reported results are misclassification of human papillomavirus exposure and insufficient adjustment. OEHHA did not reference this article. OEHHA Claim: "Positive associations were observed in two of three case-control studies and a statistically nonsignificant positive association was observed in the only cohort study conducted." (p. ES-7) -12-
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nickel; occupation in the fiuniture, textile, and shoe industries; occupational exposures to chromium, mustard gas, isopropyl alcohol, and radium; and possibly chronic sinusitis. (DeVita, et al., 1993) OEHHA Claim: "The results have been observed in studies conducted in eastern and western countries, in males and females..:'(p. 7-28) Response: OEHHA's statement misrepresents the available data, reported for only one sex in each of two countries. • Two studies present data for nonsmoking Japanese women (Hirayama; Fukuda and Shibata) and one for nonsmoking white American men (Zheng, et al.). The Japanese studies do not have data on nonsmoking men, and the U.S. study does not have data on women.
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OEHHA Claim: "The results have been observed in studies ... with some adjustment for potential confounders." (p. 7-28) Response: • OEHHA states that the risk estimates from the Hirayama study included an adjustment for husband's age. Hirayama has adopted this approach consistently in his analyses of data from this study, when, as it has been noted elsewhere, "[i]t is absolutely routine in epidemiology to standardize for the age of the subject." [emphasis added] (Lee, 1992) Hirayama's odd approach would seem to raise questions about the overall validity of his study. • Cancer of the paranasal sinuses is more common in Japan than in the United States. (DeVita, et al., 1993; for references to materials not cited by OEHHA, see Attachment A) This would imply that caution is warranted in applying the reported results of the Hirayama study (as well as the Fukuda and Shibata study) to the United States population. There may well be ethnic-specific and lifestyle risk factors that have not been considered, as have been reported for nasopharyngeal cancer. (Schottenfeld and Fraumeni, 1996) • While Hirayama writes in the paper cited by OEHHA that "[n]o other risk factors studied significantly altered the risk of nasal sinus cancer in women," he fails to provide the reader with information as to what those risk factors might be. (Hirayama, 1983) Possible risk factors for this cancer have been called "multifold," and reportedly include exposure to -8-
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Section 7.4.1. -- Cervical Cancer OEHHA Claim: Cervical cancer is listed under "effects with suggestive evidence of a causal association with ETS exposure" in Table ES. 1. (p. ES-2) Response: • Nowhere does OEHHA indicate how it reached a conclusion of "suggestive" evidence for "causality." In fact, OEHHA's statements in the text of the document do not even mention a causal relationship. • The Table ES.1 conclusion is not justified or supported by the analysis presented in Chapter 7, which only states: "There is supportive evidence from epidemiological and biochemical studies implicating a role for ETS exposure in the etiology of cervical cancer in nonsmokers." (p. 7-32) OEHHA presents no criteria for extrapolating from this position to a conclusion that there is "suggestive" evidence for "causality," nor does it define what exactly constitutes "supportive" evidence or "suggestive" evidence. • The reader of the OEHHA document is led to assume that the conclusion of "suggestive" evidence for "causality" is scientifically justified -- because it appears in the Executive Summary of the document -- when, in actuality, OEHHA provides no rationale at all for such a conclusion, and when the Executive Summary states a conclusion different from the conclusions stated in the text of Chapter 7. -10-
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• OEHHA should formally indicate how it reached the determination of "suggestive" evidence for causality that warranted the inclusion of cervical cancer in Table ES. 1. • OEHHA states in Chapter 7: "[H]uman papillomarivus [sic] ... has been accepted as the sexually transmitted etiological factor in cervical cancer." (p. 7-28) OEHHA thereby seemingly acknowledges the scientific consensus on the importance of the sexually- transmitted agent, human papillomavirus (HPV), in the etiology of cervical cancer. (See Attachment A: Winklestein, 1990; Bosch, et ai., 1995; Munoz, et al., 1996; Bosch, et al., 1996) In fact, a consensus panel of the National Institutes of Health has concluded that cervical cancer is "causally related" to infection with HPV. (McNeil, 1996) • Given the general acknowledgment of HPV as a cause of cervical cancer, it is curious that OEHHA would even evaluate ETS exposure in terms of "causality," as indicated in Table ES.1. • Moreover, a noted cancer textbook refers to active smoking as a "cofactor" for cervical cancer (DeVita, et al., 1993); given this position, it clearly seems very premature to conclude that there is suggestive evidence that ETS is causally associated with this disease, as OEHHA claims.
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Response: • Misleading Presentation ofData: Table 7.9 (p. 7-66) is incomplete, reporting data from only two of the available studies (Slattery, et al.; Coker, et al.). Nevertheless, Table 7.9 (p.7- 66) clearly shows that only one cited risk estimate from the two studies is statistically significant. Chance has thus not been excluded as an explanation for the overwhelming majority of the reportedly increased risks cited by OEHHA in that table. • The series of risk estimates OEIIHA chose to include in Table 7.9 (p. 7-66) includes the largest risk estimate reported by Slattery, et al., for nonsmokers. The only statistically significant risk estimates for ETS exposure reported in Table 6 of the Slattery, et al., paper are for the highest exposure group for "all" reported ETS exposure or for home ETS exposure. This pattern would be consistent with confounding factors being clustered in that exposure group. • Sample Size: It is also clear from Table 7.9 (p. 7-66) that the risk estimates for different ETS "exposure levels" are highly suspect, based as they are on very few cases. • Moreover, the Slattery, et al., study, which seems to be key to OEHHA's contentions, fails to even provide the numbers of cases and controls for the different levels. This incomplete reporting makes interpretation of the study's reported results quite difficult. -13-
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• However, OEHHA's treatment of confounders can certainly be further "extended." This is perhaps the most important issue in evaluating the cervical cancer studies, and OEHHA pays completely insufficient attention to it. • Inadequate Treatment of Confounders in Individual Studies: The discussion in Chapter 7, plus statements from other reviews, clearly indicates shortcomings in the available literature's treatment of confounding factors. • It has been stated about the Hirayama study: [T]here are a number of doubts about the results from this large prospective study, the most important of which appear to be incompleteness of follow-up and doubts about the validity of the statistical methods used. Failure to gather data on confounding variables relevant to specific cancers is also a problem. [emphasis added] (Lee, 1992) OEHHA even acknowledges that Hirayama's ETS data "were not adjusted for potential confounders including subjects' or husband's sexual activity" (p. 7-29). Given what is now known about the relationship between HPV and cervical cancer, this failure on Hirayama's part means that this study can be treated as providing essentially no information relevant to the question of ETS exposure and cervical cancer risk.
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OEHHA Claim: "There is supportive evidence from epidemiological ... studies implicating a role for ETS exposure in the etiology of cervical cancer in nonsmokers." (p. 7-32) Response: • Many questions remain about the ETS-cervical cancer epidemiologic studies cited by OEHHA, making any summary interpretation of their reported conclusions difficult. Many of the reported risk estimates are not statistically significant. The studies have generally (in some cases, completely) failed to adequately consider potential confounding factors. Given these limitations, OEHHA's conclusion is unwarranted. OEHHA Claim: "The current chapter extends or modifies the discussion of issues related to ETS exposure and cervical cancer (e.g., on confounders in epidemiological studies ... )" (p. iii) Response: • Compared to the cervical cancer section in the May 1994 External Review Draft, the current Chapter 7 does include some "modified" discussion of confounders, in that they are now briefly mentioned in the context of the data collected in the epidemiologic studies cited by OEHHA. 0 -14-
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192 Eur. ,1. Drug Metab. Pftarmacakinet., 1993, Na. 2 administered radioactivity. For NIC and most of its metabolites, statistically significant differences were seen in the amounts excreted in the urine between noninduced and Aroclor-induced rats. Of the metabolites not shown in Figure 3, each contributed to less than 1% of the administered radioactivity. These included the following identified metabolites NMNN'O, NN'DMNIC, PTHF, PABAc, N'MNIG and PBAc, accounting in total for about 19'0 of the administered radioactivity. A comparison of the chromatograms of the urine samples with those of the reference compounds did not indicate that DHMNIC, NMNIC, NMCOT, and DMCNO am urinary S-nicotine metabolites in the rat The urinary metabolite profile of R-nicotine in the rat is different from that of S-nicothie as indicated by results we have obtained in an ongoing study. Dif- ferences were pronounced, e.g. in the excretion of TNN'O, NNIC, and COT as the major R-nicotine me- tabolites in the nomnduced rat These results as well as the fact that S-nicotine interferes with the metabo- lisn of R-nicotine (8) make it questionable to compare the metabolite profde obtained for S-nicotine in our study with that previously published for racemic R/S- nicotine (28). Unidentified metabolites Of the unidentified toetabolites, those designated as B, C, and D showed different peak shapes in the cluoma- tograms of different urine samples- This led us to the assumption that each of these peaks represents more than one metabolite. The formation of inetabolite C, eluting immediately after 3HOC, was significantly in- creased by Aroclor induction. Metabolite A, which was also significantly enhanced by induction, is prob- ably strongly polar as indicated by its early elution in front of CNO under both ion-pair and ion-exchange chromatographic conditions. Using ion-pair HPLC, metabolite A was found to have the same retention time as DMCNO, which has been proposed to be a nicotine metabolite (29). However, due to the differ- ent retention times of metabolite A and the reference compound DMCNO in the ion-exchange HPLC, it can be concluded that DMCNO is not a NIC tnetabolite in the rat Metabolites were observed that elute at the begin- ning of the chromatograms (designated together as F). This indicates that they are even more polar than me- tabolite A. Therefore, we assumed that they represent the nicotine phase 2 metabolites which have been found in human urine (30-35) as well as in the urine of IdIC-neated rats and hamsters (34). Recently, allohydroxydemethylcotinine has been described as a nicotine metabolite found in the urine of rats 4~), and its dehydration product de~ihylco- turine-A ~ cnamine in the urine of humans (32). In order to check whether these metabolites occur in the urine of NIC-treated rats, we performed a synthesis of allohydroxydemethylcotinine according to Kyerematen by ammonolysis of the methyl ester of POBAc. Ion- pair HPLC of the reaction mixture yielded 2 major product peaks. GCIMS analysis of each of these 2 HPLC fractioas yielded similar results: for each frac- tion 2 peaks were found by GC, one showing the mass spectrum identical with that of 4-(3-pyridyl)-4oxobu- tyramide (37), the other a mass spectrum most prob- ably of demethylcotinine-A 4"s' -enamine. 4-(3-Pyci- dyl)-4oxobutyramide is the ring-opened tautomer of allohydroxydemethylcotinine and therefore - analo- gous to 5HOC and POMBAm- we do not expect that our HPLC method is able to separate these 2 com pounds. Demethylcotinine-0°'S' -enamine is the dehy- dration product of these 2 compou]3-&. However, neither of the 2 product peaks in the HPLC corre- sponded to any of the peaks found in the radiochroma- tograms of the rat urine samples. Therefore, we con- clude that neither allohydtoxydemethylcotinine, 4(3- pyridyl)-4oxobutyramide, nor demethylcotinine-E4s- enanilne are urinary metabolites of NIC in the rat Kinetics of urinary excretion The sigma minus plots of NIC and its major urinar}y metabolites (Fig. 4) are not linear over the full time range but have three different curve shapes. The con- cave curves of NIC and CNN'O are linear during the first 6 h. For this period, the urinary excretion half- tunes (tr2) were calculated to be 1.6 h for NIC and 2.1 h for CNN'O in the noninduced and() 2and 1.3 h, respectively, in the Aroclor-induced male rats. For the other urinary metabolites in Figute 4, convex curves in the noninduced and sigmeidal curves in the induced rats were observed- This was the case not only for the secondary metabolites via COT (POBAc, POMBAm, CNO, and 3HOC) and the unidentified metabolite A but also for COT and NNIC. Both COT and NNIC are formed in a two-step reaction via iminium ion in- tPSI11PfIfA}PS (38, 39) in contrast to CNN'O which is fomed by a one-step oxidation of NIC. The fact that the excretion kinetics of the unidentified metabolite A are very similar to those of the secondary melabolites via COT and in addition that its formation is enhanced by Aroclor induction in the same way as the second-
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A
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sexual, and reproductive factors to adequately allow for these differences.
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Committee. Publishing an incomplete and unreviewed ETS assessment now has the potential of eclipsing the State's stated commitment to uniform, sound risk assessment practices. • As developed more fully herein, Cal/EPA cannot take any action based on reliance upon U.S. EPA's Risk Assessment. Cal/EPA should not rely upon U.S. EPA's Risk Assessment on ETS, and in so doing, avoid the necessary and required original work by Cal/EPA. Under the California Health and Safety Code, 539660(c), Cal/EPA must evaluate the "quality of data" underlying any health evaluation. Cal/EPA cannot and should not pursue any report which relies upon EPA's Risk Assessment. • Cal/EPA's ETS risk assessment (and specifically the lung cancer sub chapter) should not be pursued because Cal/EPA lacks the requisite authority to pursue such work. Cal/EPA's risk assessment on ETS was initiated prior to the enactment of AB 13, which imposed a state-wide ban on smoking in most places in California. See Labor Code sec. 6404.5(b). The restrictions of AB 13 encompass most nonresidential areas in the state, and even where smoking is restricted to designated areas, AB 13 requires ventilation equipment meeting specified standards to ensure that the air is not recirculated but exhausted outside. Even prior to AB 13, the Proposition 65 - 4 -
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ratios are probably no more than the leftover effect of variables controlled imperfectly by logistic regression. [emphasis added] • Zang, et al., also note: The apparent association between cervical cancer and environmental smoke exposure may, in fact, be the result of increased exposure to papillomaviruses or herpes simplex virus 2 through contact with male sexual partners who smoke, since one might expect smokers to be generally more sexually active than nonsmokers, and case patients are more sexually active than control subjects. • The author of a 1992 book on ETS chronic disease claims stated about the Slattery, et al., study: [T]he major problem with interpreting this finding as cause and effect is the extreme difficulty of adequately adjusting for sexual habits... . Since the number of sexual partners is only an inaccurately measured surrogate of the true sexually related cause of cervix cancer ... the adjustment will be incomplete and leave a residual confounding effect. It is entirely plausible that the whole of the adjusted relative risk could be explained by this. [emphasis added] (Lee, 1992) • That potentially misleading results can come from studies with inadequate attention to confounding factors (e.g., Hirayama, as acknowledged by OEHHA; Slattery, as pointed out by other reviewers) seems clear when the Coker, et al. (1992), study is compared to those other studies. In that study, the authors included age, race, education, number of Pap smears, number of sexual partners, and history of genital warts as confounders in their analysis of ETS exposure and cervical cancer risk. As for their reported results, they note, "the -17-
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B
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• With respect to the Sandler, et al., study, Lee (1992) notes: Apart from ... the problems of multiple testing, it should be realized that the Sandler I study collected no data on sexual habits, which are strongly correlated both with risk of cervix cancer and with smoking habits. [emphasis added] OEHHA acknowledges this as well: "[I]nformation typically obtained in studies of a specific cancer site (e.g., sexual activity in studies of cervical cancer) was not collected." [emphasis added] (p. 7-29) As with the Hirayama study, the Sandler study can be given little weight in analyses of cervical cancer claims. • Studies report that both personal smoking behavior and ETS exposure are correlated with a number of other lifestyle factors (related to sexual activity and alcohol and drug use) that appear to be markers for an increased risk of HPV infection. Disentangling the possible effects of these potential confounders is difficult, and the epidemiologic studies to date have not adequately addressed them. • In a letter to the editor on the Slattery, et al., study, Zang, et al., note: In this report, there clearly is undermatching of control patients with regard to important risk factors including sexual activity, religious background, and education.... Since the previously mentioned risk factors are correlated highly with one another as well as with active and passive smoking, the risk estimates relating smoking and cervical cancer may be subject to substantial bias and confounding.... The effect of adjustment on odds ratios is far greater than expected in case-control studies of this sort, as, for example, the decrease from 14.84 to 2.96 for passive smoke exposure. In fact, the adjusted odds -16-
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confidence intervals associated with each of these adjusted odds ratios were wide and clearly not statistically significant." [emphasis added] • Omission of Relevant Data: In the discussion of cervical cancer, OEHHA completely overlooked two articles presenting relevant data on the potential for confounding in the epidemiologic studies. Given OEHHA's predilection for data from California elsewhere in the report, this omission seems particularly odd. • Holly, et al. (1992), report data which "confirm that cigarette smoking is strongly associated with numerous life-style and behavioral factors, many of which have been linked directly or indirectly with cervical cancer." These data were collected from women residing in the San Francisco Bay area. • In another study from the same research group, Cress, et al. (1994), investigated whether women exposed to ETS differed from non-exposed women on a number of characteristics. This study was based on self-report of ETS exposure, just as are the epidemiologic studies of cervical cancer risk. Several statistically significant differences between "ETS-exposed" and non-exposed women were reported; they included marijuana use, beer consumption, and being divorced or separated. The authors concluded: [W]omen nonsmokers exposed to passive smoke were different from those not exposed. Studies that examine the association between passive smoking and disease need to measure dietary, lifestyle, -18-
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SECTION I: OVERVIEW IlND INTRODUCTION In the January 1996 external review,draft, "Carcinogenic Effects of Exposure to Environmental Tobacco Smoke -- Excerpt: ETS and Lung Cancer" (hereinafter, the Excerpt), Cal/EPA contends that recently published epidemiologic studies support the conclusions reached in the U.S. EPA's 1993 Risk Assessment on environmental tobacco smoke (ETS). This contention is incorrect and is not supported by the data. In this written comment, it will be well documented that the present draft of the Excerpt is a superficial treatment of the claimed association between ETS exposure and lung cancer risk. (Section II) Cal/EPA selectively cites and interprets studies and data in an attempt to support a conclusion that the recent epidemiologic data agree with the positions espoused in the U.S. EPA Risk Assessment on ETS. Further, this comment will highlight and summarize a number of the unresolved criticisms of the U.S. EPA Risk Assessment v on ETS, showing that the assumption that that document was a w conclusive, thorough review of the literature is unfounded. (Section III) In particular, data on ETS chemistry, ETS exposure data, the limitations of questionnaire data, animal inhalation studies and genotoxicity data were not adequately addressed.
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PHILIP MORRIS U.S.A. COMMENTS ON: CARCINOGENIC EFFECTS OF EXPOSURE TO ENVIRONMENTAL TOBACCO SMOKE EXCERPT: ETS AND LUNG CANCER California Environmental Protection Agency Reproductive and Cancer Hazard Assessment Section Office of Environmental Health Hazard Assessment External Review Draft January 1996 April 1, 1996
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Attachment B to Comments on Chapter 7-- Carcinogenic Effects Philip Morris Comment on 1996 External Review Draft
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Scientific Advisory Panel listed ETS as a carcinogen. As a result, in those areas where limited smoking is allowed, Proposition 65 requires that warnings be posted to alert persons of their potential exposure to ETS. Given the existence of AB 13 and Proposition 65, and given the authority of the California Occupational Safety and Health Administration to generally regulate worker health and safety it is difficult to see any basis of authority for Cal/EPA to pursue a risk assessment concerning ETS. • Cal/EPA should decline to take any further action on the ETS risk assessment given the extensive regulatory work already underway at the U.S. Occupational Safety and Health Administration (OSHA). Not only does Cal/EPA lack authority to pursue an ETS risk assessment, but if there were a current compelling need to review ETS issues, that need is being met by U.S. OSHA. Federal OSHA has completed the public comment phase of the lengthiest, most extensive regulatory hearing and review of potential ETS effects ever conducted. The agency held six months of public hearings, amassed more than 110,000 comments from interested parties and scientific experts, and collected extensive studies and data regarding the claimed health effects of exposure to ETS. Unlike the brief Cal/EPA workshops, the federal OSHA hearings were conducted as part of federal OSHA's regulatory responsibility for determining - 5 -
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Finally, since Cal/EPA relies almost exclusively on the epidemiologic literature in reaching its conclusions, this comment will address a number of epidemiologic issues that were treated in insufficient detail in the Excerpt. (Section IV) Given the shortcomings of the Excerpt, this comment concludes that Cal/EPA's stated position is not adequately supported. Specifically, when all of the relevant data are considered, those data do not support the claim that ETS exposure is associated with an increased risk of lung cancer in nonsmokers. Based on the information contained herein and upon publicly available information, Cal/EPA should defer from taking any further action on this assessment of risks. • To avoid "bad science," the Cal/EPA report should await the recommendations of the OEHHA Risk Assessment Advisory Committee. Cal/EPA Secretary James Strock has been quoted as saying that "scientifically sound risk assessments are the foundation upon which wise environmental decisions are made," which implies an obligation to assure that this report is sound science. (i) The California Legislature has also recognized the significant impact of risk assessments. In 1993, the - 2 -
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39. Tredaniel, J., et al., "Environmental Tobacco Smoke and the Risk of Cancer in Adults," Eur JCancer29A(14): 2058-2068, 1993. 40. Tredaniel, J., et al., "Exposure to Passive Smoking During Pregnancy and Childhood, and Cancer Risk: The Epidemiological Evidence," Paediatr Perinat Epidemiol 8: 233-236, 1994. 41. Vaughan, T.L., et al., "Nasopharyngeal Cancre in a Low-Risk Population: Defining Risk Factors by Histological Type," Cancer Epidemiol Biomarkers Prev 5: 587-593, 1996. 42. Wells, A., "Re: 'Breast Cancer and Cigarette Smoking: A Hypothesis,"' Am JEpidemiol 135(10): 1184-1186, 1991. 43. Wilkins, J., and Geidenberger, C., "Perinatal Exposure to Environmental Tobacco Smoke and Risk of Childhood Brain Tumor," Am JEpidemiol 136(8): 1010, 1992. Abstract. 44. Wilkins, J.R., and Sinks, T., "Parental Occupation and Intracranial Neoplasms of Childhood: Results of a Case-Control Interview Study," Am JEpidemiol 132: 275-292, 1990. 45. Witorsch, P., et al., "Re: Prenatal Exposure to Parents' Smoking and Childhood Cancer," Am JEpidemiol 135: 713, 1992. 46. Woodward, A., and McMichael, A., "Passive Smoking and Cancer Risk: The Nature and Uses of Epidemiological Evidence," Eur J Cancer 27(11): 1472-1479, 1991. 47. Zang, E., et al., "Exposure to Cigarette Smoke and Cervical Cancer," JAMA 262(4): 499, 1989. -4-
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• Revise and reissue in draft all other chapters in this assessment of ETS risks, to incorporate the suggestions inherent in the foregoing as to all chapters. • Review the Congressional Research Service Report. Most of Cal/EPA's overall risk assessment does not have the benefit of the November 1995 Congressional Research Service (CRS) report on ETS which raised serious questions about the methodology of the U.S. EPA's ETS risk assessment. The CRS study was not addressed in Cal/EPA's most recently released draft excerpt.
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Legislature enacted S.B. 1082 (Cal. Health & Safety Code sec. 57004), which required the director of Cal/EPA's Office of Environmental Health Hazard Assessment (OEHHA) to convene an advisory committee "to conduct a comprehensive review of the policies, methods, and guidelines to be followed by the state for the identification and assessment of chemical toxicity." The Advisory Committee was convened in 1994. It has held seven meetings; the next meeting will be held on April 10 and 11, 1996. At the same time that the work of the Advisory Committee is nearing completion, Cal/EPA is attempting to produce a formal risk assessment concerning ETS, complete with conclusions that may be calculated to command attention in the press but may be easily misinterpreted as to their true significance. Given the universally recognized need for thorough and reasonable risk assessment guidelines,' it is difficult to see how Cal/EPA would now allow the release of any risk assessment without a determination that the assessment followed the recommendations of the Advisory 1. For example, note the current ongoing work of President Clinton's Commission on Risk Assessment and Risk Management (scheduled to issue a report on April 24, 1996) and the ongoing work of the U.S. Environmental Protection Agency to issue Revised Carcinogen Risk Assessment Guidelines. - 3 -
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• Reissue Chapter 7 of the Cal/EPA assessment in its entirety, as the new table of contents indicates some changes from the chapter on "other cancers" issued in May 1994. • Substantially broaden the scope of its literature review to include all relevant materials. In particular, Cal/EPA should consider the public record compiled in response to U.S. OSHA's rulemaking on indoor air quality, which contains a vast amount of literature as well as perhaps the most recent and comprehensive analyses of ETS issues, many of which are cited herein. • Given the unresolved criticisms of the U.S. EPA Risk Assessment on ETS, not rely on that document and its analyses, but obtain the relevant literature and re-analyze it. That is, Cal/EPA must "start from the beginning." In this regard, the agency should obtain and analyze the underlying data -- particularly the data from the Fontham, et al., study. • Abandon the selective approach taken in the present draft of the Excerpt, and clearly define the criteria used in evaluating data and delineate the rationale for its approaches, in order to reduce the appearance of subjectivity. - 7 -
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SECTION II: CAL/EPA'S REVIEW IS SUBJECTIVE, SELECTIVE AND UNSCIENTIFIC Cal/EPA's Excerpt clearly appears to be designed to support the conclusion that ETS exposure is associated with an increased lung cancer risk. The following discussion will illustrate a number of specific examples of subjectivity and selectivity in the Excerpt. A. Cal/EPA Frequently Engages in Selective Reporting Cal/EPA selectively references studies and reports data from studies in a manner that strengthens its positions. One example of this selectivity is found in the citation of the "first" studies to be published on spousal smoking and lung cancer. Cal/EPA cites only the Hirayama'-and Trichopoulos, et al.,z studies, published in 1981. (p. 8) This statement is incomplete. Also in 1981, Garfinkel published data from a large cohort study conducted in the United States.' Unlike the Hirayama and Trichopoulos studies, Garfinkel's data support no increased lung cancer risk associated with ETS exposure. The failure to include the Garfinkel (1981) study -- whether intentional or an oversight -- gives the appearance that it was omitted because it did not support the theme being developed by Cal/EPA.
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12. Hirayama, T., "Passive Smoking and Cancer: The Association Between Husbands Smoking and Cancer in the Lung of Non-snmoking Wives," in: Indoor Air Quality, ed., H. Kasuga, Berlin, Heidelberg, Springer-Verlag, 299-311, 1990. 13. Hirose, K., et al., "Subsite (Cervix/Endometrium)-Specific Risk and Protective Factors in Uterus Cancer," Japan J Cancer Res 87: 1001-1009, 1996. 14. Holly, E.A., et al., "Characteristics of Women by Smoking Status in the San Francisco Bay Area," Cancer Epidemiol Biomarkers & Prev 1: 491-497, 1992. 15. Hurley, S.F., et al., "Tobacco Smoking and Alcohol Consumption as Risk Factors for Glioma: A Case-Control Study in Melbourne, Australia," J Epidemiol Comm Health 50: 442-446, 1996. 16. Ji, B.T., et al., "Patemal Cigarette Smoking and the Risk of Childhood Cancer Among the Offspring of Nonsmoking Mothers," JNatl Cancer Inst 89: 238-244, 1997. 17. Kilpatrick, S.J., "Re: Prenatal Exposure to Parents' Smoking and Childhood Cancer," Am JEpidemio1135: 712,1992. 18. Kreiger, N., et al., "Risk Factors for Renal Cell Carcinoma: Results of a Population-Based Case-Control Study," Cancer Causes and Control 4: 101-110, 1993. 19. Layard, M., "Environmental Tobacco Smoke and Cancer: The Epidemiologic Evidence," in: Environmental Tobacco Smoke: Proceedings of the International Symposium at McGill University 1989, eds., D. Ecobichon and J. Wu, Lexington, Mass., Lexington Books, 99-115, 1990. 20. Layde, P., "Smoking and Cervical Cancer: Cause or Coincidence?" JAMA 261: 1631-1633, 1989. 21. Lee, P., "Other Cancers," in: Environmental Tobacco Smoke and Mortality, Basel, Karger, 168-183, 1992. 22. Lee, P.N., "Re: Prenatal Exposure to Parents' Smoking and Childhood Cancer," Am J Epidemiol 135: 713-714,1992. 23. Linet, M.S., Gridley, G., Cnattingius, S., Nicholson, H.S., Martinsson, U., Glimelius, B., Adami, H.O., and Zack, M., "Matemal and Perinatal Risk Factors for Childhood Brain Tumors (Sweden)," Cancer Causes and Control 7: 437-448, 1996. 24. Liu, L., et al., "Early Life Experience and Other Risk Factors for Breast Cancer in Chinese Never-Smoking Women," Epidemiology 7(4, Suppl.): S37, 1996. -2-
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25. London, S.J., et al., "Exposure to Residential Electric and Magnetic Fields and Risk of Childhood Leukemia," Am JEpidemio1134: 923-937, 1991. 26. McCredie, M., et al., "Perinatal and Early Postnatal Risk Factors for Malignant Brain Tumours in New South Wales Children," Int JCancer 56: 11-15, 1994. 27. McNeil, C., "Consensus Panel on Cervical Cancer Highlights the HPV Connection," JNatl Cancer Inst 88: 575, May 1, 1996. 28. Munoz, N., et al., "Difficulty in Elucidating the Male Role in Cervical Cancer in Colombia, a High-Risk Area for the Disease," JNatl Cancer Inst 88: 1068-1075, 1996. 29. National Cancer Institute, Office of Cancer Communications, "Abortion and Possible Risk for Breast Cancer: Analysis and Inconsistencies," Press Release, October 26, 1994. 30. Norman, M.A., et al., "Prenatal Exposure to Tobacco Smoke and Childhood Brain Tumors: Results from the United States West Coast Childhood Brain Tumor Study," Cancer Epidemiol Biomarkers & Prev 5: 127-133, 1996. 31. Preston-Martin, S., et al., "Maternal Consumption of Cured Meats and Vitamins in Relation to Pediatric Brain Tumors," Cancer Epidemiol Biomarkers & Prev 5: 599-605, 1996. 32. Ross, J.A., "Epidemiology of Childhood Leukemia, with a Focus on Infants," Epidemiol Reviews 16: 243-272, 1994. 33. Schottenfeld, D., and Fraumeni, J.F., Cancer Epidemiology and Prevention, Second Edition, New York, Oxford University Press, 1997. 34. Schymura, M.J., et al., "A Case-Control Study of Childhood Brain Tumors and Maternal Lifestyle," Am JEpidemiol 143(11, Suppl.): S8, 1996. - 35. Shu, X.O., et al., "Parental Alcohol Consumption, Cigarette Smoking, and Risk of Infant Leukemia: A Childrens [sic] Cancer Group Study," JNatl Cancer Inst 88: 24-31, 1996. 36. Shu, X.O., "A Population-Based Case-Control Study of Childhood Leukemia in Shanghai," Cancer 62: 635-644, 1988. 37. Sorahan, T., et al., "Childhood Cancer and Parental Use of Alcohol and Tobacco," Ann Epidemiol5: 354-359,1995. 38. Sorahan, T., et al., "Childhood Cancer and Parental Use of Tobacco: Deaths from 1953 to 1955," Brit JCancer 75: 134-138, 1997. -3-
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The review of workplace data by Cal/EPA appears to be an attempt by the agency to "make something out of nothing," because, as noted previously, meta-analyses of the workplace data consistently show no association. Cal/EPA's discussion of the epidemiologic data on reported ETS exposures from sources other than the spouse is also flawed, because it relies in large part on only the reported results of the four most recent studies, rather than discussing the data presented in all the studies on this topic. As reported in a recent comprehensive review of the ETS-lung cancer literature, the data on lung cancer risks for non-spousal ETS exposures, taken as a whole, provide "little or no substantial evidence of an association.j26 Cal/EPA's approach to the data effectively ignores such analyses. . B. The Excerpt Contains Misleading Statements O Ow W :- C3• , .p -- W There are a number of instances in the Excerpt where ~ . statements are framed in such a manner as to yield misleading impressions. Cal/EPA must draft its document so that its meaning is clear. For instance, on p. 8, Cal/EPA states: "In order_to gain a more accurate estimate of the association between ETS exposure - 4 -
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95°s confidence interval of 0.95-1.21, is not statistically significant. The inclusion of two additional recent studies in the meta-analysis effectively reversed the conclusion of the U.S. EPA Risk Assessment on ETS. The summary relative risk is no longer statistically significant, and, therefore, does not support a conclusion_of an association between spousal smoking and lung cancer. Gross (1995) noted that when Kabat (1990, an initial report of the study discussed in the Excerpt52), Stockwell, et al.,zz and Brownson, et al.," are included in a U.S. EPA-type -meta- analysis, a summary risk estimate of 1.12 (95% CI 0.99-1.27) is calculated -- prior to adjustment for smoking status misclassification.' Gross states: "Since any such adjustment lowers the relative risk, it implies the null hypothesis of no association cannot be rejected." In another 1995 paper, Gross provides the misclassification-adjusted summary risk estimate; even when the U.S. EPA's very low rate of 1 percent is used, the summary value is 1.07 (95o CI 0.95-1.21).e Similarly, Gori (1994) observes that, while he does not endorse the U.S. EPA approach:9 Including the two latest studies [Brownson and Stockwell] explicitly excluded from the EPA's review and using the EPA's own [adjustment] - 8 -
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and lung cancer, a meta-analysis approach has been used to pool results of comparable studies." (emphasis added) This statement implies that the spousal smoking studies are "comparable," when it has been clearly illustrated that they are not.26 Moreover, it implies that meta-analysis may be appropriately applied to these studies, which is not a universally accepted position.z' On the same page, the Excerpt states: "Most of the individual studies found a small increased risk." (p. 8) Actually, of the 40 studies presently available, l-',11-la,zo-z5,2e-5o only eight report statistically significantly increased overall risk estimates for spousal smoking and lung cancer.1•2,13, s,z5,35,a ,42 The vast majority -- 80 percent -- of the spousal smoking studies report overall risk estimates that are not statistically significant. Without statistical significance, a reportedly increased risk is compatible with the null hypothesis of no association. Thus, Cal/EPA's reference to a "small increased risk" is misleading. Another example of misleading reporting is found in Cal/EPA's treatment of the Fontham, et al., study. Cal/EPA states that cotinine measurements were obtained for 81 percent of self- respondent cases. (p. 15) The choice of this percentage is misleading. Fontham, et al. (1994), state; "Urine samples were analyzed for 356 (53.5%) of 665 cases and 1064 (83.3%) of 1278 controls."1' Thus, cotinine samples were available for only about - 5 -
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Elsewhere, Cal/EPA claims that U.S. EPA's Risk Assessment on ETS is the "most recent" meta-analysis of the_epidemiologic data on ETS exposure and lung cancer. (p. 8) This is inaccurate -- the results of several meta-analyses that fail to support U.S. EPA's conclusions have subsequently been published.4-10 Again, the omission of these references contributes to the impression that Cal/EPA is not presenting a thorough, complete representation of the scientific literature, and is instead focusing only on material that supports its conclusions. Cal/EPA is also guilty of selectively reporting data from individual epidemiologic studies. For example, in the discussion of childhood ETS exposure, Cal/EPA references a reported risk for "high exposure" during childhood of 2.07 (95% CI 1.16-3.68) from the Janerich, et al., study. This is the only statistically significant association reported in that study." Not only could a single statistically significant risk estimate have arisen by . chance, given the number of analyses reported in that paper, but taking that result out of context misrepresents the Janerich, et al., study. Cal/EPA could just as well have highlighted the workplace risk estimate from the Janerich, et al., study, 0.91 (950 CI 0.80-1.04) in the Excerpt's workplace discussion -- but it did not. _a " 'a N ;. _ - 2 -
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C. Cal/EPA Fails to Provide Justification for Considering the Recent Epidemiologic Studies Separately from the U.S. EPA Risk Assessment One of the most curious omissions in the Excerpt is its failure to discuss the outcome of a U.S. EPA-style meta-analysis in which data from the four epidemiologic studies published since 1991 are included. Cal/EPA provides no justification for this omission. Given Cal/EPA's acceptance (and even defense) of the U.S. EPA meta- analysis, the failure to consider a meta-analysis including all the studies can be interpreted as an attempt to exclude an analysis that would not support Cal/EPA's conclusions. A review of the scientific literature reveals several post-EPA meta-analyses conducted by other researchers.°,'-10 These_ meta-analyses illustrate that, if the U.S. EPA's approach is adopted, inclusion of new studies lowers the overall summary risk estimate. For instance, in a recent paper, LeVois and Layard report:' Using the EPA's_ methods and assumptions, we have calculated a summary relative risk of 1.07 from a meta-analysis of 13 U.S. female spousal smoking studies, including these two recent studies [Brownson, et al., and Stockwell, et al.] This relative risk, with W 7
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Similarly, Cal/EPA selectively reports other data on workplace ETS exposures and lung cancer risk. Only two studies have reported workplace risk estimates that are statistically significant."•13 The vast majority of workplace risk estimates are not statistically significant.11•1''25 While this is evident from the table of workplace results in the Excerpt, the text does not make this clear. Nor does the text indicate that recent meta-analyses of the workplace data report a summary risk estimate of approximately 1.0, consistent with no increase nor decrease in risk." Cal/EPA also engages in "data-dredging" in discussing the workplace data. Namely, it creates a subcategory of three studies "in which the assessment of workplace exposure to ETS was complete," (p. 18) and claims that those studies are "generally supportive of an association between workplace ETS exposure and risk of lung cancer." (p. 18) However, Table 7.7 of the Excerpt shows that only one of the three risk estimates, that of Fohtham, et al., is statistically significant. The workplace risk estimates for the other two studies cited to support Cal/EPA's proposition (Wu, 1985, and Wu-Williams, et al., 1990) are not statistically significant, and thus do not clearly support an association, contrary to the Cal/EPA's claim. - 3 -
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For example, Cal/EPA states: "Results from the largest population-based study, conducted in several metropolitan areas of the U.S. (Fontham et al., 1994) were closest to the pooled estimate of the U.S. EPA report." (p. 7) (emphasis added) The subjective phrase "closest to" has no scientific meaning. Used in this manner, it appears to be a transparent attempt to bolster the position that the newer studies support the conclusions of the U.S. EPA Risk Assessment, without conducting a statistical analysis. E. The Excerpt Contains Unreferenced and Poorly Referenced Claims In the Executive Summary of the Excerpt, Cal/EPA claims that there is "compelling biologic plausibility of an effect of ETS on lung cancer." (p. 7) No references are cited in support of this proposition. Thus, the public may take this statement at face value, when the statement is actually not supported by a review of the relevant scientific literature. Neither animal inhalation studies nor genotoxicity studies provide support for the claim, as discussed elsewhere in this submission (Section III). Similarly, Cal/EPA provides only one reference for its claim that "there is no publication bias against statistically nonsignificant results on ETS in the peer-reviewed literature." (p. 10) The issue of publication bias cannot be summed up by a - 11 -
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was chosen instead of incorporating the new studies into a meta- analysis or other, more formal, comparison. Cal/EPA should also provide further explanation for its position that the recent studies support the U.S. EPA's conclusion, given that the Congressional Research Service reached the following conclusion, based on a review of the same materia1:53 The new studies, including the very large Brownson study, did not clarify_the existence of a risk. Indeed, they complicated the interpretation of the evidence, since the two largest U.S. studies -- Fontham and Brownson - - found in one case a positive risk that was barely statistically significant and the other no risk at all. (p. 25) D. Subjective Terms Are Used Throughout the Excerpt Without Definition Throughout the Excerpt, Ca1/EPA_ employs terms like "concordance" (p. 7), "very similar to" (p. 7), "closest to" (p. 7), "consistency" (p. 27), "similar" (p. 14), and "compatible" (p. 19) when discussing the results of two or more studies or analyses. Because these terms are not defined, their scientific meaning is unknown to readers of the Excerpt, who are therefore left with the impression that these terms are subjective descriptions without a rigorous scientific basis. - 10 -
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half of all the cases in the study, according to the original report. Here, Cal/EPA's choice of the_value to cite seems_ to be intended to portray the Fontham, et al., study's_methods_ in the most favorable light. In another section discussing the Fontham, et al., study, Cal/EPA states: "[T]he U.S. multicenter study corroborated the subjects' self-reported current nonsmoking status using the urinary cotinine level." (p. 21) While Cal/EPA acknowledges that cotinine measurements assess only current smoking, it accepts the misclassification rates of 0.6 percent in cases and 2.3 percent in controls reported by Fontham, et al., as accurate. The Excerpt does not mention that Fontham, et al., measured cotinine in hospitalized cases. The vast majority of hospitals severely restrict smoking (in fact, accreditation requires that smoking be banned) ; moreover, many lung cancer patients who happen to be smokers stop smoking after diagnosis. Thus, the cotinine measurements in this study do not even give a good indication of present smoking status, let alone previous long-term smoking status. The smoking status misclassification rates portrayed by Fontham, et al., as accurate are, in reality, not representative of the true situation. As reported by Lee and Forey, a review of_the literature indicates that smoking status misclassification rates may range as high as 15 to 20 percent.51 - 6 -
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procedure, a realistic correction [for misclassification] of only 2.5% would nullify any excess risk estimates in the meta-analysis of the 13 available U.S. studies of spousal exposure. Gori states that the published literature suggests smoking status misclassification rates between two and 10 percent (average four to five percent), compared to the 1.09 percent used by U.S. EPA.9 Sugita, et al., also reanalyzed the U.S. spousal smoking data, incorporating the Stockwell, et al., and Brownson, et al., studies, and calculated a summary risk estimate for the U.S. studies of 1.10 (95% CI 0.97-1.26).10 Based on this and other summary risk estimates calculated by Sugita, et al., they conclude: From these odds ratios it has not been demonstrated scientifically whether or not ETS is a particular risk factor for lung cancer. The conclusion that the relationship between exposure to ETS and lung cancer is weak remains unchanged. (p. 180) It seems clear that Cal/EPA facilitated its arguably predetermined conclusion by simply making observations about the "concordance" or "similarity" of the recent studies to U.S. EPA's conclusions, rather than by conducting its own statistical analysis. Cal/EPA should explain why the approach of comparing the individual post-1991 studies to the U.S. EPA summary risk estimate - 9 -
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ratios [in this study] shows little indication of an association of environmental tobacco smoke with lung cancer in nonsmokers."1' After noting that "[t]here were no significant associations between spouses' smoking and risk of lung cancer in male or female subjects" in the original report of the Kabat, et al., study, Cal/EPA states: "We calculated the OR for lung cancer in males and females combined to be 1.19 (95% CI=0.76-1.87) in association with spousal ETS exposure." (p. 18) (emphasis added) Cal/EPA then summarizes its conclusions: "Although [Kabat et al.] ... interpreted their findings (analyzing men and women separately) to be unsupportive of an association between ETS exposure and risk of lung cancer, the odds ratio we calculated from their results, though not statistically significant, was in fact very similar to the pooled estimate from the U.S. EPA report." (p. 19) m. m Because the author gives no statistical or scientific ~_- justification for combining the Kabat, et al., data for men and women, this approach appears to be an attempt to "make the data fit." The decision to combine the male and female data is even more curious when one considers that the U.S. EPA Risk Assessment based its conclusions on data for women only, completely ignoring the available data on males, which, taken as a whole, support no increase in lung cancer risk in men whose wives smoked.26 Moreover, - 13 - -
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single reference; Cal/EPA should review additional literature on this issue, including the literature on meta-analysis, where much of the debate on publication bias has developed. Moreover, Cal/EPA does not address the issues of selective reporting and "data-dredging," another form of bias common in epidemiologic studies. For instance, the Fontham, et al., study reports more than 100 case-control comparisons.13 With this many comparisons, a number of statistically significant associations would arise by chance alone. This issue is not adequately addressed in the original studies, nor in the Excerpt. F. Cal/EPA's Reanalysis of Kabat, et al. (1995), Is Not Justified Cal/EPA's analysis of the 1995 Kabat, et al., case- control study" certainly gives the appearance ofa a bias toward reporting results that support the position that the recent ETS epidemiologic studies support the conclusions of the 1993 U.S. EPA Risk Assessment on ETS. In fact, the approach taken by Cal/EPA has already been deemed "controversial" by observers.54 The risk estimates based on the Kabat, et al., data, as originally reported in the publication, do not agree with Cal/EPA's position. As Kabat and colleagues stated: "[T]he pattern of odds - 12 - -- - N
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[0]ur study and others conducted during the past decade suggest a small but consistent elevation in the risk of lung cancer in nonsmokers due to passive smoking. According to a scientist who has reviewed and reanalyzed the Brownson publication and the raw data from the study, "influences directed to the identification and reporting of an association between ETS [exposure] and lung cancer may have affected the reporting of the results" from this study and others.ss G. Conclusion As the lung cancer Excerpt is written, it appears to be an attempt by Cal/EPA to make recent data agree with the conclusions of the U.S. EPA Risk Assessment on ETS. The selectivity and subjectivity of the Cal/EPA discussion lead to conclusions which simply are not supported by the data and objective, thorough scientific analyses.
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the Excerpt implies that there is some importance in the reanalysis of the Kabat, et al., yielding a risk estimate of 1.19, the same as the U.S. EPA summary risk estimate, when this simple coincidence has little, if any, real meaning. The interpretation by Kabat, et al., that their data do not support an association between ETS exposure and lung cancer risk is wholly appropriate. Kabat and colleagues report a number of risk estimates that do not achieve statistical significance_ They also exercise caution in interpreting the few statistically _ significant risk estimates in their study. However, Cal/EPA does not simply accept this interpretation, apparently since it does not support Cal/EPA's position. Interestingly, Cal/EPA makes no comment about the unsupported interpretation by Brownson, et al., of the data in their case-control study.l' et al., An examination of the reported results in the Brownson, study shows that the overall risk estimate for spousal smoking is 1.0 (95e CI 0.8-1.2), clearly inconsistent with an N O m increase in risk. The only statistically significant results in w ff .A w ~ the Brownson paper were restricted to "qualitative" indices of m exposure, where study subjects provided an "estimate [of] a perceived level" of exposure.l' Despite the vast majority of the data being consistent with no increase in risk, Brownson, et al., concluded: - 14 -
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submissions were made to the U.S. EPA public comment docket. The numerous criticisms of the 1990 draft were summarized in a document prepared for the Agency, which stated:z The predominant theme in comments related to lung cancer is that the classification of ETS as a Group A carcinogen, causally related to increased risk of lung cancer, is unwarranted. . The claim that it is biologically plausible that ETS is a lung carcinogen is contentious and unjustified in the Report. (pp. 3-4) it is not possible to summarize comprehensively in just a few pages the remarks from the thousands of pages of comments submitted. (p. 4) The criticisms -- on toxicological, chemistry, exposure, epidemiologic and statistical issues -- raised with respect to the 1990 draft of the U.S. EPA Risk Assessment were not adequately addressed in the revised draft that was issued in 1992. At that time, comments were again submitted by a number of independent scientists and other interested parties. In addition to reiterating many of the criticisms initially raised, and confirming that those criticisms were not adequately addressed in the new draft, the comments also raised new issues related to the second review draft. Following the release of the final document in January 1993, essentially identical to the heavily criticized 1992 draft, - 2 - N '
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13. Fontham, E., et al., "Environmental Tobacco Smoke and Lung Cancer in Nonsmoking Women: A Multicenter Study," Journal of the American Medical Association 271(22): 1752-1759, 1994. 14. Brownson, R., et al., "Passive Smoking and Lung Cancer in Nonsmoking Women," American Journal of Public Health 82: 1525-1530, 1992. 15. Butler, T., The Relationship of Passive Smoking to Various Health Outcomes Among Seventh-Day Adventists in California, Ph.D. Thesis, University of California, 1988. 16_ Garfinkel, L., et al., "Involuntary Smoking and Lung Cancer: A Case-Control Study," Journal of the National Cancer Institute 75(3): 463-469, 1985. 17. Kabat, G.C., et al., "Relation Between Exposure to Environmental Tobacco Smoke and Lung Cancer in Lifetime Nonsmokers," Am J Epidemiol 142: 141-148, 1995. 18. Kalandidi, A., et al., "Passive Smoking and Diet in the Etiology of Lung Cancer Among Non-Smokers," Cancer Causes and Control 1: 15-21, 1990. 19. Koo, L., et al., "Is Passive Smoking an Added Risk Factor for Lung Cancer Among Chinese Women?," Journal of Experimental and Clinical Cancer Research 3(3): 277-283, 1984. 20. Lee, P., et al., "Relationship of Passive Smoking to Risk of Lung Cancer and Other Smoking-Associated Diseases," British Journal of Cancer 54: 97-105, 1986. 21. Shimizu, H., et al., "A Case-Control Study of Lung Cancer in Nonsmoking Women," Tohoku Journal of Experimental Medicine 154: 389-97, 1988. .A 22. Stockwell, H., et al., "Environmental Tobacco Smoke and Lung Cancer Risk in Nonsmoking Women," Journal of the National Cancer Institute 84(18): 1417-1422, 1992. 23. Wu, A., et al., "Smoking and Other Risk Factors for Lung Cancer in Women," Journal of the National Cancer Institute 74(4): 747-751, 1985. 24. Wu-Williams, A., et al_, "Lung Cancer Among Women in North- East China," British Journal of Cancer 62: 982-987, 1990. - 17 -
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38. Inoue, R. and Hirayama, T., "Passive Smoking and Lung Cancer in Women," Smoking and Health 1987, eds., M. Aoki, et al., Amsterdam, Excerpta Medica, 283-285, 1988. 39. Joeckel, K.H., "Passive Smoking -- Evaluation of the Epidemiological Findings,11 VDI Reports 888: Carcinogenic Substances in the Environment, VDI Verlag, 1991. 40. Koo, L., et al., "Measurements of Passive Smoking and Estimates of Lung Cancer Risk Among Non-Smoking Chinese Females," International Journal of Cancer 39: 162-169, 1987. 41_ Lam, T., et al., "Smoking, Passive Smoking and Histological Types in Lung Cancer in Hong Kong Chinese Women," British Journal of Cancer 56(5): 673-678, 1987. 42. Lam, W., A Clinical and Epidemiological Study of Carcinoma of Lung in Hong Kong, M.D. Thesis Submitted to the University of Hong Kong, 1985. See also: Lam, T. and Cheng, K., "Passive Smoking Is a Risk Factor for Lung Cancer in Never Smoking Women in Hong Kong," Smoking and Health 1987, eds., M. Aoki, et al., Amsterdam, Excerpta Medica, 279-281, 1985. 43. Lan, Q., et al., "Risk Factors for Non-Smokers in Xuanwei County of China," Biomedical and Environmental Sciences 6: 112-118, 1993. 44. Layard, M.W., "Ischemic Heart Disease, Lung Cancer, and Spousal Smoking in the National Mortality Followback Survey," Addendum to Comments on the OSHA Proposal for Indoor Air Quality Standards, OSHA Docket H-122, Ex. 10-219, 1994. 45. Liu, Q., et al., "Indoor Air Pollution and Lung Cancer in . Guangzhou, People's Republic of China," American Journal of Epidemiology 137: 145-154, 1993. 46. Liu, Z., et a1. , "Smoking and Other Risk Factors for Lung Cancer in Xuanwei, China," International Journal of Eoidemiologv 20(1): 26-31, 1991. 47. Pershagen, G., et al., "Passive Smoking and Lung Cancer in Swedish Women," American Journal of Epidemiology 125(1): 17-24, 1987. 48. Sobue, T., et al., "Passive Smoking Among Nonsmoking Women and the Relationship Between Indoor Air Pollution and Lung Cancer - 19 -
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REFERENCES 1. Hirayama, T., "Non-Smoking Wives of Heavy Smokers Have a Higher Risk of Lung Cancer: A Study from Japan," British Medical Journal 282: 183-185, 1981. 2. Trichopoulos, D., et al., "Lung Cancer and Passive Smoking," International Journal of Cancer 27(1): 1-4, 1981. 3. Garfinkel, L., "Time Trends in Lung Cancer Mortality Among Nonsmokers and a Note on Passive Smoking," Journal of the National Cancer Institute 66(6): 1061-1066, 1981. 4. LeVois, M.E., and Layard, M.W., "Inconsistency Between Workplace and Spousal Studies of Environmental Tobacco Smoke and Lung Cancer," Reg Tox and Pharm 19: 309-316, 1994. 5. Biggerstaff, B.J., et al., "Passive Smoking in the Workplace: Classical and Bayesian Meta-Analyses," Int Arch Occub Environ Health 66: 269-277, 1994. 6. Tweedie, R.L., and Mengersen, K.L., "Lung Cancer and Passive Smoking: Reconciling the Biochemical and Epidemiological Approaches," Br J Cancer 66: 700-705, 1992. 7. Gross, A.J., "The Risk of Lung Cancer in Nonsmokers in the United States and Its Reported Association with Environmental Tobacco Smoke," Journal of Clinical Epidemiolocxv 48: 587-598, 1995. 8. Gross, A.J., "Uncertainties in Lung Cancer Risk Estimates Reported for Exposure to Environmental Tobacco Smoke," Environmetrics 6: 403-412, 1995. 9. Gori, G.B., "Reply to_the Preceding Dissents," Journal of Clinical Epidemiolocrv 47: 351-353, 1994. 10. Sugita, M., Izuno, T., and Kanamori, M., "Recalculation of Summarised odds Ratios for the Relationship Between Passive Smoking and Lung Cancer Based on Data in the EPA Report," Indoor Environment 4: 177-181, 1995. 11. Janerich, D., et al., "Lung Cancer and Exposure to Tobacco Smoke in the Household," New Enaland Journal of Medicine 323: 632-636, 1990. 12. Kabat, G. and Wynder, E., "Lung Cancer in Nonsmokers," Cancer 53(5): 1214-1221, 1984. - - - - 16 -
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Incidence -- Results of a Multicenter Case Controlled Study," Gan to Rinsho 36(3): 329-333, 1990. 49. Svensson, C., et al., "Smoking and Passive Smoking in Relation to Lung Cancer in Women," Acta Oncologica 28(5): 623-629, 1989. 50. Wang, F.-L., et al., "Childhood and Adolescent Passive Smoking and the Risk of Female Lung Cancer," International Journal of Epidemioloav 23(2); 223-230, 1994. 51. Lee, P.N., and Forey, B.A., "Misclassification of Smoking Habits as Determined by Cotinine or by Repeated Self-Report -- A Summary of Evidence from 42 Studies," J Smoking-Related Dis 6: 109-129, 1995. 52. Kabat, G., "Epidemiologic Studies of the Relationship Between Passive Smoking and Lung Cancer," Toxicolocrv Forum: 1990 Annual Winter Meeting, Washington, D.C., 187-199, February 19- 21, 1990. 53. Redhead, C.S., and Rowberg, R.E., "Environmental Tobacco Smoke and Lung Cancer Risk," CRS Report for Congress, Congressional Research Service, Library of Congress, November 14, 1995. 54. Lovett, R., "OEHHA Releases ETS Lung Cancer Chapter," Prop 65 News, March 1996, p. 5. 55. Butler, W.J., "Re-Analysis of Data Provided in Fontham et al. (1994) : No Increase in the Risk of Lung Cancer Associated with Adult ETS Exposure," OSHA Docket H-122, Ex. 537, 1996.
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25. Zaridze, D.G., and Zemlyanaya, G.M., "Indoor Air Pollution and Lung Cancer Risk in Non-Smoking Women in Moscow," Experimental Oncolow 16: 441-445, 1994. 26. Lee, P.N., Environmental Tobacco Smoke and Mortalitv, Basel, Karger, 1992. 27. Fleiss, J.L., and Gross, A.J., "Meta-Analysis in Epidemiology, With Special Reference to Studies of the Association Between Exposure to Environmental Tobacco Smoke and Lung Cancer: A Critique," J Clin Epidemiol 44: 127-139, 1991. 28. Akiba, S., et al., "Passive Smoking and Lung Cancer Among Japanese Women," Cancer Research 46: 4804-4807, 1986. 29. Brownson, R., et al., "Risk Factors for Adenocarcinoma of the Lung," American Journal of Epidemiology 125(1): 25-34, 1987. 30. Buffler, P., et al., "The Causes of Lung Cancer in Texas," Lung Cancer: Causes and Prevention, eds., M. Mizell and P. Correa, Deerfield Beach, Verlag Chimie International, 83-99, 1984. 31. Chan, W. and Fung, S., "Lung Cancer in Non-Smokers in Hong Kong," Cancer Campaign Vol. 6, Cancer Epidemioloav, ed., E. Grundmann, Stuttgart, Gustav Fischer Verlag, 199-202, 1982. 32. Correa, P., et al., "Passive Smoking and Lung Cancer," Lancet II: 595-597, 1983_ 33. Du, Y., et al., "Exposure to Environmental Tobacco Smoke and Female Lung Cancer," Indoor Air 3: 231-236, 1995. 34. Gao, Y., et al., "Lung Cancer Among Chinese Women," International Journal of Cancer 40: 604-609, 1987. 35. Geng, G., et al., "On the Relationship Between Smoking and Female Lung Cancer," Smoking and Health 1987, eds., M. Aoki, et al., Amsterdam, Excerpta Medica, 483-486, 1988. 36. Hole, D., et al., "Passive Smoking and Cardiorespiratory Health in a General Population in the West of Scotland," British Medical Journal 299: 423-427, 1989. 37. Humble, C. , et al., "Marriage to a Smoker and Lung Cancer Risk," American Journal of Public Health 77(5): 598-602, 1987. - 18 -
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a number of articles critical of the Risk Assessment have appeared in the scientific press.3-9 For instance, Huber and colleagues (1993) wrote:' [T]he EPA's risk assessment is built on the manipulation of data, ignores critical chemical analyses and key epidemiological data, violates time-honored statistical principles, fails to control adequately for important confounding influences (factors other than the one studied that may affect a result or conclusion) that provide alternative explanations for its conclusions, and violates its own guidelines for assessing and establishing risk to a potential environmental toxin. It lacks credible quality control and adequate external unbiased peer review. In short, in its report on ETS, the EPA did not comply with accepted principles of toxicology, chemistry, and epidemiology, nor with its own guidelines for undertaking cancer risk assessment. In fact, the conclusions drawn by the EPA are not even supported by the EPA's own statements. (p. 45) (emphasis added) In a 1994 commentary on the U.S. EPA Risk Assessment, Gori stated:' [T]his figure [3,000 deaths] is . . . the result of an unprecedented exercise in data o manipulation. Among other unjustifiable a, _ gambits, this EPA report stands out for its Ca unorthodox insistence on one-tailed statistics and 90% confidence intervals, for arbitrary and unproven adjustment procedures, and for a its selective use of epidemiologic evidence. w For instance, without explanation the report has summarily excluded workplace data and the latest epidemiologic studies, which together void the report's conclusions when added to the body of evidence. (emphasis added) - 3 -
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nonsmoking areas of workplaces and public places and in homes with or without smokers.1s,27,30,31,99,59,so Similarly, levels of volatile organic compounds such as formaldehyde and benzene in the presence of smoking are often indistinguishable from levels reported in nonsmoking areas.ls,az,3s,so-sz Studies that have examined ETS constituent levels of nitrosamines also report minimal contributions to overall ambient air levels in homes, offices and public places.4e,",sz - - - - D. Limitations of Exposure Estimates Based on Questionnaires Were Not Discussed by U.S. EPA The "exposure" estimates in epidemiologic studies on ETS are wholly independent of a large body of data on ambient exposure measurements to various constituents of ETS, which, taken as a whole, suggests that ETS exposures are minimal. The epidemiologic studies rely on questionnaire data, the accuracy of which depends on an individual's ability to recall past events, such as how much a husband smoked in the past 20 to 30 years.a3-ss The two bodies of data, epidemiologic/health effects, on the one hand, and exposure measurements on the other, have never been.integrated into one comprehensive study. - In a 1994 article critical of the U.S. EPA Risk Assessment, Gori wrote: - 10 -
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Furthermore, in another discussion published in 1995, Gori observed:s EPA claims to have used a weight of evidence approach. In reality, ETS has been characterized only indirectly by an analogy to active smoking that EPA itself discounts. Negative epidemiologic reports were summarily ignored. Statistical standards were relaxed. Competing and well-known risk factors were disregarded. Documented misclassification biases were discounted. By its own admission, EPA made very selective use of available studies, emphasizing only those that support its preconceived conclusions. (pp. 20-21) (emphasis added) In a technical analysis, Gross (1995) illustrated clearly that the U.S. EPA methodology for calculating deaths_purportedly attributable to ETS exposure "is far from an exact or even approximate science. ...[M]inor to moderate changes in the model v 0 m parameters lead to large changes in the estimated number of W 01 ~ [deaths]."6 (p. 411) (emphasis added) Further, he stated that "EPA has not given very much thought to the uncertainty of its estimates," and noted that the epidemiologic data on ETS and lung cancer are consistent with a risk estimate of 1.0, which, in U.S. EPA's model, would yield an estimate of zero "attributable" deaths. More recently, the Congressional Research Service (CRS) has issued two reports that raise substantial questions about the- - 4 - ' 10 , 4~
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SECTION III: CAL/EPA RELIES INAPPROPRIATELY ON THE U.S. EPA RISK ASSESSMENT ON ETS, WITHOUT SUFFICIENT ATTENTION TO THE DEMONSTRATED FLAWS IN THAT DOCUMENT Cal/EPA relies extensively on the 1993 U.S. EPA Risk Assessment on ETS, which purportedly reviewed the literature on ETS and lung cancer through 1991.1 Cal/EPA has apparently accepted the U.S. EPA Risk Assessment as conclusive. While mention is made of certain criticisms of the U.S. EPA document (e.g., pp. 9-10), Cal/EPA simply lists those criticisms, without providing sufficient rebuttal. Cal/EPA sums up its discussion of the U.S. EPA Risk Assessment by stating: "[T]he U.S. EPA's report is based on the total weight of evidence, and not on an individual study." (p. 10) The following discussion will show that (i) the criticisms leveled against the U.S. EPA report are much more severe than the superficial treatment by Cal/EPA would imply, and (ii) U.S. EPA omitted a large amount of relevant data and information in reaching its conclusions about ETS, so that the "weight of the evidence" claim is misleading. A. Criticisms of the U.S. EPA Risk Assessment Have Never Been Adequately Addressed by the Agency Criticisms have been raised about the U.S. EPA's Risk Assessment since the initial release of a draft for public review in 1990. At that time, independent scientists and interested parties raised concerns about many important issues; over 100
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differences" among the three kinds of smoke (mainstream smoke, sidestream smoke and ETS).13 Studies also indicate that constituents in ETS are hundreds to thousands of times more dilute than either sidestream or mainstream smoke.16•1'- Concentrations of ETS- constituents in real-life situations are often below the limits of detection and measurement for even the most sensitive air monitors_ Often, the contributions of ETS constituents to the ambient air are indistinguishable from background levels of the same constituents generated by other sources.ls The strategy of comparing mainstream and sidestream smoke employed by U.S. EPA in_the Risk Assessment (e.g., Chapter 3) ignores the profound effect of dilution in the ambient air upon tobacco smoke constituents. As two tobacco smoke chemists report:.l' The important question is not the ratio of sidestream/mainstream but rather what is the concentration of the constituent in the indoor environment and how does it compare to levels from sources other than ETS. Studies based solely on observations of fresh sidestream, or highly and unrealistically concentrated ETS should take into account the possible differences between these smokes and ETS found in real life situations. Similarly, the 1986 Report of the Surgeon General notes:__ "SS _ [sidestream smoke] characteristics, as measured in chambers, do not - 6 -
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U.S. EPA Risk Assessment.lo•11 In 1994, one of the authors of the 1994 CRS review, Jane Gravelle, described the conclusions of that document as follows:12 [0]ur evaluation was that the statistical evidence does not appear to support a conclusion that there are substantial health effects of passive smoking. (emphasis added) A compilation of materials criticizing or questioning the U.S. EPA Risk Assessment is submitted as Appendix I to_ this comment. B. Data on ETS Chemistry Were Omitted from the U.S. EPA Risk Assessment The U.S. EPA Risk Assessment failed to discuss or even to reference a significant amount of relevant literature on the physical and chemical properties of ETS. A number of scientists have concluded that ETS is not the same as either mainstream or sidestream smoke.l'-1' ETS is an aged and diluted mixture of sidestream and exhaled mainstream smoke; ETS is a dynamic, ever-changing mixture that undergoes chemical transformations and physical changes as it_ ages and is diluted in the air.l'-15 As one researcher in tobacco smoke chemistry has observed, "there are profound physical and quantitative chemical - 5 -
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exposure as "none or low." Similarly, 30 to 40 percent of individuals married to smokers were actually exposed to less than the average ETS levels measured among subjects with a nonsmoking partner. The researchers conclude: "Clearly, spousal smoking status would not be a reliable means to assess the ETS exposure of individuals or small groups of subjects."'0 Thus, °marriage to a smoker" may -- or may not -- imply exposure to ETS; it cannot serve as an accurate, quantitative surrogate for actual exposure measurements. 2. Attempts to "Refine" the Spousal Smoking index Do Not Yield Better Data In attempting to "refine" the spousal smoking index, a number of spousal smoking studies have employed exposure estimates beyond the simple index of "marriage to a smoker."65•'1 Questions regarding ETS exposures have included those related to the intensity of exposure (e.g., the number of,smokers in the home or the number of cigarettes smoked per day or per year), or the duration of exposure (e.g., hours of exposure, years of marriage to a smoker or years of smoke exposure).71 Estimates of the intensity or duration of spousal smoking exposure are derived by questionnaire or interview of the study subject (the case) or next of kin.7z - 13 -
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retest estimates of reliability suggest that misclassification of such exposure may be extensive. In "An Assessment of the Validity of Questionnaire Responses Provided by a Surviving Spouse,"'s Lerchen and Samet report that wives generally agree with the smoking status classification given by their spouse, but that 44 percent of the spouses could not provide a detailed smoking history of the spouse. The authors observed that wives tended to report that their spouse smoked 20 cigarettes per day, even when the husbands reported that they smoked substantially more or less. The researchers remarked that "the validity of such surrogate information, when available, is uncertain." Kolonel, et al., reported similar results in 1977.8' Sandler and Shore investigated the degree of agreement on parental smoking status from interviews of parents and their (adult) children." These authors reported reasonable agreement in responses about the smoking status of the parent, but agreement on exposure level estimates was extremely poor. The authors conclude that childhood ETS exposure information obtained via questionnaire from adults "cannot readily be used to estimate levels of exposure" to ETS, and that "data on levels of exposure were not of high enough quality to allow for detailed evaluation of dose-response."" - 16 -
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represent those of ETS, as inhaled by the nonsmoker under non- experimental conditions."le C. Data on Measurements of ETS Exposure Were Omitted from the U.S. EPA Risk Assessment Cal/EPA focuses heavily on the epidemiologic literature in the Excerpt. In failing to mention the literature on issues of ETS exposure, Cal/EPA seems to be implying that the U.S. EPA Risk Assessment provides sufficient discussion of these issues, which is not the case. The initial public review draft for the U.S. EPA Risk Assessment (1990) did not even contain an exposure assessment. Comments submitted to the public docket for the 1990 draft risk assessment observed that U.S. EPA had failed to provide an exposure assessment that considered data from the numerous published studies on actual levels of ETS constituents in the air of public places and workplaces. In-apparent response to that criticism, the ra:-- revised 1992 draft of the U.S. EPA Risk Assessment contained a o_:.. w chapter entitled "Estimation of Environmental Tobacco Smoke .a w - Exposure," but the chapter's authors failed to consider at least 35 .A w 11 pertinent exposure studies on ETS constituent levels in public places. - 7
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A major limitation of epidemiologic studies on ETS has been the unreliable estimates of dose, which compound the uncertainties of personal or proxy recall of the intensity, frequency, and duration of exposures over individual lifetimes. Even the simple dichotomous classification of exposed and non-exposed subjects presents recognized uncertainties, such as those deriving from the self- classification of some smokers as non-smokers. (p. 327) U.S. EPA relied heavily on the epidemiologic data in its Risk Assessment on ETS, without adequately exploring the limitations of questionnaire data. - 1. "Spousal Smoking" Is Not Equivalent to ETS Exposure A fundamental problem with Cal/EPA's Excerpt and with U.S. EPA's Risk Assessment is the uncritical acceptance of the risks reported in spousal smoking studies as true risks due to exposure to ETS. The central question is whether or not "ETS exposure" is the same thing as "spousal smoking"; that is, does living with a smoker imply exposure to ETS? Even though U.S. EPA concedes that spousal smoking status is a "crude measure" of ETS exposure and one that is "prone to exposure misclassification,"66 the Agency nevertheless equates spousal smoking status with household exposures to ETS.6" However, the extent to which the spousal smoking index is an accurate, - 11 -
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This means that the typical nonsmoking worker would have to spend from 200 to more than 1,000 hours in an office, restaurant or public place in order to be exposed to the nicotine equivalent of a single cigarette.3s,s5-5s - The cigarette equivalent approach has been criticized because different results would be obtained if different reference compounds are chosen. These criticisms are based simply on calculating the sidestream/mainstream ratios for the different reference compounds. Such a calculation would be accurate if there were no chemical and physical changes which occur when sidestream smoke is transformed into ETS. This, as has already been clearly pointed out earlier in this submission, is not the case. As a consequence, it would not be at all correct to state that a typical nonsmoking worker who spends 200 to 1,000 hours in an office, restaurant or public place is exposed to the smoke equivalent of one cigarette, since ETS is not at all chemically or physically identical to the smoke to which a smoker is exposed. With regard to the calculation of_cigarette equivalents based on other reference compounds, it is important to note that the vast majority of ETS constituents are found, generally in much greater amounts, in indoor environments in the absence of ETS. Researchers report that there is little difference in ambient levels of..carbon monoxide or nitrogen oxides in smoking and - 9 -
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Furthermore, during its review of the chapter in July of 1992, the U.S. EPA Science Advisory Board rejected the chapter and returned it to the author for rewriting. Nevertheless, without either an exposure assessment or recourse to any of the data pertaining to it, the Science Advisory Board endorsed U.S. EPA's estimate of exposure and risk for the entire U.S. population. A revised chapter on ETS exposure occurs in the final U.S. EPA Risk Assessment; however, the studies and data therein are not integrated into the U.S. EPA Risk Assessment. Only a small number of actual ETS exposure studies available in the published literature are even discussed in the U.S. EPA Risk Assessment. Many of the studies of exposure to ETS constituents under realistic conditions in public places, workplaces and homes omitted from the U.S. EPA Risk Assessment report minimal exposures to ETS; these reported exposures do not support the conclusions of the U.S. EPA Risk Assessment.1'.19-s2 ~ 0 For instance, studies on ambient measures of nicotine in w cr r__;. 4~- offices and restaurants report average levels of approximately 2.0 W .~. : ~ and 3.5 ug/m3 nicotine, respectively. These exposures are equivalent to 1/400 to 1/200 of the nicotine found in a single cigarette. Averages for nonsmoking areas in_workplaces with smoking restrictions are even lower, averaging less than 1 ug/m' nicotine, or about 1/1,000 of the nicotine in a single cigarette. - 8 -
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reliable and valid quantitative indicator of ETS exposure was not assessed by U.S. EPA in its Risk Assessment. The quality of the underlying exposure data from the spousal smoking studies is thus of critical importance for epidemiologic studies on ETS and chronic disease, because "the accuracy of any statistical analysis is limited by the accuracy of the information upon which it is based. "se The spousal smoking index is not an accurate marker for exposure to ETS. That "marriage to a smoker" is not equivalent to home exposures to ETS has been demonstrated in two studies. 69,70 Friedman, et al., polled nearly 38,000 nonsmokers and ex-smokers in California about ETS exposure. Nearly 35 percent of respondents classified by their spouse's smoking status reported no exposure overall to ETS; 47 percent of women married to smokers reported zero hours of exposure at home.69 The researchers concluded that "using the spouse's smoking status to classify persons resulted, as far as can be discovered with our relatively crude questionnaire, in a considerable amount of [exposure] misclassification."69 A 1994 published study conducted in the U.K. reports a similar conclusion.'0 The researchers compared subjective estimates of ETS exposure with objective measurements of airborne ETS markers via personal monitors (breathing zone measurements) . They reported that 45 percent of subjects with a smoking partner assessed their - 12 -
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196 Eur. J. Drug Maab. Phm7nacokiner., 1993, No. 2 REFERENCES 1. GaxodJ.W,JeancP.(1975):ThetmtabeBmoftob.ao alknloidt. In: Hayes WJ, ed. Eaays in Teocicology, VoL 6. New Yode Acadomic Prns, pp. 35-78. 2. Schievelbein H. (1982) : Ncotine, resorption and fate. Pha®awL Ther, 18, 233-7A8. 3. Nahyama Ii. (1988) : Nicotine metabolism in .++.mm+t• Drug Metab. Drug Intecact, 6, 95-122. 4. Benowitz Nl.. Potchet H- Janob P. (1990) : Pbvm.ookinetia, metsbolism, and phamaoodyoamics of oimtiue. Im Wonm.co¢ S. Rutull MA.Ii~ Stolemmdi LP., eda. Nicotine Psyahophemscelogy, Moleculay Celbda<, and Behaviounl Aspects. Gzfaad: University Pmn, pp. 112-157. 5. Kyencrusum G.A~ VeseB ES. (1991): Niambolism of nicotine. Drug Metab. Rev., 23. 3-41. 6. Jeaoer P. Gonod 1.W. Becix¢ AA. (1973) : Species vciation in the metabolism of R-(+)- and S{-}aiwtmc by c.C- and No:udation in vitm. Xembiotiea, 3, 573-580. 7. Narosu C.G., Oneks PA. (I988) : Species vmetion and V, cava in rats f« longtime infnsioua (Implmte5x-R). Z Vasudsaie&d, ]2,17I-178. 21. Omdy K.C, Cmo1v PA. (1984) :1Bgh-perk®aax liquid cln+am.to$>Phie method far the det®mation of N-methylated metabolitn of nicotine. 1. Oaumamg. 306, 291-301. 22. Demetcou D. Bua®eierP, VoaoYm P, Schepea 0. (I992) :'lWo xadiadaommgrythie HPLC mednds for tlx defean®[im of nieetiae metabolifts. Med. Sci. Res, 20, 873-875. 23. Kynemafea GA., D.miano MD, Dvotdulc BH. VcseB E-S. (1982) : Smol®g-iadund thffiges in nimt'tne disposi6on: appliatiaa of a new IPLC assay fQ nimtioe and its met.lnliUML Clin. PbamnmL, 32, 769-780. 24. Jmner P, Gonad J.W., Baetnn A.I-L (1973) : The absorption of nicrotiml'-N-nxide md its reduction in the gasno-iucertiasl nx% in man. Xembioua, 3, 341-349. 25. Daj.ni RM, Garecd J.W., Betten Ax (1975) : Reduction in vivu of (-)aicatino-l'-N-acide by genn-free and ormveatiooal rats Biocrm Ph®ecoL,l.i, 648-650. 26. Dusn MJ., Yu L, Savaaepcdi C, Jamb III P., Beaowi2 NJ_ (1991) : Disposition 1®etio and metabolism of nicell'-N-oxide in sabbita. Drug Mateb. Dispos, 19, . steteoselodivity in the metabolism of nicotine earotiamrs. Xenobiotio, 18,1361-1372. 8. Cuody K.C., C:oolu PA., Godin CS. (1985) : Remakable substcsu-inlubitor properties of nimtine enantiean: towecds a gui- pig lung aaumatic ambetancyr7e N-mcihylb-f-s. Biochem. Biophys. Brs. Cnmmoa,128, 312-316. 66717Z 27. SepEovie D.W., Hdey NJ. (1987) : Metafi6ltiivn of aicofine in ®o)xta avd nou->makm. Im Martin WR, Van Laan GR, Iwamoto ET. Davis L, eds. Tobacco Smoldng and Nseune. New Yox1G Plenum Ptas, pp. 375-388. 28. Kyetematen GA., Owens GF., Clmncpadhyay B., deBetbiry 9. Tumer DM (I977) : The effects of some tobacco smoke oonstiments on foreign eompound metabolism in the cat and the tat. Res. iCnom>•= Chem. PathoL PhamaeoL,16, 85-100. JD., VeseB ES. (I988) : Sexuil dimocphism of nimtine , metebolimm and distvb,IDO¢ in the caL Drug Metab. Diapos, 16, 823328. 10. McCoy GD., DeMemo GJ. (1986) : Charaaedra¢on of hamste liver niootiae metabolism. IL Differential effear of ethaaol ocphenobabital ptanentment on miaw®al N®d C vzidation. Biodxm. Pha®eroL, 35, 4590-4592 11. R-ndell U. Foth H.. Ksbl GF. (1987) : Eightfold induction of nirotine eI®ination in perfused rat liva by p:caeamuat vmh pbenobaxbitd. Biachcm. Biophys. Brz CammmL,148, 192-198. 29. Cavm A., Monji N, Ali H, Yi 1M. Bowmmm ER., )rlcYests%• A(1980) : Nmtine mn'hodies: Comp®son of Bgand specifidties of annbodies produced against twu timtine conjugetes Euc J. Biochem,104, 331-340. 30. Cutval! M., Kszcmi Vala E., Engluad G,E~e11 CR (1989): Utmaty excretion of nicotiae and ite majx mctabolites. Paper No. 54 presented at ihe 43rd Tobacco Cicmists' Reaeasda Confaenee, Ridbmond VA., USA. 12. Shinegags M.K. Tmvor AJ, Csstagmli N. (1988~ : Metabolism-depesdent covaleot binding of (S)-15-'H]nicotme to liver and lung mictosomel m~omolecules. Drug Metzb. Dispos., 16, 3974OZ 13. MeCoy GD., DeMmm GJ, Koop DR (1989): Miansomal nicotine metabolism: A campa<ison of relative activities of siz purified rabbit cytachxome P-450 isocymes. Biocbem. PharmacoL,38,1185-1188. 31. Curvall ld~ Kar~i Vain fi., Engluad G. (1991) : Conjugation pathways in nicotine medbolism In: Adlkafer F. Th,aau K, eds. Effects of Nicotine oo Biologid Sys[ems. Ba.seb Bid;heuse V eriag, pp. 69-75. 32. Kyetematm GA, Motgan ML, Qmvapadhysy B, deBefhizy J.D., Vesell ES. (1990) : Disposition of nicotine and eight metabolites in amoEecs and nonsmoke<s 1d-dOfi-c3tion in amokess of two metabolites that ste longer lived than cotinine. 14. Kyemmaten G.q, Morgan M., Warner G., Martin L.F, Vesell ES. (1990) : Metabolism of nicotine by hepatocytes. Biochem. PhasmecoL, 40, 174J-1756. 15. Foth H., Looschea H, Neurath H., Kabl G.F. (1991) : I'licotine metabolism in isolated pafuted bmg and livec of pLeuobarbital- and bemoflavone-neated rats. Amh. ToxicoL, Clia. Phsmscol. Ther., 48. 641fi51. 33. Byrd GD.. Cbang K.-M. Gceem 1.M., deBah¢y 1D. (1992) : Evidence for uanary esaeioa of glueiuenide eeajugata of nicaiue, cotinine, and trau-3'-hydmzycotinme in smokas. Drug lrletab. Dispos., 20, 192-197. 34. Rmtemeier K. Demetrieu D. Schepes G, Vonckeu P. N ~ 65, 68-72. (1993) : Aigh-perfo,mame liquid c1umemgephic deter- 16. Adir J., WBdfeuerW.,MiIkrRP.(1980):Effectofethaml pmtteatmeat on the pha®scrolcioetic of nicotine in tats. J. miouion of niwtine and its utmary metabolites via their 1,3-diethyl-2-Wiobetbituric acid deuvativd. J. Chtamamgr_ ~ PhetmacoL Exp. Tber., 212. 274279. 613,95-103. -p' 17. Foth H. Waltlnr U.L, Kshi GF. (1990): Increased hepatic nicotine elimination aftrr pheoobatbita[ induction in the conscious raL ToxicoL Appl. Phatmaml, 105. 382-392. 18. Ames BN., McCenn J., Yamasaki E. (1975) : Methods for detecting eaminogens and mutngcas with the . salmonellalmammalian-mienssome mutagenicity teot MutzL Ra., 31, 347-363. 35. Schepen G.. Demetriou D., Ruumeier K, Veackm P., Diebl B. (1992) : Nicotine phase 2 metabolites in huann utine - smteuae of inetabo)i®Dy formed tmnr-3'-hydmxycotinine gluaaonide. Med. Sci. Ibs., 20, 863-865. 36. Kyeamatea GA. Taylor LH., deBethizy 1D. VeseB ES. (1988): Phssmacroltinetics of nicetiuc and 12 metabolites in the m1 Drug Metab. Diapos.16, 125-129. Q 19. Detnetriou D., Rustemeicr K, Vomka P., Schapess G. (1993) : HPLC sepamtion of the enantiomen of nicotine and nicotine-like compounds. (Isinlity, In press. 20. Lehahazdt F: J. (1989) : A mv system for cathetmsm of the 37. Piletu A. EmeB CR. McKemis H. Bowman ERrDufre E, Holmstedt B. (1976) : Studies on the identification of tobacco alkaloids, their mammalian metebalita and teleted eempamds by gas chromatagaphy-mav specuoman]'. Beitr.
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Two recent, large-scale studies in the U.S. and the U.K. compared perceptions of individual exposures to ETS (by self- report) with actual levels of ETS constituents collected in the subjects' breathing zones by personal monitors in agreement with OSHA practice.'o•a3 In the U.S. study, greater than 50 percent of all self-reports of ETS exposures occurred at work. Actual exposure measurements, however, indicated that average exposure levels were approximately four to six times greater in venues outside the workplace.83 In the U.K. study, individuals subjectively ranked relative contributions of ETS as leisure > work > home > travel.70 Measured exposure levels, however, indicated a ranking of: home > leisure > work > travel. The authors suggest that -the discrepancy between subjective rankings and objective measurements of exposure may be due to the subject's inaccurate estimate of the time spent in each venue. Other researchers have studied variations in subjective perceptions of exposure and have reported that perceptions will vary depending or whether or not the exposure source (i.e., the smoker) can be seen.84 5. Cotinine Measurements Do Not Validate Questionnaire Responses Nicotine, because it is characteristic of tobacco smoke in the air, has been used extensively as an ambient air exposure marker -for ETS.'s,,o,sa,e5 Likewise, cotinine, one of the substances converted from nicotine by the body, has been used as a biomarker - 19 - - -
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Questionnaires on exposure to environmental tobacco smoke generally assess the strength of the source, e.g., the number of smokers, the number of cigarettes consumed and the duration of exposure. The concentration of environmental tobacco smoke, however, depends not only on the source strength, but on room size, mixing, adsorption of smoke components, and the rate of- exchange of indoor with outdoor air. Personal exposure also varies with the nonsmoker's proximity to the smoker. Questionnaires cannot comprehensively and accurately assess each of these factors. Estimates of "intensity" and "duration" of exposure also depend upon respondent memories of literally decades of potential exposure scenarios. Complete recall, of course, is impossible, and even partial recall may be faulty. For example, studies indicate that while spouses (or children) may generally agree in reporting a partner's (or parent's) smoking status (i.e., whether or_not a spouse smoked), agreement regarding the amount smoked or duration of smoking is often very poor.es,es,"" Pron, et al., examined the reliability of self-reported ETS exposure histories by interviewing and re-interviewing.study subjects.ss Consistency of responses about exposure between initial interview and re-interview was poor, and correlations between responses were low, especially for questions related to intensity and duration of exposure. The authors conclude: [T]his is the first study to assess the reliability of information reported on passive smoke exposures in personal interviews. Test- - 15 -
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There is no standardized or validated questionnaire available for use in epidemiologic studies on ETS, and no single questionnaire was used in the published studies on spousal smoking.'3 As a consequence, definitions of various ETS exposure indices have differed considerably from study to study with respect to source, intensity and duration. 3. Questionnaire Responses Are Neither Reliable Nor Accurate The questionnaire, by its very nature, can provide only a crude, qualitative estimate of exposure. Questionnaire data do not, and cannot, provide information on concentration (e.g., actual levels of airborne ETS constituents) or frequency of ETS exposure. As the U.S. National Academy of Sciences observed in 1991: "Exposures [to ETS] occur at a wide range of concentrations for highly variable periods and in numerous indoor environments. Unlike active smoking, exposure to ETS cannot now be easily assessed. ..."73 The questionnaire is thus only an indirect means of assessing exposure. This, according to a NIOSH witness at the U.S. OSHA Public Hearing, is one of "the principal weaknesses in the epidemiologic evidence to date."'4 - Where exposure is defined as "concentration over time" or "intensity, frequency and duration," the questionnaire's inherent shortcomings are obvious. Thus, Coultas, et al., note:'5 - 14 -
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(internal dose marker) for nicotine (and ETS) exposures.es-9o Several researchers, including Riboli, et al., of IARC, have used measurements of cotinine in an attempt to validate self-reports of ETS exposures.8'•90-9Z The advantage of body fluid measurements of cotinine over nicotine is that cotinine has a longer half-life (17 hours vs. 3 hours). Despite this longer half-life, however, cotinine-derived estimates are of little value in determining past (greater than one week) exposures to nicotine. As Reasor has written:93 At present, however, there is no reliable way, through the use of biological markers, to assess long-term exposure to ETS. Cotinine has even more fundamental problems when used as a biomarker for exposure to ETS,81,88,93-9, For example, an individual will metabolize and clear cotinine from his/her system at different rates at different times (intra-individual variation), and clearance rates vary considerably from individual to individual (inter-individual variation).9'•9s,se Common foods also contribute trace levels of nicotine (which are thus converted to cotinine), thereby confounding inferences about ambient nicotine (ETS) exposures.99 Moreover, body fluid levels of cotinine do not correlate well with ambient levels of nicotine (or any other constituent of ETS) , 64,70,81,93,96 and saliva, plasma and urine levels of cotinine are also poorly correlated.64,9s 0 - 20 -
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4. Questionnaire Responses Are Influenced by Perceptions Reconstruction of a life history of ETS exposure is highly influenced by recall accuracy -- imagine recalling, with any degree of accuracy, the duration, frequency and degree of personal ETS exposure over the previous week, year or decade, or during a marriage, during adulthood, etc. Studies suggest that even recent perceptions of ETS exposure may be wholly inadequate indicators of actual ETS exposures.s4,,o,e -ea O'Connor, et al., compared self-reported exposures to ETS with ambient levels of nicotine collected by personal monitoring devices.81 Thirty-six percent of women who reported ETS exposure by questionnaire were misclassified as "exposed" to ETS according to objective measures of exposure. Coultas, et al., in a study of 10 homes, found that questionnaire responses about ETS exposures "were poor predictors of concentrations of respirable particles and nicotine."64 Similarly, a workplace study conducted by Schenker, et al., reported no association between respirable nicotine concentrations and self-reported exposures to ETS.82 The authors conclude that "self-reported exposure to ETS is an inaccurate measure of passive smoking in the occupational setting." - 18 -
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Similar results were reported in a study by Coultas, et al., in 1989.63 Brownson, et al., in a 1993 study, assessed ETS exposure histories through interviews and re-interviews of cases and controls in a spousal smoking study on lung cancer.'9 Agreement for spousal smoking status after re-interview was 84 percent, indicating that 16 percent of cases and controls actually reversed responses regarding the smoking status of their spouse. The agreement rate for exposure histories in adulthood among cases was only 49 percent. Overall, the reliability of responses (assessed by agreement in responses over time) about quantities of exposure was extremely poor, and lower for cases than for controls.'9 Despite representations to the contrary,"•'9 studies indicate that the personal interview may not even guarantee a reliable answer concerning the smoking status of one's spouse, much less a reliable estimate of ETS exposure duration or intensity. Of course, the reliability of questionnaire data is determined only by agreement of respondent answers concerning estimates of exposure. The accuracy of the estimate itself is not addressed. No standard exists for validating a history of exposure to ETS, or for assessing the accuracy of_exposure estimates. _ - 17 -
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Large intra- and inter-subject variabilities in the conversion, metabolism and clearance of cotinine call into question the validity of its use in surveys that employ single-point measurements (of cotinine) to represent ETS dose.10° Dr. Paul Nelson of R.J. Reynolds recently observed: "It is likely that inter-individual variations in nicotine and cotinine metabolism or excretion would far outweigh the small incremental increase in cotinine concentration following exposure to typical levels of ETS nicotine. In other words, the variation between people is larger than the variation due to normal exposures."10' virtually every cotinine measurement survey relied upon by U.S. EPA for ETS exposure estimates was restricted to single- point measurements of cotinine.gs,a,,s ,z2 However, an individual's cotinine level at a single point in time will be determined by the timing of the specimen collection, and by the individual's own rates of uptake, metabolism and clearance. Thus, Idle observes:ss Single point cotinine concentrations can give no more than a clue of past exposure to pyridine alkaloids [such as nicotine] of unknown amounts, at an unspecified time, by an unknown route of entry and from unknown origins. Phillips, et al., reported extremely poor correlations between salivary cotinine values and 24-hour respirable particle and nicotine exposure measurements.'° The data from the Phillips, - 21 -
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would recommend against using cotinine levels as a strictly quantitative indicator of ETS. 6. A Potential Bias in ETS Questionnaire Data Self- (and surrogate) reports of exposure to ETS are neither accurate nor reliable when compared to a standard of personal measures of exposure to airborne ETS constituents. Questionnaires are limited by accuracy of recall and the individual's ability to provide comprehensive, quantitative estimates of exposure over time. Other problems beset the questionnaires used in studies on ETS. As Tager notesc105 - Among the most significant limitations of existing questionnaire data [on ETS] are the facts that many of the questions were not designed specifically to- investigate involuntary exposure, or the questionnaires have been incomplete in their probing of the circumstances of exposure (e.g. intensity, duration, specific location, etc.). These limitations have made it difficult to provide even semi-quantitative exposure estimates over time. The accuracy of an exposure estimate is of -obvious importance because of its profound effect on risk estimates in epidemiologic studies.106 Garfinkel, et al., in a spousal smoking study published in 1985, reported different risk estimates ranging from 0.46 to 3.57 depending upon who responded to questions about exposure. If a nonsmoking wife or smoking spouse responded to the - 25 -
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exposure questions, the estimated risk from spousal smoking was below unity, i.e., no increased risk. If a son or daughter responded, the estimated risk rose to over 3.00. Responses were also influenced by changing the wording of the exposure-related que s t i ons . l°6 An entire range of potential biases is introduced through the improper design and administration of specific questionnaires. One particular bias, however, is directly applicable to the ETS exposure issue. It is one that arises, in part, from the way in which ETS exposure questions_are phrased and presented -- called "recall bias. ^72•1°'-10s Recall bias in relation to ETS is a differential bias, in that cases and controls are likely to be affected differently.'2 Given the tremendous publicity generated, for example, by the U_S. EPA's classification of ETS as a "known human" carcinogen, it is without question that a nonsmoking lung cancer case could be influenced by such publicity in the effort to account for his/her disease. If ETS is mentioned or prompted in any way by the questionnaire or interviewer, the likelihood increases that ETS will be selected by the respondent as "the correct answer."10Z A control, or someone without lung cancer, is not likely to respond in that way. Tunstall-P-edoe, et al., for example, found that self- reports of exposure exhibited strong associations with symptoms, - 26 -
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et al., study indicate that some subjects who were exposed to high_ levels of particles and nicotine had no detectable cotinine levels, while some subjects exposed to low levels of ETS, as determined by personal monitor, exhibited high levels of cotinine. O'Connor, et al., reported levels of urinary cotinine that "did not differ" among ETS exposed and non-exposed.Bl Ogden, et al., reported "virtually identical median levels of salivary cotinine" for all subjects, even though nicotine exposures varied nearly three-fold between exposed and nonrexposed individuals.96 Cotinine measurements likewise do not correlate well with self-reports of ETS exposures.70,75, 00,07,102,103 Wagenknecht, eE a1., found that 58 percent of 575 study participants who reported ETS exposures of 42 hours or more had no detectable serum cotinine levels; of the 186 individuals who reported no known exposures, 23 percent had a detectable cotinine level.102 Delfino, et al., examined salivary cotinine levels and compared them with questionnaire-derived responses about ETS exposures."3 No correlations were reported. In 1994, Emmons, et al., reported that nearly half of those individuals who recalled exposure to ETS at work had nondetectable cotinine concentrations, as did 29 percent of those who reported exposures at home.87 Coultas, et al., found that both - 22 - - - - H N
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urinary and salivary cotinine levels of workers, measured post- workshift, "varied widely with self-reported exposures."'S Similar results were reported by Heller, et a1.,94 and Suadicani and co- workers.104 In the latter study, investigators reported that individuals who were classified (by self-report) as "often exposed," "occasionally exposed" and "rarely/never exposed" exhibited similar average serum cotinine levels of 25, 22, and 24 ng/ml, respectively.109 Tunstall-Pedoe and coworkers recently reported poor correlations between self-reported ETS exposure and serum cotinine level.89 They concluded that "their poor correlation with each other ... undermine[s] the validity of the two measures of passive smoking." Despite acknowledged shortcomings in the use of cotinine as a quantitative biological marker for ETS, some researchers nevertheless have used cotinine measurements in an effort to validate self-reports of ETS exposure_8s•91 Riboli, et al., for example, reported that mean urinary cotinine levels among their study population showed a linear increase with self-reports of ETS exposure, and that cotinine levels were indicative of reports concerning the duration of exposure and the number of cigarettes smoked in the presence of the subject.91 Close examination of the Riboli, et al., study data reveals, however, that 20 percent of the study population had - 23
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while the relationships were "weak or absent" for cotinine levels.89 The authors suggest that individuals with respiratory conditions may exaggerate ETS exposures, thereby creating a (recall) bias in self-reported exposure estimates. The influence upon the respondent by the way a question is phrased or asked may be extensive. It reflects, in Wynder's words, a "wish bias," a "tendency on the part of the subject or the investigator to reach a desired result.j10' Wynder writes: Research workers, like everyone else, often develop an affection for their own hypotheses and may prefer to see them supported rather than refuted. This may lead to incomplete review of the literature in which papers failing to support the hypothesis may be ignored or more subtly, may be rejected because they are considered to be of worse quality than papers that support it. Sometimes hypotheses are based on a single piece of evidence . . . The wishes of the investigator may also enter into the collection of the data. Greater care and thoroughness may be given to collection of the data from the cases than from the controls. The foregoing analysis demonstrates that ETS exposure and the surrogate "spousal smoking history" are not the same thing. A spouse living with a smoker may or may not be exposed at home, and he or she may or may not_be_exposed elsewhere. If an individual is truly exposed to ETS, the questionnaire response provides no information on the concentration or frequency of exposure. Thus, - 27 - -
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nondetectable cotinine levels, and that a predicted increase of 5 ng/ml urinary cotinine could be calculated from self-reported exposures ranging anywhere from six to 83 cigarettes per day.91 Riboli, et al., do not indicate the existence of any overlap in cotinine measures among the groups examined.91 ..Other researchers, however, have reported wide variations of cotinine measures within specific levels of reported exposure.a3,-,o,e. For example, Phillips, et al., reported "considerable variation in the direct measurements [of cotinine] corresponding to the higher grades of subjective [ETS] assessment.p70 Even proponents of the use of cotinine to validate self- reports of ETS exposures realize the limitations of the method.63 Cummings, et al., provide the following caveats to their study:63 Cotinine was chosen as a biological marker of ETS exposure because it is specific to tobacco smoke. However, cotinine levels in body fluids may not only reflect environmental exposure to tobacco smoke, but also factors that influence uptake and metabolism of nicotine. And: The relatively modest correlation between reported ETS exposure and urinary cotinine indicates that other factors such as differing metabolic rates and body size may have a confounding effect on the relationship between cotinine levels and questionnaire measures of ETS exposure. In view of this finding, we - 24 -
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if meaningful at all, the surrogate "spousal smoking" may be more of a measure of the many lifestyle factors surrounding marriage to a smoker than it is of ETS exposure; indeed, a history of spousal smoking may have a number of implications for disease that have nothing whatsoever to do with exposure to ETS.11o E. Relevant Animal Inhalation Studies Were Essentially Overlooked in the U.S. EPA Risk Assessment U.S. EPA's risk assessment overlooked the vast majority a m of relevant animal data,' citing only a few studies. This omission ~; severely undermines the claim that the U.S. EPA's conclusions are CR ".` based on the "weight of the evidence." As the following discussion will show, if Cal/EPA were to review all the relevant animal data, it would be clear that those data provide no support for a claim that ETS exposure is associated with lung cancer. The data in animal inhalation studies reported to date provide no support for a claim that there is "biological plausibility" for the position that ETS exposure is causally related to lung cancer. These data are not referenced in the Excerpt. ~ - 28 -
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lung tumors in the strain of mouse used, which failed to show any significant difference between unexposed animals and animals exposed to a surrogate for ETS. The strain used lives less than one year and.is known to develop lung tumors within four to six months after the beginning of exposure to various chemicals._ In the Witschi, et al., study, these mice were exposed (whole-body) to concentrations of aged sidestream smoke well in excess of measured "real-world" levels. (While not equivalent to ETS, sidestream smoke has been used as a surrogate in animal studies.) Despite the demonstrated sensitivity of this strain of mouse, the authors reported the following: "The number of animals bearing lung tumors was the same in smoke-exposed as in filtered air-exposed animals as was the average number of tumors per lung." These data are compatible with the conclusion that sidestream smoke, under the test conditions, is not a lung carcinogen in this strain. In a recent review of the relevant literature, Rodgman cautions:13 Classifying a substance as tumorigenic or `carcinogenic' can be misleading. Often, these terms are overinterpreted. One must be aware of the precise meaning and limitations of the terms tumorigenicity and carcinogenicity when applied to specific - 33 -
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The "carcinogenic agents" supposedly identified in tobacco smoke (e.g., the "list" of suspected carcinogens referred to in the U.S. EPA Risk Assessment) either are not suspected pulmonary carcinogens or have not been unequivocally demonstrated as tumorigenic to human tissue or to the lung tissue of experimental animals.13•111•112 In conjunction with U.S. OSHA's lengthy rulemaking process (which is still underway), several reviewers have examined and summarized the available animal inhalation studies relevant to U.S. OSHA's claim that animal inhalation studies provided supporting data for an ETS-lung cancer relationship. These reviewers unanimously conclude that the animal studies do not support the claimed carcinogenicity of_ETS.113"11' One reviewer, Christopher R.E. Coggins, Ph.D., concluded that animal studies using close surrogates for ETS "show no meaningful toxicological changes, even at massive exaggerations of real-world ETS concentrations.j113 Similarly, Gordon Newell, Ph.D., told U.S. OSHA that studies using a number of animal species have "failed to support the hypothesis that fresh tobacco smoke causes lung cancer" in those species.114 Moreover, Gio B. Gori, Sc.D., noted in his submission to U.S. OSHA's rulemaking record:i15 - 29 -
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authors noted that "[n]o smoke-exposure-related histopathological changes were observed in trachea and lungs." In the subchronic study of Coggins, et al.,123 aged and diluted sidestream smoke was used as a surrogate for ETS. This substance may be a more appropriate approximation of ETS than are other forms of tobacco smoke. Effects (hyperplasia and inflammation) were reported only in animals exposed to particle concentrations some 100 times higher than typical real-world concentrations. Coggins, et al.,124 also report the same minor, completely reversible histopathological changes. The changes did, not progress over longer periods of exposure, and once again occurred only at particle concentrations some 100-fold higher than real-world levels. In a 14-day inhalation study, one would not expect lung tumors to develop. Thus, the relevance of the work of Coggins, et al., to the discussion of cancer is limited. Nevertheless, Coggins, et al. show only minor, reversible cellular changes following intense exposure to a surrogate for ETS.1zs Only one animal inhalation study to date has used exposures to a surrogate for ETS of a duration sufficient for it to be considered a chronic study.126 Witschi,_- et_-al., recently reported the results of a study, specifically designed to produce - 32 - -
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Experimental data offer no plausible argument to classify ETS as a human risk ... The arbitrariness of a nriori assumptions of ETS- related human risks is further underscored by equivocal and uninterpretable epidemiologic reports. Overall, tobacco smoke inhalation studies have not produced an increased-incidence of lung tumors in experimental animals compared to controls; the relevance of other routes of exposure (e.g., skin painting) is questionable.116•117 The vast majority of the available data from-animal inhalation studies using surrogates for ETS deal with subchronic exposures, which are of minimal relevance to the question of the claimed "carcinogenicity" ra :: m r w m ~ of ETS. Nevertheless, none of the subchronic studies report data w N =_ n~ . supporting any permanent changes following subchronic exposure of animals to sidestream smoke at levels exceeding those encountered in "real-life" situations. Brief summaries of the subchronic studies follow. In two 1987 papers, Haley, et al., present preliminary reports on an American Health Foundation study, in which hamsters were exposed to mainstream or sidestream smoke 7 days/week for 18 months.118•119 Apparently, however, no final report has been published. In those reports, the authors note that smoke-exposed animals were living longer than were sham or cage control animals. No additional information was presented. - 30 -
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compounds and must exercise considerable care in the use of these and related terms. * * * Many of these 43_MS and/or tobacco components [claimed to be carcinogens] should be excluded from the list on the basis of published data on their tumorigenicity (or lack of it) in laboratory animals at levels determined in MS, their lack of tumorigenicity in most instances on inhalation, and the equivocal evidence of their tumorigenicity in humans at levels in MS. In this major review, Rodgman also writes: [I]nhalation studies from 1936 to date involving lifetime exposure of laboratory animals to whole cigarette MS have consistently failed to induce squamous cell carcinoma . . . The failure to produce in MS-exposed laboratory animals the tumor type reported to be associated with smoking in humans is important not only with regards to the biological properties of MS itself but also with respect to that of diluted MS delivered to the caged animals. . . . If, as Stewart and Herrold (1962) noted, these smoke-inhalation experiments more closely resembled passive smoke (or ETS) exposure than human exposure during actual smoking, then substantial evidence is available to demonstrate that exposure to 'passive smoke' (or ETS), more concentrated than that encountered in the human situation, is ineffective in induction of the tumor type supposedly associated with cigarette smoking in humans . . . - 34 -
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von Meyerinck and colleagues (1989) describe a study in which rats and hamsters of both sexes were exposed to sidestream smoke at a concentration of 4 mg/m3 TPM and 25 to 30 ppm carbon monoxide for 10 hours/day, five days/week for 90 days.'-2o These authors note about their exposure system: "The levels in the exposure chamber were at least 1 and in some instances 2 orders of magnitude higher than reported for smoke-polluted rooms under real- life conditions." (Elsewhere, the authors described these conditions as "unrealistically high."lz1) One hundred animals of each species were exposed, 115 of each species were sham controls, and 100 of each species were room controls. The authors reported minor, completely reversible histopathological changes in the nasal cavity in rats only, and no alterations in any other part of the respiratory tract. Male rats and male hamsters were nose-only exposed to fresh sidestream smoke (FSS) for seven hours/day, seven days/week for 90 days, in a study reported in 1994 by Teredesai and Pruehs.122 One group of 20 animals was exposed to FSS with a total particulate matter (TPM) concentration of 2 ug/L, one to FSS with TPM of 6 ug/L, and one served as a sham exposure group. Histopathological changes described as "slight" were reported in the nose and larynx of exposed rats, "mainly in the high FSS concentration group." These changes were reversible following cessation of exposure. The - 31 -
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Studies have reported that various constituents and extracts of ETS collected from indoor air are capable of inducing mutations in the Ames assay."3-1'9 However, the significance of such reported findings has not been established. Virtually all air samples, whether in the presence or absence of smoking, can be shown to be mutagenic in various bioassays. Indeed, many substances, including foods and other "natural" materials, have been shown to exhibit mutagenic and/or carcinogenic properties.i'e Of relevance, Sonnenfeld and Wilson report that sidestream smoke exhibits reduced activity as it ages and becomes- diluted, that is, as it becomes ETS.149 These authors report on an experiment in which cultured mouse fibroblast-like cells were exposed to mainstream or sidestream smoke of various ages. In this report, cytotoxicity (cell mortality) is used as a measurement of DNA damage sufficient to cause cell death. The authors write: Aging of SS smoke resulted in a rapid decline in the mortality generated by the smoke. As calculated from the linear regression curve, an increase in age of SS smoke of 30 [seconds] after generation would have resulted in a total loss of cytotoxic effects. (emphasis added) Another area of relevant research comprises those studies that have compared the mutagenicity of body fluids of nonsmokers exposed to ETS and nonsmokers not exposed to ETS. Several of these - 39 - -
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The above discussion illustrates that the available animal inhalation data provide no support for the claimed "biological plausibility" that ETS exposure is associated with an increased lung cancer risk. The unreferenced statement made in the Excerpt is not representative of the actual state of the scientific literature, and as such, results in a misleading impression being conveyed to readers of the Excerpt. Cal/EPA should review the relevant animal inhalation studies and the critiques/discussions already in the U.S. OSHA rulemaking record as it revises this Excerpt. F. Data on Genotoxicity and Related Endpoints Were Essentially Overlooked in the U.S. EPA Risk Assessment The U.S. EPA Risk Assessment fails to reference a number of actual studies comparing levels of mutagens and other genotoxic markers in the body fluids of exposed and non-exposed nonsmokers.lz'- 136 The results of those studies suggest no statistically significant increases in mutagenic activity in the body fluids of nonsmokers exposed to realistic levels of ETS compared with nonsmokers who are not exposed. -- When considering data on genotcxicity, it is important to put the genetic changes reported in such studies into context. All forms of life are constantly exposed to physical and chemical - 35
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agents in the environment (e.g., radiation) and to endogenous (internal) agents with the ability to cause changes in DNA. According to Bruce Ames, developer of the Ames assay for mutagenicity, human exposure to potentially mutagenic or carcinogenic substances is much greater than generally appreciated, i.e., the environment can be thought of as "filled with potential carcinogens."13' DNA has been called an "unstable" molecule, and it has been noted that endogenous DNA damage may occur at the rate of 100,000 base pairs per cell, per day.13',1'e Thus, DNA is not completely stable; changes are regularly occurring, but for the most part, do not result in heritable effects on the organism. As toxicologist Christopher R.E. Coggins of R.J. Reynolds stated in testimony before U.S. OSHA, "Toxicologically, I'm not sure that we really- know what mutagenesis really means because of ... DNA repair. "13s - - - Therefore, conclusions about genotoxicity obtained from in vitro systems, while certainly providing some information about the substance being tested, must nevertheless be put into the proper biological context. The magnitude of a genotoxic response in the whole organism may be substantially different than that observed in a bioassay.1'0•1" As Ames and Gold noted:14° [H]umans have numerous inducible defense systems against mutagenic carcinogens, such as DNA repair, antioxidant defenses, glutathione - 36 -
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unresolved questions and uncertainties remain. Moreover, more recent data on exposure, animal inhalation studies and genotoxicity do not conclusively support U.S. EPA's conclusions, contrary to the statements made by Cal/EPA. A large number of the submissions and articles critical of U.S. EPA's risk assessment are now part of the U.S. OSHA rulemaking record. U.S. OSHA also relied heavily on the U.S. EPA Risk Assessment, and commenters have called upon OSHA to critically evaluate its conclusions.
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transferases, and so forth ...[L]ow doses of carcinogens appear to be both much more common and less hazardous than is generally thought. The difficulty of extrapolating from in vitro genotoxicity to in vivo carcinogenicity is illustrated by data presented in the recent animal inhalation study of Witschi, et al., which used sidestream smoke."6 Other studies suggest that tobacco smoke condensates may be_mutagenic when tested using in vitro systems; however, there are no such studies using ETS condensates. In their recently published chronic inhalation study, Witschi and colleagues reported no differences in the total number of animals with tumors or in the average number of tumors per lung in the smoke-exposed animals when compared to filtered air-exposed control animals -- consistent with sidestream smoke not being carcinogenic under the test conditions -- even though they did report positive results for molecular biomarkers. Thus, these data suggest that the relationship of mutagenicity to "carcinogenicity" is not clear- cut. In another study, Nikula, et al. (1995), investigated the inhalation carcinogenicity of two substances that were essentially equivalent, except that one contained mutagens and the other did not.142 If mutagenic properties were relevant to carcinogenicity, it would be expected that the substance with mutagens would have - 37 -
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All probands revealed measurable and varying SSB levels. Since DNA is an unstable molecule and estimated endogenous damage exceeds 100000 affected base pairs per cell per day, we assume that SSB base levels reflect unrepaired lesions. Active smoking caused an increase in SSBS in peripheral blood lymphocytes. This effect could not be found in passive smokers. ETS exposure in this study consisted of five smokers each smoking 24 cigarettes in eight hours in an exposure chamber. This study provides no support for claimed genotoxic effects of ETS, even at a high exposure level. This review of data from studies in which genotoxicity was assessed in persons actually exposed to ETS thus provides little, if any, support for the contention that ETS is genotoxic at levels encountered in workplaces and other indoor environments. As Doolittle stated in his submission to U.S. OSHA:1ss The hypothesis that ETS causes lung cancer is not supported by any of the available genotoxicity data. There is no evidence that ETS at or near ambient levels of exposure produces genotoxicity. The available published evidence comes to the opposite conclusion, namely ETS is not genotoxic. G. Conclusion In conclusion, the U.S. EPA Risk Assessment on ETS is not the definitive document portrayed by Cal/EPA. A large number of - 45 _
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produced a stronger carcinogenic response. However, as the authors noted, the two substances yielded "very similar" responses in the test system. Thus, these data, too, suggest that positive genotoxicity results cannot necessarily be correlated with carcinogenicity. In its Proposed Rule, U.S. OSHA discussed a number of studies in which cigarette smoke or cigarette_smoke condensate was tested in the Ames Salmonella tvohimurium assay, and an increased mutation rate was reported. OSHA's inclusion of studies dealing with mainstream and sidestream smoke revealed the misconception pervading the Proposed Rule that ETS, mainstream, and sidestream smoke are equivalent. - Moreover, OSHA omitted at least one relevant study from this discussion in the Proposed Rule. In 1991, Bombick, et al., reported on a cellular smoke exposure technique using rat liver cells and the Ames Salmonella assay.135 After a three-hour exposure using ETS at a concentration of 1.5 mg total particulate matter/m3, the authors report: Using the neutral red cytotoxicity and Ames mutagenesis assays there were no differences observed in the ETS-exposed cells and their respective room air controls, indicating that ETS was biologically inactive as tested. (emphasis added) - 38 -
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Citing the high variability of measures of urinary mutagenicity and questions about the relevance of increased urinary mutagenicity to cancer risk, the authors write:13s The data suggest that nonsmokers in real-life situations take up very low doses of ETS constituents, and detoxification of the genotoxic substances inhaled is effective. And : 137 Whether ETS exposure can lead to an elevated urinary mutagenicity is a matter of controversy. In most investigations no significant increase has been observed. ... ... The results of our investigations, as well as those of other authors, suggest that urinary mutagenicity, which would be a potential marker for ETS particle exposure, remains unchanged after ETS exposure. The few studies reporting statistically significant increases in urinary mutagenicity among individuals exposed to ETS do not employ realistic levels of exposure to ETS, nor do they control adequately for the presence of mutagens in the diet of the study subjects.lsz-.sn For instance, in the Bos, et al., study, the exposure condition consisted of the smoking of 157 cigarettes over six hours in a room with "poor ventilation."152 The relevance of such an exposure to "real-life" conditions is certainly - 41 -
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studies report no significant difference in mutagenic actlvlty,129,130,134,136,150,151 For instance, in a study by a team of German researchers, ten nonsmokers were exposed to ETS, generated by human smokers, for eight hours under two exposure conditions.l3e The two experiments were characterized by CO levels of 10 ppm and 25 ppm, respectively; according to the authors, both exposure regimes represent higher exposures than "real-life" situations. Elsewhere, they describe Experiment 2 as "far from being realistic,"134 and bearing "no relation to a real-life situation."13s In addition, the authors controlled for the effect of mutagens from the diet by keeping their subjects on a diet low in polycyclic-aromatic hydrocarbons. Urine samples from both smokers and nonsmokers were tested in the Ames Salmonella assay. The authors report:lao - - All urine extracts of ETS exposed non-smokers were found to be negative in the mutagenicity test when applying the [criterion] of Ames (doubling of spontaneous mutation rate). Thus, even at exposure levels higher than would be expected on average, no increase in mutagenicity could be measured. These data do not support claims that ETS exposure is associated with an increase in mutagenic activity; moreover, because the samples come from exposed humans, the influence of physiological processes following exposure is indirectly taken into account. - 40 -
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questionable. With respect to diet, Bartsch, et al., acknowledge, concerning their study, thatl.' Urinary mutagenicity is influenced also by dietary habits; although we collected information on diet, the dimension of the study (particularly as far as passive smokers are concerned) does not allow adequate statistical treatment of this potential confounding factor. Other related studies have examined levels of various DNA changes in nonsmokers exposed to ETS.131,139,151,155-15fi Based on the data presented in these studies, nonsmokers exposed to ETS do not appear to exhibit increased DNA adduct formation, nor do studies report increased levels of chromosomal changes in cells of nonsmokers exposed to ETS. Discussion of these studies follows. Collman, et al., collected data from 16 nonsmokers, 15 "passive smokers" (currently living with one or more smokers)_, and 13 current smokers, all women.lss Sister-chromatid exchange (SCE) frequencies in lymphocytes (a type of white blood cell) were compared with and without coincubation with a chemical that enhanced the frequency of SCEs._ Based on both assays, the authors report that "the frequency of SCEs in_persons passively exposed to smoke was not higher than in nonsmokers." - 42 -
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In a report by Husgafvel-Pursiainen, peripheral blood lymphocytes were examined for SCE frequency, a sensitive test."' This test uses cells from the exposed individual, rather than another organism, and also considers repair mechanisms, thus being a better representation of actual events at the cellular level. Study groups consisted of 12 smoking waiters and waitresses, 20 nonsmoking waiters and waitresses who were occupationally exposed to ETS, and 14 nonexposed office workers. The author reports that "[t]he mean SCE level in exposed non-smokers did not differ from that observed in the non-exposed group." _ Although no ETS measurements from the restaurants were reported, the author characterizes them as "heavily polluted," and the exposure as "long-term." This study, which reports data from persons exposed in a "real-life" situation, does not support claims of the genotoxicity of ETS. Chromosomal aberrations (CAs) and SCEs were examined in peripheral blood lymphocytes from nine smoking waiters, 16 nonsmoking waiters exposed to ETS at work, and seven reportedly nonexposed nonsmokers by Sorsa, et a1."s The authors report that "[n]o significant differences were seen between the groups or subgroups in the 2 parameters." Thus, no "genotoxic" effects_could be detected in persons exposed to ETS at "real-world" occupational exposures. - 43 -
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REFERENCES 1. U.S. Environmental Protection Agency, Office of Research and Development, Respiratory Health Effects of Passive Smoking: Lung Cancer and Other Disorders, EPA/600/6-90/006F, 1993. 2. Anonymous, "A Brief Summary of the Public Comments Submitted to the U.S. EPA in Response to the External Review Draft EPA/6oo/6-9o/006A, Health Effects of Passive Smoking: Lung Cancer in Adults and Respiratorv Disorders in Children," made available at Science Advisory Board meeting, December 4 and 5, 1990. 3. Huber, G.L., Brockie, R.E., and Mahajan, V., "Smoke and Mirrors: The EPA's Flawed Study of Environmental Tobacco Smoke and Lung Caricer " Regulation 1993(3): 44-54, 1993. 4. Gori, G.B., "Science, Policy, and Ethics: The Case of Environmental Tobacco Smoke,-" Journal of Clinical Epidemiology 47: 325-334, 1994. 5. Gori, G.B., "Policy Against Science: The Case of Environmental Tobacco Smoke," Risk Analysis 15: 15-22, 1995. 6. Gross, A.J., "Uncertainties in Lung Cancer Risk Estimates Reported for Exposure to Environmental Tobacco Smoke," Environmetrics 6: 403-412, 1995. 7. Gross, A.J., "The Risk of Lung Cancer in Nonsmokers in the United States and Its Reported Association with Environmental Tobacco Smoke," Journal of Clinical Epidemioloav 48: 587-598, 1995. 8. Gori, G.B., "Reply to the Preceding Dissents," Journal of Clinical Epidemiologv 47: 351-353, 1994. 9. Sugita, M., Izuno, T., and Kanamori, M., "Recalculation of Summarised Odds Ratios for the Relationship Between Passive Smoking and Lung Cancer Based on Data in the EPA Report, " Indoor Environment 4: 177-181, 1995. 10. Gravelle, J.G., and Zimmerman, D., "Cigarette Taxes to Fund Health Care Reform: An Economic Analysis," CRS Report for Congress, Congressional Research Service, Library of Congress, March 8, 1994. 11. Redhead, C.S., and Rowberg, R.E., "Environmental Tobacco Smoke and Lung Cancer Risk," CRS Report for Congress, Congressional Research Service, Library of Congress, November 14, 1995. - 47 -
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Holz, et al., report that DNA adduct levels were compared in monocytes (a type of white blood cell) of smokers and "heavily exposed passive smokers," who had been exposed in a chamber."' DNA adducts above background were reported in smokers; they disappeared in less than 40 hours. The authors report no above-background adduct levels in study subjects exposed to ETS. In a study by Gorgels, et al., 50 self-reported ETS- exposed men ("passive smokers"; average 72.8 hours exposure per_ week) were compared with 56 self-reported low ETS-exposed men (average 5.1 hours per week).158 SCEs in cultured lymphocytes were examined; the authors report that "[n]o difference was observed between low exposed non-smokers and the passive smokers." They conclude: our results are in accordance with previous smaller studies in less homogeneous populations of non-smokers. These studies also failed to demonstrate even a tendency for an association between passive smoking and SCE levels. Five male smokers, five male nonsmokers, and five male nonexposed nonsmokers were compared in Holz and colleagues' 1993 paper.138 The endpoint examined was DNA single-strand breaks (SSBs), "considered to be an important parameter of genotoxic stress," in lymphocytes. The authors write: - 44 -
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69. Friedman, G., Pettiti, D., and Bawol, R., "Prevalence and Correlates of Passive Smoking," American Journal of Public Health 73(4): 401-405, 1983. 70. Phillips, K., Howard, D., Browne, D., and Lewsley, J., "Assessment of Personal Exposure to Environmental Tobacco Smoke in British Nonsmokers," Environment International 20(6): 693-712, 1994. 71. Lee, P., Environmental Tobacco Smoke and Mortality, Karger, Basel, Switzerland, 1992, pp. 85-110. 72. Kilpatrick, S., "Misclassification of Environmental Tobacco Smoke Exposure: Its Potential Influence on Studies of Environmental Tobacco Smoke and Lung Cancer," Toxicoloay Letters 35(1987): 163-186, 1987. _ 73. National Research Council, National Academy of Sciences, "Current and Anticipated Applications," in Human Exposure Assessment for Airborne Pollutants: Advances and Opportunities, Washington, D.C., National Academy Press, 1991, pp. 207-218. 74. Steenland, K., Testimony Before OSHA on Indoor Air Quality Proposed Rule, Transcript p. 1,963, 1994. 75. Coultas, D., Samet, J., McCarthy, J., and Spengler, J., "A Personal Monitoring Study to Assess Workplace Exposure to Environmental Tobacco Smoke," American Journal of Public Health 80(8): 988-990, 1990. 76. Lerchen, M., and Samet, J., "An Assessment of the Validity of Questionnaire Responses Provided by a Surviving Spouse," American Journal of Epidemiologv 123(3): 481-489, 1986. 77. Sandler, D., and Shore, D., "Quality of Data on Parents' Smoking and Drinking Provided by Adult Offspring," American Journal of Epidemiology 124(5): 768-778, 1986. 78. Pershagen, G., "Validity of Questionnaire Data on Smoking and Other Exposures, with Special Reference to Environmental Tobacco Smoke," European Journal of Respiratory Diseases 65(Suppl. 133): 76-80, 1984. 79. Brownson, R., Alavanja, M., and Hock, E., "Reliability of Passive Smoke Exposure Histories in a Case-Control Study of Lung Cancer," International Journal of Epidemiology 22: 804- 808, 1993. - 53 -
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12. Gravelle, J.G., and Zimmerman, D., Statement before the Subcommittee of Clean Air and Nuclear Regulation, Committee on Environment and Public Works,--United States Senate, on Environmental Tobacco Smoke, May 11, 1994. 13. Rodgman, A., "Environmental Tobacco Smoke," Recr Tox and Pharm 16: 223-244, 1992. 14. Baker, R. and Proctor, C., "The Origins and Properties of Environmental Tobacco Smoke," Env Int (16): 231-245, 1990. 15. Reasor, M., and Will, J., "Assessing Exposure to Environmental Tobacco Smoke: Is It Valid to Extrapolate from Active Smoking?" Journal of Smoking-Related Diseases 2(1): 111-127, 1991. 16. Guerin, M., et al., The Chemistry of Environmental Tobacco Smoke: Composition and Measurement. Chelsea, Michigan, Lewis Publishers, 1992. 17. Nystrom, C., et al., "Assessing the Impact of Environmental Tobacco Smoke on Indoor Air Quality: Current Status." In: Proceedings of the ASHRAE Conference, IAO '86. April 20-23, 1986, Atlanta, Georgia, 213-244, 1986. 18. U.S. Department of Health and Huinan Services, Public Health Service, Office on Smoking and Health, The Health Consequences of Involuntary Smoking: A Report of the Surgeon General, DHHS Publication No. (CDC) 87-8398, Washington, D.C., U.S. Government Printing Office, 1986. 19. Proctor, C. and Dymond, H., "The Measurement of ETS Through Adsorption/Desorption Procedures." In: Indoor Air Quality. H. Kasuga (ed.). Springer-Verlag, Berlin, Heidelberg, 82-89, 1990. 20. Rawbone, R., "The Aging of Sidestream Tobacco Smoke Components in Ambient Environments." In: Indoor Air Quality. H. Kasuga (ed.). Springer-Verlag, Berlin, Heidelberg, 55-61, 1990. 21. Piade, J., et al., "Assessment of ETS Impact on Office Air Quality." In: Indoor Air Quality. H. Kasuga (ed.). Springer-Verlag, Berlin, Heidelberg, 112-119, 1990. 22. Scherer, G., et al., "Importance of Exposure to Gaseous and Particulate Phase Components of Tobacco Smoke in Active and Passive Smokers," Occup Env Health (62): 459-466, 1990. - 48 -
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23. Sterling, T., et al., "Indoor Byproduct Levels of Tobacco Smoke: A Critical Review of the Literature," J Air Pollut Control Assoc 32(3): 250-259, 1982. 24. Sterling, T., et al., "Environmental Tobacco Smoke and Indoor • Air Quality in Modern Office Work Environments," J Occup Med 29(1): 57-62. 25. Pedelty, J. and Holcomb, L., "A Computer Simulation of Indoor Air Quality Which Models Changes in Point Sources and Ventilation," Environ Technol Letters 11: 1053-1062, 1990. 26. Adlkofer, F. "Biological Effects After Exposure to ETS." In: Indoor Air Ouality. Buenos Aires, The National Academy of 27. Sciences of Buenos Aires, 61-78, 1989. Kirk, P., et al., "Environmental Tobacco Smoke in Indoor Air." In: Indoor and Ambient Air Oualitv. R. Perry and P._ Kirk (eds.). London, Selper Ltd., 99-112', 1988. 28. Carson, J. and Erikson, C., "Results from Survey of Environmental Tobacco Smoke in Offices in Ottawa, Ontario," Environ Technol Letters 9: 501-508, 198 8._ 29. Sterling, T. and Mueller, B., "Concent rations of Nicotine, RSP, CO and COZ in Nonsmoking Areas of Offices Ventilated by Air Recirculated from Smoking Designated Areas," Am Ind Hya Assoc J 49(9): 423-426, 1988. 30. Cox, B. and Whichelcw, M., °Carbon Monoxide Levels in the Breath of Smokers and Nonsmokers: Effect of Domestic Heating Systems," J Epidemiol Communitv Health 39: 75-78, 1985. ~ 31_ Good, B., et al., "Effect of Cigarette Smoking on Residential °` NOz Levels," Environ Int 8: 167-175, 1982. co ? ~ 32.. Godish, T., "Formaldehyde Exposures from Tobacco Smoke: A 07 RaviaTa.T1 A.7PA 79(Rl e l(144-1(14`i_ 19R9_ 41 ~ 10 33. Proctor, C., "The Analysis of the Contribution of ETS to Indoor Air." In: Indoor and Ambient Air Ouality. R. Perry and P. Kirk (eds.). London, Selper_Ltd., 57-66, 1988. 34. Eatough, D., et al., "Assessing Exposure to Environmental Tobacco Smoke." In Indoor and Ambient Air Quality. R. Perry and P. Kirk (eds.). London, Selper Ltd., 131-140, 1988. - 49 -
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35. Proctor, C., et al., "Measurements of Environmental Tobacco Smoke in an Air-Conditioned Office Building,° Environ Technol Letters (10): 1003-1018, 1989. 36. Proctor, C., "A Multi-Analyte Approach to the Measurement of Environmental Tobacco Smoke." In: Indoor Air Quality and Ventilation. F. Lunau and G. Reynolds (eds.). London, Selper Ltd. 427-436, 1990. 37. Jenkins, R., et al., "Development and Application of a Thermal Desorption-Based Method for the Determination of Nicotine in Indoor Environments." In: Indoor and Ambient Air Ouality. R. Perry and P. Kirk (eds.) . London, Selper Ltd., 493-496, 1988. 38. Muramatsu, M., et al., "Estimation of Personal Exposure to Ambient Nicotine in Daily Environment,_"_ Arch Occup Environ, Health 59: 545-550, 1987. 39. Thompson, C., et al., "A Thermal Desorption method for the Determination of Nicotine in Indoor Environments," Environ Sci Technol 23: 529-435, 1989. 40. Hosein, R., "The Relationship Between Pollutant Levels in Homes and Potential Sources." In: Transactions: Indoor Air Oualitv in Cold Climates, Hazards and Abatement Measures. D. Walkinshaw (ed.). Pittsburgh, Air Pollution Control Association, 250-260, 1986. 41. Quackenboss, J. and Lebowitz, M., "Indoor-Outdoor Relationships for Particulate Matter: Exposure Classifications and Health Effects," Environ Int 15: 353-360, 1989. 42. Sterling, T., "ETS Concentrations Under Different Conditions of Ventilation and Smoking Regulation." In: Indoor and o ` Ambient Air Ouality. R. Perry and P. Kirk (eds.). London, m ;: . Selper Ltd., 89-98, 1988. G5 . Q, -.. ~ 43. First, M., "Constituents of Sidestream and Mainstream Tobacco w tn ~ Smoke and Markers to Quality Exposure to Them." In: I ndoor _ _ Air and Human Health. Chelsea, Michigan, Lewis Publishers, 195-203, 1985. 44. Bouhuys, A., et al., "Do Present Levels of Air Pollution Outdoors Affect Respiratory Health?," Nature 276: 466-471, 1978. - 50 -
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101. Nelson, P.R., "Comments on the Use of Nicotine as an Environmental Tobacco Smoke Biomarker," OSHA Docket H-122, Ex. 9-47,553, 1994. 102. Wagenknecht, L., Manolio, T., Sidney, S., Burke, G., and Haley, N., "Environmental Tobacco Smoke Exposure as Determined by Cotinine in Black and White Young Adults: The CARDIA Study," Environmental Research 63: 39-46, 1993. 103. Delfino, R., Ernst, P., Jaakkola, M., Solomon, S., and Becklake, M., "Questionnaire Assessments of Recent Exposure to Environmental Tobacco in Relation to Salivary Cotinine," European Respiratory Journal 6: 1104-1108, 1993. 104. Suadicani, P., Hein, H., and Gyntelberg, F., "Serum Validated Tobacco Use and Social Inequalities of Risk of Ischaemic Heart Disease," International Journal of Epidemiology 23: 293-300, 1994. 105. Tager, I., "Health Effects of Involuntary Smoking in the Workplace," NY State J of Med :27-30, 1989. 106. Garfinkel, L., Auerbach, 0., and Joubert, L., "Involuntary Smoking and Lung Cancer: A Case-Control Study," Journal of the National Cancer Institute 75: 463-469, 1985. 107. Wynder, E., Higgins, I., and Harris, R., "The Wish Bias," Journal of Clinical Eoidemiolocrv 43(6): 619-621, 1990. 108. Wynder, E., "Epidemiological Issues in Weak Associations," International Journal of-Epidemiology 19(3) (Suppl. 1): S5-S7, ~ 4 1990. w ~ . 109. Wynder, E. and Kabat, G., "Environmental Tobacco Smoke and w Lung Cancer: A Critical Assessment," in Indoor Air Ouality, u~ . ~ H. Kasuga (ed.), Springer-Verlag, Berlin, 1990, pp. 5-15. a 110. Thornton, A., et al., "Differences Between- Smokers, Ex- smokers, Passive Smokers, and -Non-Smokers," Journal of Clinical Enidemiology 47(1): 1143-1162, 1994. 111. Aviado, D., "Suspected Pulmonary Carcinogens in Environmental Tobacco Smoke," Environ. Tech. Letters, 9: 539-544, 1988. 112. Aviado, D., "Health Effects of 50 Selected Constituents of Environmental Tobacco Smoke," Indoor Air Quality, ed. H. Kasuga, Springer-Verlag, Berlin Heidelberg: 383-389, 1990. - 56 -
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P., "Carcinogen Hemoglobin Adducts, Urinary Mutagenicity, and Metabolic Phenotype in Active and Passive Cigarette Smokers," Journal of the National Cancer Institute 82: 1826-1831, 1990. 155. Sorsa, M., et al., "Cytogenetic Effects of Tobacco Smoke Exposure Among Involuntary Smokers," Mut Res 222: 111-116, 1989. 156. Collman, G.W., Lundgren, K., Shore, D., Thompson, C.L_, and Lucier, G.W., "Effects of alpha-Naphthoflavone on Levels of Sister Chromatid Exchanges in Lymphocytes from Active and Passive Cigarette Smokers: Dose-Response Relationships," Cancer Research 46: 6452-6455, 1986. 157. Husgafvel-Pursiainen, K., "Sister-Chromatid Exchange and Cell Proliferation in Cultured Lymphocytes of Passively and Actively Smoking Restaurant Personnel," Mutation Research 190: 211-215, 1987. 158. Gorgels, W.J.M.J., van Poppel, G., Jarvis, M.J., Stenhuis, W., and Kok, F.J., "Passive Smoking and Sister-Chromatid Exchanges in Lymphocytes," Mutation Research 279: 233-238, 1992. 159. Doolittle, D.J., "Comments on Environmental Tobacco Smoke and Genotoxicity," OSHA Docket H-122, Ex. 9-47,553, 1994.
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45. Binder, R., et al., "Importance of the Indoor Environment in Air Pollution Exposure," Arch Environ Health 31(6): 277-279, 1976. 46. Lebowitz, M., et al., "Respiratory Symptoms and Peak Flow Associated with Indoor and Outdoor Air Pollutants in the Southwest," J. Air Pollut Control Assoc 35: 1154-1158, 1985. 47. McAughey, J., et al., "Risk Assessment of Exposure to Indoor Air Pollutants," Env Int (il): 295-302, 1990. 48. Stehlik, G., et al., "Concentration of Dimethylnitrosamine in the Air of Smoke-Filled Rooms," Ecotoxicol Environ Safetv 6: 495-500, 1982. 49. Sega, K., and Fugas, M., "Nitrogen Dioxide Concentrations in Residences." In: Indoor and Ambient Air Oualitv. R. Perry and P. Kirk (eds.). London, Selper Ltd., 493-496, 1988. 50. Bayer, C. and Black, M., "Thermal Desorption/Gas Chromatographic/Mass Spectrometric Analysis of Volatile Organic Compounds in the Offices of Smokers and Nonsmokers," Biomed and Envir Mass Spect 14(8): 363-367, 1987. 51. Adlkofer, F., et al., "Significance of Exposure to Benzene and Other Toxic Compounds Through Environmental Tobacco Smoke," J Cancer Res Clin Oncol (116): 591-598, 1990. 52. Godish, T., "Residential Formaldehyde: Increased Exposure Levels Aggravate Adverse Health Effects," J. of Environ Health 53(3): 34-35, 1990. 53. Hugod, C., et al., "Exposure of Passive Smokers to Tobacco Smoke Constituents," Int Arch Occup Environ Health (42): 21- 29, 1978. 54. Proctor, C., et al., "Measurement of Environmental Tobacco Smoke in an Air-Conditioned Office Building." In: Present and Future of Indoor Air Quality. C.J. Bieva, et al. (eds.) Brussels, Elsevier Science Publishers, 169-172, 1989. 55. Hedge, A., et al., "Effects of Restrictive Smoking Policies on . Indoor Air Quality and Sick Building Syndrome: A Study of 27 ~' Air-Conditioned Offices," Indoor Air '93 1: 517-522, 1993. - 56. Oldaker, G., et al., "Investigations of Ventilation, Smoking Activity and Indoor Air Quality at Four Large Office Buildings," IAO '92: Environments for People, Atlanta, ASHRAE, 1992: 248-257. - 51 -
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143. Lofroth, G., et al., "Passive Smoking and Urban Air Pollution: Salmonella/Microsome Mutagenicity Assay of Simultaneously Collected Indoor and Outdoor Particulate Matter," Env Sci Res 27: 515-525, 1983. 144. Alfheim, I., and Ramdahl, T., "Contribution of Wood Combustion to Indoor Air Pollution as Measured by Mutagenicity in Salmonella and Polycyclic Aromatic Hydrocarbon Concentration,° Environ Mutaa 6: 121-130, 1984. - 145. Lewtas, J., et al., "Human Exposure to Mutagens from Indoor Combustion Sources." In: Indoor Air '87. B. Seifert, et al., (eds.). Berlin, Institute for Water, Soil and Air Hygiene, 473-482, 1987. 146. Ling, P., et al., "Mutagenic Determination of Passive Smoking," Tox Letters 35: 147-151, 1987. 147. Lofroth, G., et al., "Public Exposure to Environmental Tobacco Smoke," Mutat Res 202: 103-110, 1988. 148. Ames, B.N., Profet, M., and Gold, L.S., "Dietary Pesticides (99.99°s All Natural)," Proc Natl Acad Sci USA 87: 777-7781, 1990. 149. Sonnenfeld, G., and Wilson, D.M., "The Effect of Smoke Age and Dilution on the Cytotoxicity of Sidestream (Passive) Smoke," Toxicoloav Letters 35: 89-94, 1987. 150. Scherer, G., Conze, C., Tricker, A.R., and Adlkofer, F., "Uptake of Tobacco Smoke Constituents on Exposure to Environmental Tobacco Smoke (ETS)," Clinical Investigator 70; 352-367, 1992. 151. Husgafvel-Pursiainen, K., et al., "Passive Smoking at Work: Biochemical and Biological Measures of Exposure to Environmental Tobacco Smoke," Int Arch Occup Environ Health 59: 337-345, 1987. 152. Bos, R.P., Theuws, J.L.G., and Henderson, P.T., "Excretion of_ Mutagens in Human Urine After Passive Smoking," Cancer Letters 19: 85-90, 1983. 153. Mohtashamipur, E.; Muller, G., Norpoth, K., Endrikat, M., and Stucker, W., "Urinary Excretion of Mutagens in Passive Smokers," Toxicology Letters 35: 141-146, 1987. 154. Bartsch, H., Caporaso, N., Coda, M., Kadlubar, F., Malaveille, C., Skipper, P., Talaska, G., Tannenbaum, S.R., and Vineis, - 60 -
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132. Sorsa, M., et al., "Cytogenetic Effects of Tobacco Smoke Exposure Among Involuntary Smokers," Mutation Res 222(2): 111-116, 1989. 133. Husgafvel-Pursiainen, K., "Sister-Chromatid Exchange and Cell Proliferation in Cultured Lymphocytes of Passively and Actively Smoking Restaurant Personnel," Mutation Res 190: 211-215, 1987. 134. Scherer, G., Westphal, K., Biber, A., Hoepfner, I., and Adlkofer, F., "Urinary Mutagenicity After Controlled Exposure to Environmental Tobacco Smoke," Toxicology Letters 35: 135- 140, 1987. 135. Bombick, D., et al., "Assessment of the Biological Activity of Mainstream or Environmental Tobacco Smoke (ETS) Using a Cellular Smoke Exposure Technique," Abstracts of the Twenty- Second Annual Scientific Meeting of the Environmental Mutaaen Society, Kissimmee, Florida: April 1991. Abstract. 136. Scherer, G., Westphal, K., Adlkofer, F., and Sorsa, M., "Biomonitoring of Exposure to Potentially Genotoxic Substances from Environmental Tobacco Smoke," Environment International 15: 49-56, 1989. 137. Ames, B.N., "Mutagenesis and Carcinogenesis: Endogenous and Exogenous Factors," Environmental and Molecular Mutaaenesis 14 (Suppl. 16): 66-77, 1989. 138. Holz, 0., Meissner, R., Einhaus, M., Koops, F., Warncke, K., Scherer, G., Adlkofer, F., Baumgartner, E., and Rudiger, H.W., "Detection of DNA Single-Strand Breaks in Lymphocytes of Smokers," International Archives of _ Occupational and Environmental Health 65: 83-88, 1993. 139. Coggins, C.R.E., Testimony Before OSHA on Indoor Air Quality Proposed Rule, Transcript p. 11,684, 1994. 140. Ames, B.N., and Gold, L.S., "Pesticides, Risk, and Applesauce," Science 244: 755-757, 1989. 141. Ashford, J., Testimony Before OSHA on Indoor Air Quality Proposed Rule, Transcript p. 726, 1994. 142. Nikula, K.J., Snipes, M.B., Barr, E.B., Griffith, W.C., Henderson, R.F., and Mauderly, J.L., "Comparative Pulmonary Toxicities and Carcinogenicities of Chronically Inhaled Diesel_ Exhaust and Carbon Black in F344 Rats," Fund Appl Toxicol 25: 80-94, 1995. - 59 -
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80. Kolonel, L., et al., "Adequacy of Survey Data Collected from Substitute Respondents," Am J Epidemiology 106: 476-484, 1977. 81. O'Connor, T., Leaderer, B., Holford, T., and Bracken, M., "Measurement of Exposure to Environmental Tobacco in Pregnant Women Using Questionnaire, Personal Monitoring and Urine Cotinine: A Problem in Exposure Modelling," Proceedings of Indoor Air '93 3: 373-378, 1993. 82. Schenker, M., Hammond, K., Samuels, S., Kado, N., and Woskie, S., "Assessment of Environmental Tobacco Smoke Exposure in Epidemiologic Studies," Chest 91(2): 313-314, 1987. 83. Jenkins, R., et al., "Determinations of Personal Exposure of Nonsmokers to Environmental Tobacco Smoke in the United States," in International Svmposium on Lifestvle Factors and Human Lung Cancer, China, 1994. 84. Moschandreas, D., and Relwani, S., "Perception of Environmental Tobacco Smoke Odors: An Olfactory and Visual Response," Atmospheric Environment 26B(3): 263-269, 1992. 85. Holcomb, L., "Indoor Air Quality and Environmental Tobacco Smoke; Concentration and Exposure," Environment International 19: 9-40, 1993. 86. Cummings, K., Markello, S., Mahoney, M., Bhargaa, A., McElroy, P., and Marshall, J., "Measurement of Current Exposure to Environmental Tobacco Smoke," Archives of Environmental Health 45(2): 74-79, 1990. N , 87. Emmons, K., Abrams, D., Marshall, R., Marcus, B., Kane, M., o e- Novotny, T., and Etzel, R., "An Evaluation of the Relationship Between Self-Report and Biochemical Measures of Environmental Tobacco Smoke Exposure," Preventive Medicine 23: 35-39, 1994. 0+` w ~- r.,o tn ' . ~ 88. Etzel, R., "A Review of the Use of Saliva Cotinine as a Marker of Tobacco Smoke Exposure," Preventive Medicine 19: 190-197, 1990. 89. Tunstall-Pedoe, H., et al., "Passive Smoking by Self-Report and Serum Cotinine and the Prevalence of Respiratory and Coronary Heart Disease in the Scottish Heart Health Study," J Epi Comm Health 49: 139-143, 1995. 90. Riboli, E., Haley, N., Tredaniel, J., Saracci, R., Preston- Martin, S., and Trichopoulos, D., "Misclassification of Smoking Status Among Women in Relation to Exposure to - 54 -
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57. Sterling, T., et al., "Environmental Tobacco Smoke and Indoor Air Quality in Modern Office Work Environments," Journal of Occupational Medicine 26(1): 57-62, 1987. 58. Hinds, W., and First, M., "Concentrations of Nicotine and Tobacco Smoke in Public Places," New England Journal of Medicine 292(16): 844-845, 1975. 59. Holcomb, L., "Indoor Air Quality and Environmental Tobacco Smoke: Concentration and Exposure," Environment International 19: 9-40, 1993. 60. Girman, J., and Traynor, G., "Indoor Concentrations," JAPCA 33(2): 89, 1983. 61. Klus, H., et al., "Tobacco Specific Volatile N-Nitrosamines in Environmental Tobacco Smoke of Offices," Indoor Environ 1: 348-350, 1992. 62. Tricker, A., et al., "Tobacco-Specific and Volatile N- Nitrosamines in Environmental Tobacco Smoke," Proceedinas of Indoor Air '93 3: 47-52, 1993. 63. Coultas, D., et al., "Questionnaire Assessment of Lifetime and Recent Exposure to Environmental Tobacco Smoke," American Journal of Epidemioloav 130 (2) : 338-347, 1989. 64. Coultas, D., et al., "Variability of Measures of Exposure to Environmental Tobacco Smoke in the Home," American Review of Respiratorv Disease 142: 602-606, 1990. 65. Pron, G., et al., "The Reliability of Passive Smoking Histories Reported in a Case-Control Study of Lung Cancer," American Journal of Epidemiology 127(2): 267-273, 1988. 66. Farland, W., Statement, U.S. House of Representatives, House Agriculture Subcommittee on Specialty Crops and Natural Resources, Committee on Agriculture, Washington, D.C., July 21, 1993, p.9. 67. Axelrad, R., et al., "Setting the Record Straight: Secondhand w m Smoke is a Preventable Health Risk." Tobacco Control 3:- 263- ~ 267, 1994. w cn ~ 68. Sorahan, T., and Gilthorpe, M., "Non-differential Misclassification of Underestimation of Exposure Risk: An Always Leads to an Incorrect Conclusion," Occupational and Environmental Medicine 61: 839-840, 1994. - 52 -
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Specifically, Gori (1995) comments about the ETS epidemiology:' The weak data on ETS are compatible with either a slight increase or a decrease of risk, but are impotent to certify either conclusion on a scientific basis. In the unlikely hypothesis that ETS were a risk, it would have to be small beyond detection because of the inevitable and excessive noise to signal ratios of its epidemiology. (p. 20) (emphasis added) 1. Cal/EPA's Discussion of the Potential Impact of Confounders in the Incomplete Spousal Smoking Studies Is In the Excerpt, Cal/EPA essentially dismisses the possibility that confounding factors could contribute to the increased lung cancer risk reported in some of the spousal smoking studies. (p. 22) Cal/EPA cites only a few studies other than some of the spousal smoking studies themselves, in a review that is clearly incomplete and selective. The use of spousal smoking status as a proxy for ETS exposure introduces substantial uncertainties into estimates of risk. Spousal smoking (marriage to a smoker) does not measure ETS exposure alone, but rather encompasses numerous variables that may be related to lung cancer risk.e-12 These other variables, or - 2 -
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113. Coggins, C.R.E., "Comments on Environmental Tobacco Smoke Work with Experimental Animals," OSHA Docket H-122, Ex. 10-170, 1994. 114. Newell, G., "Re: Filing of Testimony and Evidence -- Hearing on Proposed Rule on Indoor Air Quality," OSHA Docket H-122, Ex. 10-206, 1994. - 115. Gori, G.B., "Comment on the Proposed Rule on Occupational Exposure to Indoor Air Pollutants Issued by the Occupational Safety and Health Administration," OSHA Docket H-122, Ex. 9- 47,592, 1994. 116. Philip Morris, Submission on OSHA's Proposed Rule on Indoor Air Quality, OSHA Docket H-122, Ex. 9-47,500, 1994. 117. Rodgman, A., "The Chemical Composition of Environmental Tobacco Smoke: Some Comments on the Occupational Safety and Health Administration's Notice on 'Indoor Air Quality' (OSHA, 1994)," OSHA Docket H-122, Ex. 9-47,532, 1994. 118. Haley, N.J., Adams, J.D., Axelrad, C.M., and Hoffmann, D., "Sidestream Smoke Uptake by Syrian Golden Hamsters in an Inhalation Bioassay." In: Indoor Air '87: Proceedings of the 4th International Conference on Indoor Air ouality and Climate (VOl. 2). B. Seifert, et al. (eds.) . Berlin, Institute for Water, Soil and Air Hygiene, 68-75, 1987. 119. Haley, N.J., Adams, J.D., Alzofon, J., and Hoffmann, D., "Uptake of -Sidestream Smoke by Syrian Golden Hamsters," Toxicoloay Letters 35: 83-88, 1987. 120. von Meyerinck, L., Scherer, G., Adlkofer, F., Wenzel-Hartung, R., Brune, H., arid Thomas, C., "Exposure of Rats and Hamsters to Sidestream Smoke from Cigarettes in a Subchronic Inhalation Study," Experimental Pathology 37: 186-189, 1989. 121. Adlkofer, F., Scherer, G., Wenzel-Hartung, R., Brune, H., aind Thomas, C., "Exposure of Hamsters and Rats to Sidestream Smoke of Cigarettes: Preliminary Results of a 90-Day-Inhalation Study." In: Indoor and Ambient Air oualitv. R. Petty and P.W. Kirk (eds.). London, Selper Ltd., 252-258, 1988. 122. Teredesai, A., and Pruehs, D., "Histopathological Findings in the Rat and Hamster Respiratory Tract in a 90-Day Inhalation Study Using Fresh Sidestream Smoke of _the Standard Reference Cigarette 2R1." In: Toxic and Carcinogenic Effects of Solid Particles in the Respiratory Tract. U. Mohr, D.L. Dungworth, - 57 -
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Environmental Tobacco Smoke," European Respiratory Journal 8: 285-290, 1995. 91. Riboli, E., et al., "Exposure of Nonsmoking Women to Environmental Tobacco Smoke: A 10-Country Collaborative Study," Cancer Causes and Control 1(3): 243-252, 1990. 92. Jarvis, M., Tunstall-Pedoe, H., Feyerabend, C., Vesey, C., and Salloojee, Y., "Biochemical Markers of Smoke Absorption and Self Reported-Exposure to Passive Smoking,° Journal of Epidemiology and Community Health 38: 335-339, 1984. 93. Reasor, M., "Biological Markers in Assessing Exposure to Environmental Tobacco Smoke," Environmental Tobacco Smoke: Proceedings of_ the International Svmbosium at McGill University, D. Ecobichon and J. Wu (eds.), Lexington Books, Toronto, 1990: 69-77. 94. Heller, W., Sennewald, E., Gostomzyk, J., Scherer, G., and Adlkofer, F., "Validation of ETS Exposure in a Representative Population in Southern Germany," Proceedings of Indoor Air '93 3: 361-365, 1993. 95. Idle, J., "Commentary: Titrating Exposure to Tobacco Smoke. Using Cotinine - A Minefield of Misunderstandings," Journal of Clinical Epidemioloav 43(4): 313-317, 1990. 96. Ogden, M., Davis, R., Maiolo, K., Stiles, M., Heavner, D., Hege, R., and Morgan,_W., "Multiple Measures of Personal ETS Exposure in a Population-Based Survey of Nonsmoking Women in Columbus, Ohio," Proceedings of Indoor Air '93 1: 523-528, 1993. 97. Roberfroid, M., "Correlating Exposure to Environmental Tobacco Srcioke Exposure With Increased Incidence of Lung Cancer in Non- smokers: Is Cotinine a Valid Marker?" The Cancer Journal 7(3): 108-114, 1994. 98. Swan, G., Habina, K., Means, B., Jobe, J., and Esposito, J., "Saliva Cotinine and Recent Smoking - Evidence of a Nonlinear Relationship," Public Health Reports 106(6): 779-783, 1993. 99. Davis, R., "Dietary Nicotine: A Source of Urinary Cotinine," Fundamentals of Chemical Toxicology 29(12): 821-827, 1991. 100. Seaton, M., and Vesell, E., "Variables Affecting Nicotine Metabolism," Pharmac Ther 60: 461-500, 1993. - 55 -
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Since diet is associated with lung cancer risk, it could well be a confounder in the spousal smoking studies which have failed to adequately account for diet in their analyses. The magnitude of the risks reported for some dietary exposures suggests that if only a few cases in a study had such exposures, it could have a significant impact on the risk estimates attributed to ETS exposure. The following are brief comments on some of the recent literature that illustrates the correlation between diet and other lifestyle factors and household smoking status. • A 1992 British study examined the consumption of fried foods, fats, fruits, vegetables, and sweets in smokers, nonsmokers, and exsmokers.22 The authors reported that_nonsmokers who live in smoking households "have a diet more like smokers," and that "diet could be an important confound in epidemiological studies of ETS." The authors also noted; Our analysis showed that non-smokers in smoking households ate fried food more often, more chips [french fries] , less fruit in winter, more butter and less margarine high in polyunsaturates than non-smokers in non- smoking households. As we have pointed out, these habits are thought to increase the probability of cancer. These results suggest that it is wise to show caution when interpreting the disease patterns - 5 -
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higher risk of disease." (emphasis added) The authors also point out the importance of considering these factors, particularly when investigating weak associations in= epidemiology, such as those claimed for ETS. • Matanoski, et al. (1995), report on analyses of data from the NHANES I study, comparing nutritional and behavioral characteristics between nonsmoking women whose husbands smoked and nonsmoking women whose husbands were nonsmokers.29 Nonsmoking women with smoking spouses were statistically significantly more likely to have an urban residence, to consume beef and the skin on poultry (both suggestive of increased fat intake), to drink alcohol, and to consume less of certain vitamins and other nutrients than did nonsmoking women married to nonsmokers. The authors summarize the > -. ra ~ rn W importance of their findings as follows: ~' ~ .. (n cn . co [E]xposure to household tobacco smoke may not represent just a_single_exposure but a complex of factors, many of which, such as low vitamin intake and high alcohol intake, have been shown to influence the risk of cancer. While the Matanoski, et al., study is referenced in the Cal/EPA Excerpt, the treatment is superficial and does not give adequate attention to the importance of this study's findings. - - 7 -
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J.L. Mauderly, and G. Oberdorster (eds.). Washington, ILSI Press, 629-635, 1994. 123. Coggins, C.R.E., et al., "Fourteen-Day Inhalation Study in Rats, Using Aged and Diluted Sidestream Smoke from a Reference Cigarette. I. Inhalation Toxicology and Histopathology," Fundamental and Applied Toxicoloav 19: 133-140, 1992. 124. Coggins, C.R.E., et al., "Subchronic Inhalation Study in Rats Using Aged and Diluted Sidestream Smoke from a Reference Cigarette," Inhalation Toxicoloav 5: 77-96, 1993. 125. Brown, B.G., Bombick, B.R., McKarns, S.C., Lee, C.K., Ayres, P.H., and Doolittle, D.J., "Molecular Toxicology Endpoints in Rodent Inhalation Studies," Experimental Toxicolocry and Patholoav 47: 183-191, 1995. 126. Witschi, H., et al., "Six-Month Exposure of Strain A/J Mice to Cigarette Sidestream Smoke: Cell Kinetics and Lung Tumor Data," Fund Appl Toxicol 26: 32-40, 1995. 127. Martin, F., et al., "Urinary Excretion of Hydroxy- Phenanthrenes After Intake of Polycyclic Aromatic Hydrocarbons," Environ Int (15): 41-47, 1989. 128. Hoepfner, H., et al., "Hydroxy-Phenanthrenes in the Urine of Non-Smokers and Smokers," Toxicoloay Letters (35): 67-71, 1987.. 129. Scherer, G., Westphal, K., Sorsa, M., and Adlkofer, F., "Quantitative and Qualitative Differences in Tobacco Smoke Uptake Between Active and Passive Smoking." In: Indoor and Ambient Air Oualitv. R. Perry and P.W. Kirk (eds.). London, Selper Ltd., 189-194, 1988. 130. Scherer, G., Westphal, K., and Adlkofer, F., "Urinary Mutagenicity, Hydroxyphenanthrene, and Thioether Excretion After Exposure to Environmental Tobacco Smoke." In: Indoor Air Ouality. H. Kasuga (ed.). Berlin, Heidelberg, Spri_nger- Verlag, 138-146, 1990. 131. Holz, 0., Krause, T., Scherer, G., Schmidt-Preuss, U., and Rudiger, H.W., "32P-Postlabelling Analysis of DNA Adducts in Monocytes of Smokers and Passive Smokers," International Archives of Occupational and Environmental Health_62: 299- 303, 1990. - 58 -
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SECTION IV: CAL/EPA'S TREATMENT OF THE EPIDEMIOLOGIC DATA OVERLOOKS A NUMBER OF IMPORTANT ISSUES The following discussion will address a number of problems with Cal/EPA's treatment of the epidemiologic literature on ETS exposures and lung cancer in the Excerpt. A selection of relevant references is submitted as Appendix II. A. Cal/EPA Fails to Acknowledge the Magnitude of the Limitations of Weak Association Epidemiology Members of the scientific community have criticized the epidemiologic studies on ETS exposure and lung cancer for failing to consider certain factors, namely bias and confounding, that could affect the validity of the studies' risk estimates. The impact of such factors is particularly important in studies that, like these studies, report risk estimates that are "weak."1•z A weak association is represented by a risk estimate of less than 2.0 or perhaps even less than 3.0.3fi As Wynder notes:' [E]pidemiology has problems when the associations are of a low order of magnitude. In such instances, findings in the literature are, in general, inconsistent. . . When risks are small, and especially when effects occur many years after their causes, detecting them, estimating their magnitude, and assessing their importance for the community in light of other relevant factors pose problems of study design, data collection, analysis, and interpretation which can be exceedingly difficult. (p. 139)
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confounding factors, are factors associated with the exposure being studied (here, spousal smoking status) and with an increased risk of the outcome under consideration (in these studies, lung cancer). Confounders may seriously affect estimations of risk purportedly due to ETS exposure. As noted in a recent review:l' Because the relative risks or odds ratios for human diseases reported to be associated with ETS exposure are typically no larger than the risks for confounding lifestyle factors, epidemiological studies of' the association between ETS exposure and chronic disease should be designed to maximize data quality and statistical power. The ETS and chronic disease epidemiology studies conducted to date have not adequately controlled for all of the known confounding variables. Few of the spousal smoking studies upon which Cal/EPA relies have taken even some of the many potential confounding factors into account. Therefore, the possible impact of__ confounders on risk estimates attributed to ETS is of paramount importance. Since actual ETS exposures are not measured in these studies, where reported spousal smoking status is used as a surrogate for ETS exposure, the risk estimates claimed for_ETS are instead risk estimates for "marriage to a smoker." A number of 3
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have to apply to all the spousal smoking studies; if they influenced only a few of the studies, U.S. EPA's and Cal/EPA's position would be seriously affected. For instance, recent publications have reported the following lung cancer risk factors: body weight," history of radiation therapy,34 occupation,35 previous lung disease36 and family history of lung cancer." Moreover, in some cases, the risks reported for these other factors are far in excess of the risk estimates suggested for spousal smoking.3° As pointed out in the Report of the Independent Working Group to the Health Care Committee of the Australian National Health & Medical Research Council (NH&MRC),.while it is difficult for epidemiologic methods to detect very low relative risks, it is o conceivable that factors having low relative risks could still impact the outcome of a study.38 The Report notes: [T]hree factors, each having a relative risk of 1.1, if present together would induce (assuming no interaction positively or negatively between them) an observed relative risk of_ 1.33. It is impossible that confounding risk factors of this magnitude would be detected in the studies we have examined. Hence although these risk factors would not be detected, they could, in aggregate, produce a relative risk at least as large as that observed for exposure to ETS. - 9 -
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potential lung cancer risk factors are associated with household smoking status.14•15 One of the most important potential confounders of the claimed ETS-lung cancer relationship is dietary differences between "smoking" and "nonsmoking" households. [A "smoking" household is one in which at least one person smokes; a "nonsmoking" household contains no smokers.] In epidemiologic studies, when a nonsmoking case or control reports marriage to a smoker, this would correspond to living in a smoking household. Diet appears to be a true confounder of the spousal smoking-lung cancer relationship.ls•1' Diet satisfies the first necessary condition for being a confounder by being associated with lung cancer risk, as has been shown in a number of studies..a, s Second, dietary differences are associated with household smoking status; that is, the diets of nonsmokers living with smokers differ from the diets of nonsmokers living with nonsmokers. Data show that smokers' diets are generally different from nonsmokers' diets.20-28 In particular, the data, already suggestive of differences in diet according to household smoking status, have been dramatically strengthened by the addition of several recent studies.a,as-az Taken as a whole, the data strongly support an association between household smoking status and diet. - 4 -
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of non-smokers in smoking households. Studies to date have failed to take into account the effect that differences in dietary and lifestyle behaviour between 'smoking' households and 'non-smoking' households may have on the incidence of cancer or heart disease. In a 1993 paper, the same authors reported that, in addition to having higher intakes of saturated fats, never smokers living in smoking households consumed fats more often, drank more alcohol, and ate fewer root vegetables and cereal than did never smokers living in nonsmoking households.23 • Thornton, et al. (1994), examined 33 lifestyle factors in a survey of 9,003 British adults.e They report: [L]ike current smokers, passive smokers tended to be less educated; of lower social class; work in `risky' occupations; drink more alcohol; do nothing to keep healthy; take longer before their first meal of the day; eat more fried foods and bread; eat less cereal, fruits, salads and low fat/polyunsaturated spread; drink more tea (but not more coffee); use more sugar in tea and coffee; not cut down on fatty foods; and be more neurotic and extrovert. Thornton, et al., summarize their study as follows: "It has not perhaps been documented clearly before that smokers and non-smokers differ in so many lifestyle characteristics and that these are nearly always in the direction of predicting a - 6 -
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relative risk estimate to include potential contributive effects (including confounding) from both spousal and social exposure. [T]he absence of dose from the 'risk equation' necessitates reliance upon the recall of exposures that may have taken place decades earlier, often by a surrogate respondent. Moreover, recent publications have raised additional questions about the Fontham, et al., study.36,48,49 _Perhaps the most serious questions about the study are raised in a post-hearing brief submitted to the U.S. OSHA record that points out what appears to be a significant misinterpretation.`F9 In that submission, William Butler focuses on the data in the Fontham, et al., study concerning women who reported both childhood and adult ETS exposure. Fontham, et al., originally presented risk estimates for adult ETS exposure regardless of childhood ETS exposure status. Butler divided the study subjects into four categories: (i) neither childhood nor adult ETS exposure (the reference group); (ii) childhood but not adult exposure; (iii) adult but not childhood exposure; and (iv) both childhood and adult exposure. He calculated a statistically significantly negative lung cancer risk for women with childhood but not adult exposure (OR = 0.35, 95o CI 0.12-0.99), which, he proposes, is a result of some bias in study design or data collection. Butler states that "Fontham et al.'s - 16 -
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Similarly, a 1992 review also addressed the possibility that confounding factors may have a combined effect on estimations of lung cancer risk:zs In the absence of calculations of lung cancer risk when multiple factors apply, one can only speculate on the combined effect on an individual who, for example, might have a family history of lung cancer (RR = 2-4), lived in an urban area (RR = 1.2-2.8), worked in an occupation associated with elevated lung cancer risk (RR = 2 or more), was among the physically less active groups of the population (RR = 2) and, if a female, had the risk associated with a short menstrual cycle (RR = 2.2). Until epidemiologic studies fully account for the possible impact of confounders and independent risk factors on estimates of lung cancer risk for spousal smoking, those risk estimates must be viewed with caution and carefully interpreted. There is no indication in the Excerpt that Cal/EPA has done this. As Gio Batta Gori, Sc.D., writes in a comment in the U.S. OSHA public record:ll - [A]ttributions of epidemiologic risk to ETS cannot be rationally sustained unless confounders and biases have been convincingly controlled, and adjustments have been objectively justified. _ - 10 -
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• Emmons and colleagues (1995) also compared the diets of persons living with smokers and living with nonsmokers.30 Their data further support the differences between smoking and nonsmoking households: persons living with smokers had less healthy diets overall, consuming more fat and less fiber, fewer fruits and vegetables, and fewer micronutrients than did nonsmokers who lived with nonsmokers. All the reported differences were statistically significant. The importance of the potential impact of dietary confounding on reported estimates of lung cancer risk from the spousal smoking studies was described by Layard in a submission to U.S. OSHA's rulemaking record.10 Dietary confounding alone could easily be large enough to explain the summary U.S. spousal smoking-lung cancer relative risk of 1.09 from [Layard's] meta-analysis of female studies. Other data suggest the importance of numerous factors associated with cancer risk that may affect the reported results of the spousal smoking studies. Few, if any, of these risk factors have been considered in the epidemiologic studies on lung cancer, nor has U.S. EPA or Cal/EPA convincingly shown that these other confounders or independent risk factors for-lung cancer may be discounted. These potential confounders or risk factors would not - 8 -
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2. Cal/EPA Has Inappropriately Dismissed the Potential Impact of Several Sources of Bias in the Spousal Smoking Studies When interpreting the results of epidemiologic studies, the potential effects of biases must be considered. Bias refers to factors in the design, conduct, analysis, or interpretation of an epidemiologic study that erroneously lead to the appearance of a stronger or weaker association than in fact exists. Bias (and confounding) becomes particularly important when dealing with weak associations, i.e., risk estimates of 3.0 or less.'-3-e,e One important type of bias particular to the spousal smoking studies is smoking status misclassification bias..o,3s Smoking status misclassification occurs when smokers erroneously report themselves as nonsmokers in response_ to study questionnaires. None of the spousal smoking studies to date has been able to discount smoking status misclassification as a potential source of bias.'o It has been implied that the Fontham, et al., study41 is superior to the other spousal smoking studies because it had accounted for- smoking status misclassification. This is an incorrect portrayal. While Fontham and colleagues did the best they could to exclude current active smokers from among cases and controls, based on cotinine measurements, cotinine does not allow - 11 - -_ . _-
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the determination of past smoking status.12 Moreover, Fontham, et al., measured cotinine in hospitalized cases. The vast majority of hospitals severely restrict smoking (in,_fact, accreditation requires that smoking be banned); moreover, many lung cancer patients who happen to be smokers stop smoking after diagnosis. Thus, the cotinine measurements in this study did not even give a good indication of present smoking status, let alone previous long- term smoking status. The smoking status misclassification rates portrayed by Fontham, et al., as accurate are, in reality, not representative of the true situation.hz Smoking status misclassification could have a dramatic impact on the reported risk estimates in the Fontham, et al., study, and in other spousal smoking studies. For instance, a recent Congressional Research Service (CRS) Report on ETS calculated that, in the Fontham, et a1.,41 and Brownson, et al.,43 studies, smoking status misclassification rates of less than 10 percent alone would account for all the reported elevation in risk at the highest exposure levels, and that misclassification rates of less than three percent would mean that those risk estimates would not achieve statistical significance at the 95 percent leve1.44 Such misclassification rates are certainly possible, according to a recent review of the literature on this subject.92 - 12 -
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In addition to smoking status misclassification, another potentially important source of bias in the epidemiologic studies on ETS is misclassification of disease status, i.e., diagnosis of lung cancer. In many of the spousal smoking studies, disease diagnosis is haphazard and incomplete. Even in some of the better- designed studies, the possibility that tumors appearing in the lung may have metastasized from other sites remains likely. None of the studies confirmed lung tumor diagnosis via autopsy. In a recent study, Kaye, et al., reported that in "emotionally charged situations," misclassification of disease diagnosis could inflate cancer risk estimates by some 30 percent.45 Self-reports of cancer were compared with medical diagnoses of cancer for two groups of-people. One group lived.,in a community with a hazardous waste treatment facility (test population); the other (control population) did not. The risk estimate for malignant tumors for the test population decreased by 31 percent when the more precise medical diagnoses were used instead of self- reports of having had cancer. The authors conclude: This study demonstrates the importance of verifying reported cases of disease, even a disease as well defined as cancer, in emotionally charged situations such as living in communities surrounding hazardous waste sites. If reported cases of cancer had not been verified, it would have incorrectly - 13 -
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"no biomarker of lifetime tobacco use."91 Moreover, only slightly more than half (54a) of cases had cotinine determinations, so that even recent active smoking was not excluded for nearly half of the cases. 41 While the Cal/EPA Excerpt repeatedly stresses that the Fontham, et al., study is a multicenter case-control study, if the characteristics of the study population are examined, it is seen that the vast majority -- 81 percent and 86 percent, respectively - - of cases and controls come from two areas in California (Los Angeles and the San Francisco Bay area) . Given this breakdown, the "multicenter" label is fairly misleading. Moreover, the authors provide no breakdown of the data by study center, and it is not possible to ascertain whether the reported risks were consistent across the centers. Heterogeneity in the data among study sites would argue against combining the data as was_done by Fontham, et al. o+ . Although Fontham, et al., state that "dietary cholesterol" was considered as a potential confounder, they do not provide sufficient information in the study publication describing this factor. Apparently, Fontham, et al., did not consider dietary saturated fat intake, recently reported by Alavanja, et al_.,_ to be associated with relative risks as high as 6.0 to 11.0.21 The risk estimate was highest for nonsmoking women with adenocarcinoma; over - 18 -
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failure to mention this fact makes their analysis incomplete and their interpretation misleading." Fontham, et al., included cases and controls from the group with the decreased risk in the comparison group for their analysis of adult lung cancer risk. According to Butler, the underlying bias became a "source of artificially inflated statistical estimates that incorrectly indicate a positive association between adult ETS exposure and lung cancer." According to Butler, Fontham, et al., appeared to recognize that there was an "interaction" between childhood and adult exposure in the 1994 paper, but did not mention the bias identified in his reanalysis. He states that adjustment for the bias would be "expected to reduce" the risk estimates for spousal, household, workplace, and social ETS exposures. Butler then notes that Fontham, et al., have not provided sufficient data in their publications for these adjustments to be made, and suggests that the raw data from the study would be needed and should be released. A number of other issues have been raised concerning this study. For instance, as noted-earlier, despite the Fontham, et al., study's use of cotinine to assess current tobacco use, the authors themselves acknowledge that misclassification of ever smokers as lifetime never smokers is "problematic" because there is - 17 - - --
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epidemiology, it should review the criticisms that have been raised concerning the Fontham, et al., study, and address them. C. The Argument that a Number of the Spousal Smoking Studies Demonstrate a "Dose-Response" Relationship Is Scientifically Flawed and Cannot Be Used to Support Cal/EPA's Conclusions In the Excerpt, Cal/EPA refers to reportedly increased risks in the "high exposure" subgroups from some of the spousal - smoking studies and to claimed positive trends in risk estimates. (e.g., p. 19, p. 26) By so doing, Cal/EPA invokes the argument that these studies exhibit a "dose-response." The "dose-response" argument is based on the claim that positive results of statistical tests for trend on epidemiologic data show that lung cancer risk increases with increasing reported exposure; that is, that those tests demonstrate that a "dose- response" has occurred. Those who adopt this argument further suggest that positive tests for trend satisfy the criterion for__ dose-response used in evaluating a causal relationship in epidemiology. As will be shown below, however, such claims about "dose-response" based on the epidemiologic studies do not withstand critical scientific scrutiny. - 20 -
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appeared that community A had almost twice the rate of cancer as community B and that an association existed between living in community A and having cancer. Given the increasing public attention paid to ETS, and the claims that ETS exposure is causally associated with lung cancer, ETS could be considered an "emotionally charged" issue. While this might not affect the earlier studies on ETS, it is certainly of- potential importance for studies conducted in the last few years. B. A Number of Unresolved Questions Exist Concerning the Fontham, et al., Study, Rendering Its Interpretation Less Clear than Cal/EPA Portrays Cal/EPA offers essentially no critical comment on the Fontham, et al., (1994) spousal smoking study.41 In fact, Cal/EPA appears to favor this study, commenting that its reported results are "closest to" those reported by the U.S. EPA in its Risk Assessment on ETS. (p. 7, 19) Moreover, Cal/EPA claims that this study "successfully addressed" the many weaknesses inherent in the spousal smoking study design. (p. 27) Despite the contentions to the contrary in the Cal/EPA Excerpt, the Fontham, et al., (1994) study is still subject to the same limitations as the other spousal smoking studies. A number of criticisms of the Fontham study have been submitted to the.public record at U.S. OSHA.iz,'6," For instance, Sears and Steichen listed - 14 -
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1. The ETS Epidemiologic Studies Contain No Actual Exposure Data; The Purported "Dose" Levels Cannot Be Assumed to Represent Dose at All The ETS epidemiologic studies do not measure actual exposures to ETS in the home or workplace. Rather, the studies rely on imprecise estimates based on individual recall. Other recent studies clearly illustrate that questionnaire responses are not accurate representations of actual ETS exposures, and are subject to bias,so-sz As Wynder and Hoffmann53 note: [I]n all ETS-lung cancer studies in 'never- smokers,' assessment of - their lifetime exposure remains problematic as long as reliable biomarkers of uptake are lacking. Perhaps the greatest uncertainty about the epidemiological data is due to the unreliable information obtained by questioning volunteers in regard to their smoking habits. H. Daniel Roth, Ph.D., noted in his submission to U.S. OSHA: "The ETS exposure-data in the overwhelming majority of studies are far too weak for drawing epidemiological conclusions.j54 If the data are weak in terms of_ETS exposure as a whole, they are likely even more limited when dealing with reported "specifics," such as levels of perceived exposure (including the_number of cigarettes smoked per_day or the duration of a smoking history). - 21 -
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75% of the cases in the Fontham, et al., study were adenocarcinomas. Alcohol consumption, another potential confounder, was also not mentioned. Recent studies have reported that smokers, and the persons living in their households, are likely to consume more fatty foods and more alcohol.a•29 The "adjustment" of the reported risk estimates is difficult to interpret, as the adjustment takes into account both study design variables (e.g., subject age) and potential confounders. For instance, the workplace risk estimate in the final report of the Fontham, et al., study was 1.12 before "adjustment" for several factors.41 The "crude" risk estimate,was not statistically significant. After adjustment, however, Fontham,' et al., reported a statistically significant workplace_ risk estimate of 1.39. As was pointed out in submissions to the U.S. OSHA rulemaking record, the direction of the change was opposite what would be expected, and of a magnitude greater than many of the other adjustments in the paper.1z•46 Fontham, et al., do not provide adequate discussion of this unexpected outcome of adjustment, e.g., they do not explain which factor(s) had the most impact. The above discussion illustrates that there are many unresolved issues concerning the Fontham, et al., study. For the Cal/EPA Excerpt to accurately reflect the__nature of the ETS O m. - 19 -
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nine major categories of problems with the Fontham, et al., study, which they characterized as "significant design and execution flaws."12 These included: [T] he study population has no male representation and is not even representative of the nonsmoking U.S. female population, under-representing rural subjects and massively over-representing minorities (especially Asians); [TIhe percentage of adenocarcinoma is unusually high, possibly a reflection of abnormal demographics in the study population; [T] he phenomenon that urinary _cotinine analyses fail to detect active smoking cases onlv, suggests that misclassification is more prevalent among the lung cancer cases than among the controls, leading to an inflated relative risk point estimate; [T]he failure to promulgate the use of colon- cancer controls to account for recall bias results in over-estimation of risk; [T]he use of- frequency-only matching within age categories, combined with the high sensitivity of cancer incidence to age differences, likely introduces a bias resulting in inflated estimates of risk; [T]he categorization of individuals by broad race groupings fails too account for_important lifestyle differences, especially among the large Asian subset of this study; [T]he inability of the standard linear logistic regression approach to fully account for strongly-coupled confounding variables . . . results in inaccurate estimation of risk; [T] he non-independence of the spousal-, workplace- - and social-exposure study subpopulations forces Fontham's workplace - 15 -
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An examination of the ETS-lung cancer epidemiologic studies reveals that they use a variety of definitions to "quantify" spousal smoking. For instance, some use number of cigarettes smoked per day without consideration of duration, while others use duration regardless of amount smoked. In addition, the studies do not use the same intervals to categorize amount smoked, e.g., the "highest exposure" in one study may be >20 cigarettes per day, while in another study, it might be >40 cigarettes per day. Thus, the highest exposure category in the first would be an intermediate category in the second. As noted in the 1995 CRS Report : 44 One implication of the potential disparity between the different types of exposure measurements is that combining risk [estimates from] several studies at the highest exposure levels probably yields misleading results. (p. 31) The authors of the CRS Report note elsewhere that U.S. EPA w" (J+._ 4. calculated just such an overall risk estimate for the highest W Cn ;: --.t , exposure levels in the spousal smoking studies. W__ Given the limitations inherent in exposure estimates based on personal recall and recall by surrogate respondents, claims that the "exposure level" data in the ETS epidemiologic studies can be used to illustrate dose-response are based on a weak and inadequate foundation. Uncertainty exists with respect to both - 22 °
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the actual exposures encountered by study participants and the degree to which study subjects may be misclassified according to exposure. These limitations alone suggest that it would be prudent to exercise great caution in making interpretations about "dose- response" from these data. 2. Claims that the Spousal Smoking Studies Exhibit Dose-Response Relationships Are Most Commonly Based on the Results of Statistical Tests for Trend; Such Tests Are Clearly Not Tests for pose-Response and Should Not Be Interpreted as Such Epidemiologists frequently imply that the results of a statistical test for trend (e.g., the Mantel extension test) provide evidence of a dose-response relationship. According to Maclure and Greenland, this overstates the evidence for dose- response, particularly if "dose-response" is considered to be a monotonic relationship, that is, one in which risk increases with each increment of exposure.55 In fact, Maclure and Greenland point out: Tests for overall trend, such as the Mantel extension test, are widely but erroneously believed to be tests of the hypothesis that a monotonic dose-response relation exists -- that is, a relation in which risk continues to increase with each increment of exposure. (emphasis added) - 23 -
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the spousal smoking studies have at least one category with less_ than 20 cases. Peter Lee has noted that dose-response relationships are frequently reported by those studies that report an association in the first place, an observation that would follow from the mathematical considerations described above.20 Moreover, Maclure and Greenland also note another limitation of the Mantel extension test: that it "assumes a particular dose-response model as part of its justification.11ss (authors' emphasis) They continue: [The Mantel extension test] is not a test of the appropriateness of_that model: instead, it is a test for magnitude of trend given that the shape of _the dose-response relation implied by the linear-logistic model is appropriate. Similarly, J. Lee, et al., note that a number of possible dose-response models could be applied to the ETS-lung cancer data.36 Noting the inconsistencies among the various studies in terms of the data reported, they point out that the analyst's choice of model to apply to the data can affect the conclusions: Any conclusions about dose response relationships should not simply be a reflection about the type of dose response model chosen by the analyst. If_ a linear model is fitted to data which truly have a threshold then it is highly likely that the - 25 -
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Statistical assessments of "trend" are not equivalent to developing a dose-response relationship.3e - Maclure and Greenland point out several major statistical problems with applying the Mantel extension test to questions of dose-response.ss First, they demonstrate mathematically that when there are few subjects in one of the exposure categories, the Mantel extension test is essentially algebraically identical to a test for overall association (the Mantel-Haenszel test) . They explain the interpretation error that can arise from this misconception as follows: Numerous articles can be found in which authors conclude that the extension test for trend in risk over a trichotomous exposure, when there are few subjects in one of the exposure categories, is telling something extra. In fact, it is little more than a restatement of the results of the Mantel- Haenszel test of the collapsed dichotomous table. (emphasis added) That is, mathematical considerations dictate that, if the subdivisions ("exposure categories") include one or more with few subjects, the Mantel extension test will have a positive result in a study reporting an increased risk when only-two_categories are considered. This is frequently interpreted as evidence of dose- response, when it is essentially only an artifact of the mathematics involved. The issue is relevant because a number of - 24
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Tweedie and Mengersen illustrate this using hypothetical data,ss where all the risk estimates are equal at different dose levels, an exercise also carried out by Maclure and Greenland.ss Tweedie and Mengersen also use actual data from one of the spousal smoking studies, where "we see a raised relative risk but no discernible increase of effect with increasing dose," to illustrate the same point. A "raised relative risk," as discussed later in this section, may well be due to the effects of bias and confounding; because of the nature of the test for trend, the spuriously elevated risk can lead to an improper claim of "dose- response." - Similarly, the authors of the CRS Report comment that, of the spousal smoking studies they reviewed,44 All of the trend analyses include zero exposure. If the trend was linear down to zero exposure, then including that level in the trend analysis would yield_ the same results as when excluded. If there was a threshold effect, then a trend test which included the zero exposure level might show a trend even if-an analysis which included only exposures above zero did not show such a trend. In other words, a sharp rise at some exposure level above zero could incorrectly be interpreted as a dose response trend over all exposure levels. (emphasis added) (p. 29) So, if an elevated risk is reported (overall or for one exposure category) , and if the nonexposed group is included in a - 28 -
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Recall bias is also likely to produce an artificial dose- _ response trend in the spousal smoking data. Layard points out that cases may be motivated to try to explain their disease, which could lead to more complete recall or exaggeration of high exposure levels by cases than by controls.5e This differential would produce apparently elevated risk estimates at high exposures. Layard also notes that, under this scenario, one would expect to see a reduction in risk (i.e., a risk estimate below 1.0) at low reported exposures, just as is seen when the data from the U.S. case-control studies on women are combined in a meta-analysis. As noted earlier, the spousal smoking studies are not measuring ETS exposures, but rather are-essentially addressing risks associated with "marriage to a smoker." Spousal smoking status carries with it a number of associated lung cancer risk factors that may be associated with amount smoked.20 As Layard writes:10 [M]any potential confounders of reported spousal smoking-lung cancer associations, as well as smoking-status misclassification bias, are correlated with spousal smoking in a dose- dependent fashion, and such correlations could account for apparent dose-response trends. - 31 -
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actually not be statistically significantly different from one another. [A]ssessment of this [dose-response] relationship should be independent of the assessment of a possible overall association, and hence . . . the unexposed group should be excluded from the analysis. The rationale for this exclusion is that RRs greater than unity for exposures greater than zero may not differ from each other and yet the test for trend will be significant. In this case a trend may not be present in the RR for exposures above zero. (authors' emphasis) Thus, inclusion of the unexposed group in a test for trend can lead to an artifactual conclusion of a statistically significant trend. Moreover, because there is no error term associated with the control value (i.e., the unexposed group), the control value will have an infinite statistical impact on the reported relationship. In a 1995 paper, Tweedie and Mengersen evaluate "dose- response," taking into account "the-possible confounding effect that inclusion of the unexposed group may have.j56 In agreement with J. Lee, et al., and with Maclure and Greenland, they note that °an observed dose-response relationship may--be in fact simply evidence of overall association but not of increasing (or decreasing) risk with increasing dose." - 27 -
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of unexplained bias and confounding. For instance, Springall writes in his submission to U.S. OSHA that data exist suggesting that spousal concordance (the tendency for smokers to marry smokers) increases with the amount smoked and therefore, "an enhanced tendency to a false dose-response exists in the presence of [smoking status] misclassification."14 As smoking status misclassification has been adequately excluded in none of the spousal smoking studies, it is certainly possible that misclassification may contribute to the claimed dose-response relationships reported.20 As noted earlier, only very small rates of smoking status misclassification could account for the risk estimates reported at high exposures by Fontham, et al.,41 and Brownson, et a1.," demonstrating the clear potential for this form of bias to have an effect in this context. Another source of bias, exposure misclassification, may also contribute to the reportedly higher risks at higher exposure levels. As the authors of the CRS Report note:44 The more specific the question about exposure, the more precise the measure, but the less accurate the recall. That is, there is likely to be a very small error rate in reporting marriage to a smoker, but there could be a significant error in reporting actual amounts of exposure, such as numbers of cigarettes smoked by a spouse,_ particularly in the past. (emphasis added) F+ - 30 -
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That is, if the effects of confounding factors and biases were indeed highest in the highest exposure group, the risk estimates for those groups would be expected to be larger. Thornton, et al., report data from their survey of risk factors in a British population that support such a relationship.e The highest prevalence of many of the risk factors (dietary and behavioral) they investigated was found in smokers of 20 or more cigarettes per day. This study also showed that_persons exposed to ETS had higher prevalences of risk factors than did non-exposed persons. Thus, these data are strongly suggestive of a relationship such as that described by Layard." As most studies of risk factor clustering have not focused on associations with amount smoked, this area requires further research. This explanation for the increased risks reported at higher spousal exposures nevertheless remains a viable one. 4. A More Rigorous Analysis of the Data Does Not N Support Made the Dose-Response Claims that Have Been O a~ . w . m .p n eneral, laims about dose-r sponse are tenuously w cn m w i :: - based on qualitative reviews of data from the spousal smoking : studies. Moreover, they accept the equivalence between a positive test for trend and dose-response, which, as described herein, is inappropriate and misleading. The weakness of such claims is clearly demonstrated when Tweedie and Mengersen's more rigorous - 32 -
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straight line will be judged to provide an adequate fit. However this does not prove that the dose response is linear and any interpretations based on extrapolation could be quite erroneous. (emphasis added) Another problem with the Mantel extension test is that it does not take incremental increases in risk at each exposure category into account.55 _ The nature of the _ test is such that a single increase in risk at one exposure category could be sufficient to produce a statistically significant test for trend. Specifically, Maclure and Greenland state: The heart of the dose-response hypothesis is found in the words . . . 'continuously increasing risk.' Not only does the first small dose of exposure influence risk, but additional doses further increase risk relative to the effect of the previous dose. All dose increments are hypothesized to have effects, not just one of them. Because the extension test can yield a small p value if only one dose increment had an effect, it does not test the hypothesis of interest.ss (emphasis added) Another limitation in using a test for trend was noted by S. Lee, et al., who pointed out that inclusion of the unexposed, or reference, group in the test for trend can result in a statistically significant outcome, even though_the only difference is between nonexposed and exposed.3e That is, the different exposure levels (the point of a dose-response analysis) may - 26 -
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test for trend, the result of the test will be statistically significant, even though risk does not increase with each increment of "dose." Thus, the use of a test for trend does.not reflect dose-response, but simply the fact that, mathematically, the test is being "driven" by some reportedly elevated risk. 3. Claims of Dose-Response in the ETS Lung Cancer Studies Reflect an Increased Risk at the Highest Exposure Levels; Such Risk Estimates Do Not Reflect ETS Exposure, But Rather the Influence of Other Factors Associated with Spousal Smoking The preceding discussion illustrates that a single elevated risk estimate can drive a so-called dose-response assessment, when a test for trend is inappropriately used to reach a conclusion about dose-response. This explanation could certainly -- account for the "positive" trends reported in the ETS epidemiology. N . As Paul Switzer, Ph.D., a statistician at Stanford University, mo w m ; notes:s' - - -- a' m cn w o' tW]ithout the highest spousal exposure group there would be very few individual studies with statistically significant effects or significant dose-response relationships. Why, then, are the risk estimates elevated at the highest reported spousal exposure levels? The most likely explanation relates to the fact that the elevated risk estimates are the result - 29 -
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Tweedie and Mengersen report that when the unexposed group is excluded, for all three tests, only one study fits one of the models; no other studies show a statistically significant "dose-response." Conversely, depending on the model, when the unexposed group is included, anywhere from two to all of the studies are statistically significant (at least at the 10 percent level). Thus, these analyses illustrate the sensitivity of conclusions about dose-response to the method of analysis. In addition to the individual-studies analysis, Tweedie and Mengersen analyze the data from the case-control studies using meta-analytic techniques. Again, three models are used, and the results differ by model and, particularly, by whether the_unexposed group is,included. In conclusion, Tweedie and Mengersen note that "a simple assessment of point estimates -- without consideration of their accuracy as expressed by associated confidence intervals and without a more rigorous method of synthesising the results from individual studies -- can be quite misleading." Overall, Tweedie and Mengersen conclude,ss From the meta-analysis of studies of lung cancer and exposure to ETS in non-smoking females given here, our conclusion is that, - 34 -
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REFERENCES 1. Wynder, E.L., "Workshop on Guidelines to the Epidemiology of Weak Associations: Introduction," Preventive Medicine 16: 139-141, 1987. 2. Gori, G.B., "Science, Policy, and Ethics: The Case of Environmental Tobacco Smoke," J C1in Epidemiol 47: 325-334, 1994 3. Wynder, E.L., and Kabat, G.C., "Environmental Tobacco Smoke and Lung Cancer: A Critical Assessment." In: Indoor Air Ouality. H. Kasuga (ed.). Berlin, Heidelberg, Springer- Verlag, 5-15, 1990. 4. Rylander, R., "Prologue," International Journal of Epidemioloav 19(3, Suppl. 1): S3-S4, 1990. 5. Taubes, G., "Epidemiology Faces Its Limits," Science 269: 164-169, 1995. 6. Cole, P., "Introduction," Chapter 1. In: Statistical Methods in Cancer Research, Vol. 1 -- The Analysis of Case-Control Studies. IARC Scientific Publications No. 32. N. Breslow and N. Day (eds.). Lyon, IARC, pp. 14-40, 1980. 7. Gori, G.B., "Policy Against Science: The Case of Environmental Tobacco Smoke," Risk Analysis 15: 15-21, 1995 8. Thornton, A., et al., "Differences Between Smokers, Ex- Smokers, Passive Smokers and Non-Smokers," J Clin Epidemiol 47: 1143-1162, 1994. 9. Sterling, T.D., Weinkam, J.J., and Rosenbaum, W.L., "Comments on the Notice of Proposed Rulemaking Issued by the U.S. Occupational Safety and Health Administration Addressing Indoor Air Quality in Indoor Work Environments," OSHA Docket H-122, Ex. 10-71, 1994. 10. Layard, M.W., "Comments on the OSHA Proposal for Indoor Air Quality Standards," OSHA Docket H-122, Ex. 9-47,603, 1994. 11. Gori, G.B., "Comment on the Proposed Rule on Occupational Exposure to Indoor Air Pollutants Issued by the Occupational Safety and Health Administration," OSHA Docket H-122, Ex. 9- 47,592, 1994. - 36 -
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despite a significant observed relative risk associated with overall exposure, there is little increase in relative risk with increasing dose. One explanation that accounts for this somewhat unexpected situation, of an overall observed increased relative risk but little indication of positive dose response, is that some bias may be inflating all of the observed risks. (emphasis added) 5. Conclusion: Claims About a "Dose-Response" Relationship Derived from the ETS Epidemiologic Studies Are Unfounded and Should Not Be Relied Upon by Cal/EPA The foregoing discussion clearly illustrates that the claims that data in some of the epidemiologic studies on spousal smoking and lung cancer support a dose-response relationship are based on a misinterpretation of the use and meaning of statistical tests for_trends. Positive trend-results can be explained due to spuriously elevated risk estimates arising from uncontrolled confounding and biases. Moreover, the results of more appropriate, more rigorous statistical analyses reveal inconsistencies among and within studies in terms of "dose-response." Because so many questions remain, "dose-response" claims should not be used by Cal/EPA to support its analysis of ETS.
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analysis of the "dose-response" data, published in the peer- reviewed literature, is considered.5s Tweedie and Mengersen's 1995 paper compares several techniques for assessing dose-response in epidemiologic studies. It includes both reanalyses of the dose-response data from the individual studies and a meta-analytic assessment. The authors__ comment that they "provide a more rigorous approach than the purely qualitative assessments which have often been used in the literature. 1"s As an initial step, Tweedie and Mengersen recalculate tests for dose-response for all the spousal smoking studies reporting sufficient data, using three alternate methods. The first (the Armitage method) simply tests whether the reported risks at different categories are significantly different_ The authors_ note that its "major benefit" is that no assumption about the pattern of -dose-response is made. (Since the shape of the dose- response, if any, is unknown, using models that impose a shape will influence the outcomes of the test.) The other two methods fit models, one exponential and one linear, to the data, i.e., each entails a different assumption about the nature of the dose- response. For each of the three methods, the authors perform one test including the unexposed group, and one test excluding the unexposed group. - 33
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47. Valberg, P.A., "A Review of the Scientific Bases for the Environmental Tobacco Smoke Components of OSHA's Proposed Rule on Indoor Air Quality," OSHA Docket H-122, Ex. 9-40,401, 1994. 48. Kirkland, L.R., "Environmental Tobacco Smoke and Lung Cancer in Nonsmoking Women," JAMA 273: 519-520, 1995. 49. Butler, W.J., "Re-Analysis of Data Provided in Fontham et al. (1994): No Increase in the Risk of Lung Cancer Associated with Adult ETS Exposure," OSHA Docket H-122, Ex. 537, 1996. 50. Emmons, K.M., et al. "An Evaluation of the Relationship Between Se1f-Report and Biochemical Measures of Environmental Tobacco Smoke Exposure," Prev. Med. 23: 35-39, 1994. 51. Tunstall-Pedoe, H., et al., "Passive Smoking by Self Report and Serum Cotinine and the Prevalence of Respiratory and Coronary Heart Disease in the Scottish Heart Health Study," J Eoi Comm Health 49: 139-143, 1995. 52. Phillips, K., "Assessment of Personal Exposures to Environmental Tobacco Smoke in British Nonsmokers," Env Int 20: 693-712, 1994. - 53. Wynder, E.L., and Hoffmann , D., "Smoking and Lung Cancer: Scientific Challenges and Opportunities," Ca ncer Res 54: 5284-5295, 1994. -- - - -- - - - 54. Roth, H.D., "Comments on OSHA's Analysis of the ETS Lung Cancer and Heart Disease Data," OSHA Docket H-122, Ex. 9- 40,328, 1994. 55. Maclure, M., and Greenland, S., "Tests for Trend and Dose Response: Misinterpretations and Alternatives," Am J Epidemiol 135: 96-104, 1992. 56 "Meta-Analytic Approaches and Mengersen K L Tweedie R L . , . ., , . ., to Dose-Response Relationships, with Application in Studies of a m = Lung Cancer and Exposure Statistics in Medicine 14: to Environmental Tobacco Smoke," 545-569, 1995. w rn.- -ta C 7. witzer, P., "Comments on Proposed Standard for Indoor Air .J Ctf b . Quality in Work Environments," OSHA Docket H-122, Ex. 9- 47,595, 1994. - 58. Layard, M.W., "The OSHA Proposal for Indoor Air Quality Standards: Post Hearing Comments," OSHA Docket H-122, Ex. 407, 1996. - 40 -
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McKneally, M.F., "Dietary Beta Carotene and Lung Cancer Risk in U.S. Nonsmokers," Journal of the National Cancer Institute 86(1): 33-38, 1994. 25. Candelora, E.C., Stockwell, H.G., Armstrong, A.W., and Pinkham, P.A., "Dietary Intake and Risk of Lung Cancer in Women Who Never-Smoked," Nutrition and Cancer 17: 263-270, 1992. 26. Katzenstein, A.W., "Environmental Tobacco Smoke and Lung Cancer Risk; Epidemiology in Relation to Confounding Factors," Environment International 18: 341-345, 1992. 27. McPhillips, J.B., et al., "Dietary Differences in Smokers and Nonsmokers from Two Southeastern New England Communities," J Am Diet Assoc 94: 287-292, 1994. 28. Woodward, M., et al., "Deficient Health Knowledge, Diet, and Other Lifestyles in Smokers: Is a Multifactorial Approach Required?" Prey Med 23: 354-361, 1994. 29. Matanoski, G., et al., "Characteristics of Nonsmoking Women in NHANES I and NHANES I Epidemiologic Follow-up Study with Exposure to Spouses Who Smoke," Am J Epidemiol 142: 149-157, 1995. 30. Emmons, K.M., et al., "Dietary Intake and Exposure to Environmental Tobacco Smoke in a Worksite Population," Eur J Clin Nutr 49: 336-345, 1995. 31. Butler, W.J., "Correlates of Household and Workplace Exposure to ETS Among Self-Reported Never-Smoking Females: Estimates from the Third National Health and Nutrition Examination Survey (NHANES III)," OSHA Docket H-122, Ex. 536, 1996. 32. Lee, P., "'Marriage to a Smoker' May Not Be a Valid Marker of Exposure in Studies Relating Environmental Tobacco Smoke to Risk of.Lung Cancer in-Japanese Non-Smoking Women," Int Arch Occup Environ Health 67: 287-294, 1995. 33. Chyou, P.-H., et al., "A Prospective Study of Weight, Body Mass Index and Other Anthropometric Measurements in Relation to Site-Specific Cancers," Int J Cancer 57:, 313-317, 1994. 34. Kabat, G.C., "Previous Cancer and Radiotherapy as Risk Factors for Lung Cancer in Lifetime Nonsmokers," Cancer Causes and Control 4: 489-495, 1993. - 38 -
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12. Sears, S.B., and Steichen, T.J., "Comments on Environmental Tobacco Smoke Preliminary Quantitative Risk Assessment," OSHA Docket H-122, Ex. 9-47,553, 1994. 13. Smith, C.J., Sears, S.B., Walker, J.C., and DeLuca, P.O., "Environmental Tobacco Smoke: Current Assessment and Future Directions," Toxicologic Patholoqy 20(2): 289-303, 1992. 14. Springall, A., "Comments on the Epidemiological Evidence Associating Environmental Tobacco Smoke with Lung Cancer and Heart Disease," OSHA Docket H-122, Ex. 9-36,330, 1994 15. Butler, W.J., "Workplace Exposure to ETS and Lung Cancer: A More Detailed Presentation of the Data from a Negative Study, Brownson et-al., (1992)," OSHA Docket H-122, Ex. 9-47,597, 1994. 16. Roe, F.J.C., "Diet Is Important in Assessing Lung Cancer Risk Factors in Nonsmokers," Nutrition and Cancer 22: 203-206, 1994. 17. Strubelt, O., "Diet and Smoking Habits," BMJ 308: 1042-1043, 1994. 18. Austoker, J., "Diet and Cancer," BMJ 308: 1610-1614, 1994. 19. Fraser, G.E., et al., "Diet and Lung Cancer in California Seventh-Day Adventists," Am J Epi 133: 683-693, 1991. 20. Lee, P.N., "A Commentary on Some of the Evidence Provided by OSHA in Support of Their Proposed Rules," OSHA Docket H-122, Ex. 9-47,513, 1994. 21. Alavanja, M.C.R., Brown, C.C., Swanson, C., and Brownson, N 0 R.C., "Saturated Fat Intake and_ Luiig Cancer Risk Among Nonsmoking Women in Missouri," Journal of the National Cancer m Institute 85(23): 1906-1916, 1993. 4~- W 22. Thompson, D.H., and Warburton, D.M., "Lifestyle Differences [n co m Between Smokers, Ex-Smokers, and Non-Smokers, and Implications for Their Health," Psychology and Health 7: 311-321, 1992. 23. Thompson, D.H., and Warburton, D.M., "Dietary and Mental Health Differences Betiveen Never-Smokers Living in Smoking and Non-Smoking Households," Journal of Smoking-Related Disorders 4(3): 203-211, 1993. 24. Mayne, S.T., Janerich, D.T., Greenwald, P., Chorost, S., Tucci, C., Zaman, M.D., Melamed, M.R., Kiely, M., and - 37 -
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35. Brownson, R.C., et al., "Occupational Risk Factors for Lung Cancer Among Nonsmoking Women: A Case-Control Study in Missouri (United States)," Cancer Causes and Control 4: 449- 454, 1993. 36. Wu, A.H., et al., "Previous Lung Disease and Risk of Lung Cancer Among Lifetime Nonsmoking Women in the United States," Am J Epi 141(11); 1023-1032, 1995. 37. Wu, A.H., et al., "Family History of Cancer and Risk of Lung Cancer Among Lifetime Nonsmoking Women in the United States," Am J Epi 143: 535-542, 1996. 38. Lee, J., et al., Health-Asuects of Environmental Tobacco Smoke: An Evaluation of the Scientific Literature. Submission to the Health Care Committee of the NH&MRC by an independent group, November 1994. 39. Ashford, J.R., "Commentary on the Report: Department of Labor Occupational Safety and Health Administration, RIN 1218-AB37, Indoor Air Quality," OSHA Docket H-122, Ex. 9-26,467, 1994. 40. Starr, T.B., Comments on OSHA Proposed Rule, OSHA Docket H- 122, Ex. 10-195, 1994. - 41. Fontham, E., et al., "Environmental Tobacco Smoke and Lung Cancer in Nonsmoking Women: A Multicenter Study,11 Journal of the American Medical Association 271(22): 1752-1759, 1994. 42. Lee, P.N., and Forey, B.A., "Misclassification of Smoking Habits as Determined by Cotinine or by Repeated Self-Report -- A Summary of Evidence from 42 Studies," Journal of Smoking- Related Disorders 6: 109-129, 1995. 43. Brownson, R., et al., "Passive Smoking and Lung Cancer in Nonsmoking Women," American Journal of Public Health 82: 1525-1530, 1992. 44. Redhead, C.S., and Rowberg, R.E., "Environmental Tobacco Smoke and Lung Cancer Risk," A Report of the Congressional Research Service, November 14, 1995. 45. Kaye, W.E., et al., "Recall Bias in Disease Status Associated with Perceived Exposure to. Hazardous Substances," Ann Eoidemiol 4: 393-397, 1994. - 46. Philip Morris, Submission on OSHA's Proposed Rule on Indoor Air Quality, Section V: "Material Impairment: Lung Cancer," OSHA Docket H-122, Ex. 9-47,500, 1994. - 39 -
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38. Gravelle, J.G., and Zimmerman, D., "Cigarette Taxes to Fund Health Care Reform: An Economic Analysis," CRS Report for Congress, Congressional Research Service, Library of Congress, March 8, 1994. 39. Gravelle, J.G., and Zimmerman, D., Statement before the Subcommittee of Clean Air and Nuclear Regulation, Committee on Environment and Public Works, United States Senate, on Environmental Tobacco Smoke, May 11, 1994. 40. Gross, A.J., "The Risk of Lung Cancer in Nonsmokers in the United States and Its Reported Association with Environmental Tobacco Smoke," Journal of Clinical Epidemiology 48: 587-598, 1995. 41. Gross, A.J., "Uncertainties in Lung Cancer Risk Estimates Reported for Exposure to Environmental Tobacco Smoke," Environmetrics 6: 403-412, 1995. 42. Haley, N.J., et al., "Sidestream Smoke Uptake by Syrian Golden Hamsters in an Inhalation Bioassay." In: Indoor Air '87: Proceedings of the 4th International Conference on Indoor Air Quality and Climate (Vol. 2). B. Seifert, et al. (eds.). Berlin, Institute for Water, Soil and Air Hygiene, 68-75, 1987. 43. Haley, N.J., et al., "Uptake of Sidestream Smoke by Syrian Golden Hamsters," Toxicology Letters 35: 83-88, 1987. 44. Hoepfner, H., et al., "Hydroxy-Phenanthrenes in the Urine of Non-Smokers and Smokers," Toxicology Letters (35): 67-71, 1987. - 45. Hole, D., et al., "Passive Smoking and Cardiorespiratory Health in a General Population in the West of Scotland," British Medical Journal 299: 423-427, 1989. 46. Holz, 0., et al., "'ZP-Postlabelling Analysis of DNA Adducts in Monocytes of Smokers and Passive Smokers," International Archives of Occupational and Environmental Health 62: 299-303, 1990. 47. Holz, 0., et al., "Detection of DNA Single-Strand Breaks in Lymphocytes of Smokers," International Archives of Occupational and Environmental Health 65: 83-88, 1993. 48. Huber, G.L., Brockie, R.E., and Mahajan, V., "Smoke and Mirrors: The EPA's Flawed Study of Environmental Tobacco Smoke and Lung Cancer," Regulation 1993(3): 44-54, 1993. 49. Husgafvel-Pursiainen, K., "Sister-Chromatid Exchange and Cell Proliferation in Cultured Lymphocytes of Passively and Actively Smoking Restaurant Personnel," Mutation Res 190: 211-215, 1987. -4-
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50. Husgafvel-Pursiainen, K., et al., "Passive Smoking at Work: Biochemical and Biological Measures of Exposure to Environmental Tobacco Smoke," Int Arch Occup Environ Health 59: 337-345, 1987. 51. Inoue, R. and Hirayama, T., "Passive Smoking and Lung Cancer in Women," Smoking and Health 1987, eds., M. Aoki, et al., Amsterdam, Excerpta Medica, 283-285, 1988. 52. Joeckel, K.H., "Passive Smoking -- Evaluation of the Epidemiological Findings," VDI Reports 888: Carcinogenic Substances in the Environment, VDI Verlag, 1991. 53. Kabat, G., "Epidemiologic Studies of the Relationship Between Passive Smoking and Lung Cancer," Toxicology Forum: 1990 Annual Winter Meeting, Washington, D.C., 187-199, February 19-21, 1990. 54. Kabat, G.C., "Previous Cancer and Radiotherapy as Risk Factors for Lung Cancer in Lifetime Nonsmokers," Cancer Causes and Control 4: 489-495, 1993. 55. Kaye, W.E., et al., "Recall Bias in Disease Status Associated with Perceived Exposure to Hazardous Substances," Ann Epidemiol4: 393-397, 1994. 56. Kilpatrick, S., "Misclassification of Environmental Tobacco Smoke Exposure: Its Potential Influence on Studies of Environmental Tobacco Smoke and Lung Cancer," Toxicology Letters 35(1987): 163-186, 1987. 57. Kirkland, L.R., "Environmental Tobacco Smoke and Lung Cancer in Nonsmoking Women," JAMA 273: 519-520, 1995. 58. Lam, T., et al., "Smoking, Passive Smoking and Histological Types in Lung Cancer in Hong Kong Chinese Women," British Journal of Cancer 56(5): 673-678, 1987. 59. Lam, W., A Clinical and Epidemiological Study of Carcinoma ofLung in Hong Kong, M.D. Thesis Submitted to the University of Hong Kong, 1985. See also: Lam, T. and Cheng, K., "Passive Smoking Is a Risk Factor for Lung Cancer in Never Smoking Women in Hong Kong," Smoking and Health 1987, eds., M. Aoki, et al., Amsterdam, Excerpta Medica, 279-281, 1985. 60. Lan, Q., et al., "Risk Factors for Non-Smokers in Xuanwei County of China," Biomedical and Environmental Sciences 6: 112-118, 1993. 61. Layard, M. W., "Comments on the OSHA Proposal for Indoor Air Quality Standards," OSHA Docket H-122, Ex. 9-47,603, 1994. ~ ~ -5-
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89. Rylander, R., "Prologue," International Journal of Epidemiology 19(3, Suppl. 1): S3-S4, 1990. 90. Scherer, G., et al., "Uptake of Tobacco Smoke Constituents on Exposure to Environmental Tobacco Smoke (ETS)," Clinical Investigator 70: 352-367, 1992. 91. Scherer, G., Westphal, K., and Adlkofer, F., "Urinary Mutagenicity, Hydroxyphenanthrene, and Thioether Excretion After Exposure to Environmental Tobacco Smoke." In: Indoor Air Quality. H. Kasuga (ed.). Berlin, Heidelberg, Springer-Verlag, 138-146, 1990. 92. Scherer, G., et al., "Biomonitoring of Exposure to Potentially Genotoxic Substances from Environmental Tobacco Smoke," Environment International 15: 49-56, 1989. 93. Scherer, G., et al., "Urinary Mutagenicity After Controlled Exposure to Environmental Tobacco Smoke," Toxicology Letters 35: 135-140, 1987. 94. Scherer, G., et al., "Quantitative and Qualitative Differences in Tobacco Smoke Uptake Between Active and Passive Smoking." In: Indoor and Ambient Air Quality. R. Perry and P. W. Kirk (eds.). London, Selper Ltd., 189-194, 1988. 95. Sears, S.B., and Steichen, T.J., "Comments on Environmental Tobacco Smoke Preliminary Quantitative Risk Assessment," OSHA Docket H-122, Ex. 9-47,553, 1994. 96. Smith, C.J., et al., "Environmental Tobacco Smoke: Current Assessment and Future Directions," Toxicologic Pathology 20(2): 289-303, 1992. 97. Sobue, T., et al., "Passive Smoking Among Nonsmoking Women and the Relationship Between Indoor Air Pollution and Lung Cancer Incidence -- Results of a Multicenter Case Controlled Study," Gan to Rinsho 36(3): 329-333, 1990. 98. Sonnenfeld, G., and Wilson, D.M., "The Effect of Smoke Age and Dilution on the Cytotoxicity of Sidestream (Passive) Smoke," Toxicology Letters 35: 89-94, 1987. 99. Sorsa, M., et al., "Cytogenetic Effects of Tobacco Smoke Exposure Among Involuntary Smokers," Mutation Res 222(2): 111-116, 1989. 100. Springall, A., "Comments on the Epidemiological Evidence Associating Environmental Tobacco Smoke with Lung Cancer and Heart Disease," OSHA Docket H-122, Ex. 9-36,330, 1994. 101. Starr, T.B., Comments on OSHA Proposed Rule, OSHA Docket H-122, Ex. 10-195, 1994. -8-
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13. Biggerstaff, B.J., et al., "Passive Smoking in the Workplace: Classical and Bayesian Meta- Analyses," Int Arch Occup Environ Health 66: 269-277, 1994. 14. Bombick, D., et al., "Assessment of the Biological Activity of Mainstream or Environmental Tobacco Smoke (ETS) Using a Cellular Smoke Exposure Technique," Abstracts of the Twenty-Second Annual Scientific Meeting of the Environmental Mutagen Society, Kissimmee, Florida: April 1991. Abstract. 15. Bos, R.P., Theuws, J.L.G., and Henderson, P.T., "Excretion of Mutagens in Human Urine After Passive Smoking," Cancer Letters 19: 85-90, 1983. 16. Brown, B.G., et al., "Molecular Toxicology Endpoints in Rodent Inhalation Studies," Experimental Toxicology and Pathology 47: 183-191, 1995. 17. Brownson, R.C., et al., "Occupational Risk Factors for Lung Cancer Among Nonsmoking Women: A Case-Control Study in Missouri (United States)," Cancer Causes and Control 4: 449-454, 1993. 18. Butler, W.J., "Correlates of Household and Workplace Exposure to ETS Among Self- Reported Never-Smoking Females: Estimates from the Third National Health and Nutrition Examination Survey (NHANES III)," OSHA Docket H-122, Ex. 536, 1996. 19. Butler, W.J., "Re-Analysis of Data Provided in Fontham et al. (1994): No Increase in the Risk of Lung Cancer Associated with Adult ETS Exposure," OSHA Docket H-122, Ex. 537, 1996. 20. Butler, W.J., "Workplace Exposure to ETS and Lung Cancer: A More Detailed Presentation of the Data from a Negative Study, Brownson et al., (1992)," OSHA Docket H-122, Ex. 9- 47,597, 1994. O 21. Candelora, E.C., et al., "Dietary Intake and Risk of Lung Cancer in Women Who Never " Nutrition and Cancer 17: 263-270 1992 Smoked m w`- o+ - , . , -4+ w,. cn 22. Chyou, P.-H., et al., "A Prospective Study of Weight, Body Mass Index and Other Anthropometric Measurements in Relation to Site-Specific Cancers," Int.7 Cancer 57: 313- 317, 1994. 23. Coggins, C.R.E., "Comments on Environmental Tobacco Smoke Work with Experimental Animals," OSHA Docket H-122, Ex. 10-170, 1994. 24. Coggins, C.R.E., Testimony Before OSHA on Indoor Air Quality Proposed Rule, Transcript p. 11,684, 1994. -2-
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38. Wang, S.Y., et al., "A Comparative Study of the Risk Factors for Lung Cancer in Guangdong, China," Lung Cancer 14(Suppl. 1): S99-S 105, 1996. 39. Wang, T.J., Zhou, B.S., and Shi, J.P., "Lung Cancer in Nonsmoking Chinese Women: A Case-Control Study," Lung Cancer 14(Suppl. 1): S 93-S98, 1996. 40. Witschi, H., et al., "The Carcinogenicity of Environmental Tobacco Smoke," Carcinogenesis 18: 575-586,1997. 41. Xu, Z.Y., et al., "Lifestyle, Environmental Pollution and Lung Cancer in Cities of Liaoning in Northeastern China," Lung Cancer 14(Suppl. 1): S 149-S 160, 1996. 42. Yu, S.Z., and Zhao, N., "Combined Analysis of Case-Control Studies of Smoking and Lung Cancer in China," Lung Cancer 14(Suppl. 1): S 161-5170, 1996. 43. Ziegler, R.G., et al., "Importance of alpha-Carotene, beta-Carotene, and Other Phytochemicals in the Etiology of Lung Cancer,".Iournal of the National Cancer Institute 1996. - 88: 612-615.
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Attachment C to Comments on Chapter 7 -- Carcinogenic Effects Literature Cited in Philip Morris Comment on 1996 External Review Draft But Not in OEHHA's Final Review Draft 1. Adlkofer, F., et al., "Exposure of Hamsters and Rats to Sidestream Smoke of Cigarettes: Preliminary Results of a 90-Day-Inhalation Study." In: Indoor and Ambient Air Quality. R. Perry and P.W. Kirk (eds.). London, Selper Ltd., 252-258, 1988. 2. Alavanja, M.C.R., et al., "Saturated Fat Intake and Lung Cancer Risk Among Nonsinoking Women in Missouri," Journal of the National Cancer Institute 85(23): 1906-1916, 1993. 3. Altheim, L, and Ramdahl, T., "Contribution of Wood Combustion to Indoor Air Pollution as Measured by Mutagenicity in Salmonella and Polycyclic Aromatic Hydrocarbon Concentration," Environ Mutag 6: 121-130, 1984. 4. Ames, B.N., "Mutagenesis and Carcinogenesis: Endogenous and Exogenous Factors," Environmental and Molecular Mutagenesis 14 (Suppl. 16): 66-77, 1989. 5. Ames, B.N., and Gold, L.S., "Pesticides, Risk, and Applesauce," Science 244: 755-757, 1989. 6. Ames, B.N., Profet, M., and Gold, L.S., "Dietary Pesticides (99.99% All Natural)," Proc Natl Acad Sci USA 87: 777-7781, 1990. 7. Ashford, J.R., "Commentary on the Report: Department of Labor Occupational Safety and Health Administration, RIN 1218-AB37, Indoor Air Quality," OSHA Docket H-122, Ex. 9- 26,467, 1994. 8. Ashford, J., Testimony Before OSHA on Indoor Air Quality Proposed Rule, Transcript p. 726, 1994. 9. ' Austoker, J., "Diet and Cancer," BMJ308: 1610-1614,1994. 10. Aviado, D., "Health Effects of 50 Selected Constituents of Environmental Tobacco Smoke," ~ IndoorAir Quality, ed. H. Kasuga, Springer-Verlag, Berlin Heidelberg: 383-389, 1990. W o+ 11. Aviado, D., "Suspected Pulmonary Carcinogens in Environmental Tobacco Smoke," Enviro. rw Tech Letters 9: 539-544, 1988. •o co 12. Bartsch, H., et al., "Carcinogen Hemoglobin Adducts, Urinary Mutagenicity, and Metabolic Phenotype in Active and Passive Cigarette Smokers," JNCl82: 1826-1831. 1990.
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114. Valberg, P.A., "A Review of the Scientific Bases for the Environmental Tobacco Smoke Components of OSHA's Proposed Rule on Indoor Air Quality," OSHA Docket H-122, Ex. 9-40,401, 1994. 115. von Meyerinck, L., et al., "Exposure of Rats and Hamsters to Sidestream Smoke from Cigarettes in a Subchronic Inhalation Study," Experimental Pathology 37: 186-189, 1989. 116. Wang, F.L., et al., "Childhood and Adolescent Passive Smoking and the Risk of Female Lung Cancer," International Journal of Epidemiology 23(2): 223-230, 1994. 117. Witschi, H., et al., "Six-Month Exposure of Strain A/J Mice to Cigarette Sidestream Smoke: Cell Kinetics and Lung Tumor Data," Fund Appl Toxicol 26: 32-40, 1995. 118. Woodward, M., et al., "Deficient Health Knowledge, Diet, and Other Lifestyles in Smokers: Is a Multifactorial Approach Required?" Prev Med 23: 354-361, 1994. 119. Wu, A.H., et al., "Previous Lung Disease and Risk of Lung Cancer Among Lifetime Nonsmoking Women in the United States," Am JEpi 141(11): 1023-1032, 1995. 120. Wynder, E., "Epidemiological Issues in Weak Associations," International Journal of Epidemiology 19(3) (Suppl. 1): S5-S7, 1990. 121. Wynder, E.L., "Workshop on Guidelines to the Epidemiology of Weak Associations: Introduction," Preventive Medicine 16: 139-141, 1987. 122. Wynder, E., Higgins, L, and Harris, R., "The Wish Bias," Journal of Clinical Epidemiology 43(6): 619-621, 1990. 123. Wynder, E.L., and Hoffinann, D., "Smoking and Lung Cancer: Scientific Challenges and Opportunities," Cancer Res 54: 5284-5295, 1994. 124. Wynder, E.L., and Kabat, G.C., "Environmental Tobacco Smoke and Lung Cancer: A Critical Assessment." In: Indoor Air Quality. H. Kasuga (ed.). Berlin, Heidelberg, Springer-Verlag, 5-15, 1990. 125. Zaridze, D.G., and Zemlyanaya, G.M., "Indoor Air Pollution and Lung Cancer Risk in Non- Smoking Women in Moscow," Experimental Oncology 16: 441-445, 1994. -10-
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25. Lei, Y.X., et al., "Some Lifestyle Factors in Human Lung Cancer: A Case-Control Study of 792 Lung Cancer Cases," Lung Cancer 14(Suppl. 1): 5121-5136, 1996. 26. Liao, M.L., et al., "A Study of the Association Between Squamous Cell Carcinoma and Adenocarcinoma in the Lung, and History of Menstruation in Shanghai Women, China," Lung Cancer 14(Suppl. 1): 5215-5221, 1996. 27. McCormick, J., "Medical Hubris and the Public Health: The Ethical Dimension," Journal of Clinical Epidemiology 49: 619-621, 1996. 28. Nilsson, R., "Environmental Tobacco Smoke and Lung Cancer: A Reappraisal," Ecotoxicology and Environmental Safety 34: 2-17, 1996. 29. Roe, F.J.C., "Laboratory Studies in the Prediction of Lung Cancer Risk," Indoor & Built Environment 5: 196-204, 1996. 30. Rosenbaum, W.L., Sterling, T.D., and Weinkam, J.J., "A Critical Examination of OSHA's Assessment of Risk Associated with Workplace Exposure to Environmental Tobacco Smoke," Regulatory Toxicology and Pharmacology 23: 233-240, 1996. 31. Rylander, R., et al., "Lung Cancer, Smoking and Diet Among Swedish Men," Lung Cancer 14(Suppl. 1): S75-S83, 1996. 32. Schwartz, A.G., Yang, P., and Swanson, G.M., "Familial Risk of Lung Cancer Among Nonsmokers and Their Relatives," American Journal ofEpidemiology 144: 554-562, 1996. 33. Shen, X.B., et al., "Analysis and Estimates of Attributable Risk Factors for Lung Cancer in Nanjing, China," Lung Cancer 14(Suppl. 1): S 107-S 112, 1996. 34. Steimnetz, K.A., and Potter, J.D., "Vegetables, Fruit, and Cancer Prevention: A Review," Journal of the American Dietetic Association 96(10): 1027-1039, 1996. 35. Sterling, T.D., et al., "An Alternative Explanation for the Apparent Elevated Relative Mortality and Morbidity Risks Associated with Exposure to Environmental Tobacco Smoke," Journal of Clinical Epidemiology 49: 803-808, 1996. 36. Sugita, M., Izuno, T., and Kanamori, M., "Per Capita National Income and Summarized Odds Ratio for Epidemiologic Studies on Relationship Between Passive Smoking and Lung Cancer," Proceedings of Indoor Air `96 2: 331-336, 1996. 37. Tweedie, R.L., et al., "Bayesian Meta-Analysis, with Application to Studies of ETS and Lung Cancer," Lung Cancer 14(Suppl. 1): S171-S194, 1996. -3-
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25. Coggins, C.R.E., et al., "Fourteen-Day Inhalation Study in Rats, Using Aged and Diluted Sidestream Smoke from a Reference Cigarette. I. Inhalation Toxicology and Histopathology," Fundamental and Applied Toxicology 19: 133-140, 1992. 26. Coggins, C.R.E., et al., "Subchronic Inhalation Study in Rats Using Aged and Diluted Sidestream Smoke from a Reference Cigarette," Inhalation Toxicology 5: 77-96, 1993. 27. Cole, P., "Introduction," Chapter 1. In: Statistical Methods in Cancer Research, Vol. 1-- TheAnalysis ofCase-Control Studies. IARC Scientific Publications No. 32. N. Breslow and N. Day (eds.). Lyon, IARC, pp. 14-40, 1980. 28. Collman, G.W., et al., "Effects of alpha-Naphthoflavone on Levels of Sister Chromatid Exchanges in Lymphocytes from Active and Passive Cigarette Smokers: Dose-Response Relationships," Cancer Research 46: 6452-6455, 1986. 29. Doolittle, D.J., "Comments on Environmental Tobacco Smoke and Genotoxicity," OSHA Docket H-122, Ex. 9-47,553, 1994. 30. Du, Y., et al., "Exposure to Environmental Tobacco Smoke and Female Lung Cancer," IndoorAir3: 231-236, 1995. 31. Emmons, K.M., et al. "An Evaluation of the Relationship Between Self-Report and Biochemical Measures of Environmental Tobacco Smoke Exposure," Prev Med 23: 35-39, 1994. 32. Emmons, K.M., et al., "Dietary Intake and Exposure to Environmental Tobacco Smoke in a Worksite Population," Eur J Clin Nutr 49: 336-345, 1995. 33. Fraser, G.E., et al., "Diet and Lung Cancer in California Seventh-Day Adventists," Am JEpi 133: 683-693, 1991. 34. Geng, G., et al., "On the Relationship Between Smoking and Female Lung Cancer," Smoking and Health 1987, eds., M. Aoki, et al., Amsterdam, Excerpta Medica, 483-486, 1988. 35. Gorgels, W.J.M.J., et al., "Passive Smoking and Sister-Chromatid Exchanges in Lymphocytes," Mutation Research 279: 233-238, 1992. 36. Gori, G.B., "Comment on the Proposed Rule on Occupational Exposure to Indoor Air Pollutants Issued by the Occupational Safety and Health Administration," OSHA Docket H- 122, Ex. 9-47,592, 1994. 37. Gori, G.B., "Policy Against Science: The Case of Environmental Tobacco Smoke," Risk Analysis 15: 15-22, 1995. ro 9 .. -3-
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102. Sterling, T.D., Weinkam, J.J., and Rosenbaum, W.L., "Comments on the Notice of Proposed Rulemaking Issued by the U.S. Occupational Safety and Health Administration Addressing Indoor Air Quality in Indoor Work Environments," OSHA Docket H-122, Ex. 10-71, 1994. 103. Strubelt, 0., "Diet and Smoking Habits," BMJ 308: 1042-1043, 1994. 104. Sugita, M., Izuno, T., and Kanamori, M., "Recalculation of Summarised Odds Ratios for the Relationship Between Passive Smoking and Lung Cancer Based on Data in the EPA Report," Indoor Environment 4: 177-181, 1995. 105. Switzer, P., "Comments on Proposed Standard for Indoor Air Quality in Work Environments," OSHA Docket H-122, Ex. 9-47,595, 1994. 106. Taubes, G., "Epidemiology Faces Its Limits," Science 269: 164-169, 1995. 107. Teredesai, A., and Pruehs, D., "Histopathological Findings in the Rat and Hamster Respiratory Tract in a 90-Day Inhalation Study Using Fresh Sidestream Smoke of the Standard Reference Cigarette 2R1: " In: Toxic and Carcinogenic Effects of Solid Particles in the Respiratory Tract. U. Mohr, D.L. Dungworth, J.L. Mauderly, and G. Oberdorster (eds.). Washington, ILSI Press, 629-635, 1994. 108. Thompson, D.H., and Warburton, D.M., "Lifestyle Differences Between Smokers, Ex- Smokers, and Non-Smokers, and Implications for Their Health," Psychology and Health 7: 311-321, 1992. 109. Thompson, D.H., and Warburton, D.M., "Dietary and Mental Health Differences Between Never-Smokers Living in Smoking and Non-Smoking Households," Journal of Smoking- Related Disorders 4(3): 203-211, 1993. 110. Thornton, A., et al., "Differences Between Smokers, Ex-smokers, Passive Smokers, and Non-Smokers," Journal of Clinical Epidemiology 47(1): 1143-1162, 1994. 111. Tunstall-Pedoe, H., et al., "Passive Smoking by Self Report and Serum Cotinine and the Prevalence of Respiratory and Coronary Heart Disease in the Scottish Heart Health Study," JEpi Comm Health 49: 139-143, 1995. 112. Tweedie, R.L., and Mengersen, K.L:, "Lung Cancer and Passive Smoking: Reconciling the Biochemical and Epidemiological Approaches," Br J Cancer 66: 700-705, 1992. 113. Tweedie, R.L., and Mengersen, K.L., "Meta-Analytic Approaches to Dose-Response Relationships, with Application in Studies of Lung Cancer and Exposure to Environmental Tobacco Smoke," Statistics in Medicine 14: 545-569, 1995. -9-
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Attachment D to Comments on Chapter 7-- Carcinogenic Effects New Literature Since OEHHA's January 1996 External Review Draft on Lung Cancer 1. Agapitos, E., et at., "Epithelial, Possibly Precancerous, Lesions of the Lung in Relation to Smoking, Passive Smoking, and Socio-Demographic Variables," Scandinavian Journal of Social Medicine 24(4): 259-263, 1996. 2. Alavanja, M.C.R., et al., "Estimating the Effect of Dietary Fat on the Risk of Lung Cancer in Nonsmoking Women," Lung Cancer 14(Suppl. 1): S63-S74, 1996. 3. Armitage, A.K., et al., "Environmental Tobacco Smoke -- Is It Really a Carcinogen?" Medical Science Research 25: 3-7, 1997. 4. Auvinen, A., et al., "Indoor Radon Exposure and Risk of Lung Cancer: A Nested Case- Control Study in Finland," Journal of the National Cancer Institute 88: 966-972, 1996. 5. Barry, D., "Differential Recall Bias and Spurious Associations in Case/Control Studies," Statistics in Medicine 15: 2603-2626, 1996. 6. Barry, D., "The Assessment of Exposure to Environmental Tobacco Smoke," Environment International 23: 17-31, 1997. 7. Cardenas, V.M., et al., "Environmental Tobacco Smoke and Lung Cancer Mortality in the American Cancer Society's Cancer Prevention Study II," Cancer Causes and Control 8: 57- 64, 1997. 8. Chappell, W.R., and Gratt, L.B., "A Graphical Method for Pooling Epidemiological Studies," American Journal of Public Health 86: 748-750, 1996. 9. Charlton, B.G., "The Scope and Nature of Epidemiology," Journal of Clinical Epidemiology 49: 623-626, 1996. 10. Coggins, C.R.E., "The OSHA Review of Animal Inhalation Studies with Environmental Tobacco Smoke," Inhalation Toxicology 8: 819-830, 1996. 11. Coggon, D., "Epidemiology in the Assessment of Small Risks," Transactions of the Institution of Chemical Engineers 73 (Part B, Suppl.): S36-S38, 1995. 12. Dai, X.D., ct al., "The Etiology of Lung Cancer in Nonsmoking Females in Harbin, China," Lung Cancer 14(Suppl. 1): 585-591, 1996.
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62. Layard, M.W., "Ischemic Heart Disease, Lung Cancer, and Spousal Smoking in the National Mortality Followback Survey," Addendum to Comments on the OSHA Proposal for Indoor Air Quality Standards, OSHA Docket H-122, Ex. 10-219, 1994. 63. Layard, M.W., "The OSHA Proposal for Indoor Air Quality Standards: Post Hearing Comments," OSHA Docket H-122, Ex. 407, 1996. 64. Lee, J., et al., Health Aspects of Environmental Tobacco Smoke: An Evaluation of the Scientific Literature. Submission to the Health Care Committee of the NH&MRC by an independent group, November 1994. 65. Lee, P.N., Environmental Tobacco Smoke and Mortality, Basel, Karger, 1992. 66. Lee, P.N., "A Commentary on Some of the Evidence Provided by OSHA in Support of Their Proposed Rules," OSHA Docket H-122, Ex. 9-47,513, 1994. 67. Lee, P., "`Marriage to a Smoker' May Not Be a Valid Marker of Exposure in Studies Relating Environmental Tobacco Smoke to Risk of Lung Cancer in Japanese Non-Smoking Women," Int Arch Occup Environ Health 67: 287-294, 1995. 68. Lee, P.N., and Forey, B.A., "Misclassification of Smoking Habits as Determined by Cotinine or by Repeated Self-Report -- A Summary of Evidence from 42 Studies," JSmoking-Related Dis 6: 109-129, 1995. 69. Lewtas, J., et al., "Human Exposure to Mutagens from Indoor Combustion Sources." In: Indoor Air '87. B. Seifert, et al., (eds.). Berlin, Institute for Water, Soil and Air Hygiene, 473-482, 1987. 70. Ling, P., et al., "Mutagenic Determination of Passive Smoking," Tox Letters 35: 147-151, 1987. 71. Liu, Q., et al., "Indoor Air Pollution and Lung Cancer in Guangzhou, People's Republic of China," American Journal of Epidemiology 137: 145-154, 1993. 72. Liu, Z., et al., "Smoking and Other Risk Factors for Lung Cancer in Xuanwei, China," International Journal ofEpidemiology 20(1): 26-31, 1991. 73. Lofroth, G., et al., "Passive Smoking and Urban Air Pollution: Salmonella/Microsome Mutagenicity Assay of Simultaneously Collected Indoor and Outdoor Particulate Matter," Env Sci Res 27: 515-525, 1983. 74. Lofroth, G., et al., "Public Exposure to Environmental Tobacco Smoke," Mutat Res 202: 103-110, 1988. -6-
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13. Doull, J., Rozman, K.K., and Lowe, M.C., "Hazard Evaluation in Risk Assessment: Whatever Happened to Sound Scientific Judgment and Weight of Evidence?" Drug Metabolism Reviews 28(1&2): 285-299, 1996. 14. Du, Y.X., et al., "An Epidemiological Study of Risk Factors for Lung Cancer in Guangzhou, China," Lung Cancer 14(Suppl. 1): S9-S37, 1996. 15. Federal Focus, Iric.; Principles for Evaluating Epidemiologic Data in Regulatory Risk Assessment, (Developed by an Expert Panel at a Conference in London, England, October 1995), Washington, D.C., August 1996. 16. Gao, Y.T., "Risk Factors for Lung Cancer Among Nonsmokers with Emphasis on Lifestyle Factors," Lung Cancer 14(Suppl. 1): S39-S45, 1996. 17. Gratt, L.B., and Chappell, W.R., "US Worker Lung Cancer Risk from Environmental Tobacco Smoke: Material Health Impairment Unlikely," Proceedings ofIndoor Air '96 2: 343-348, 1996. 18. Jahn, 0., "Environmental Tobacco Smoke, a Lung Carcinogen?" (uncertified translation), Wiener Klinische Wochenschrift 108(18): 570-573, 1996. 19. Jenkins, R.A., et al., "Exposure to Environmental Tobacco Smoke in Sixteen Cities in the United States as Detennined by Personal Breathing Zone Air Sampling," Journal of Exposure Analysis and Environmental Epidemiology 6: 473-502, 1996. 20. Kabat, G.C., "Aspects of the Epidemiology of Lung Cancer in Smokers and Nonsmokers in the United States," Lung Cancer 15: 1-20, 1996. 21. Kawachi, I., and Colditz, G., "Invited Commentary: Confounding, Measurement Error, and Publication Bias in Studies of Passive Smoking," American Journal of Epidemiology 144: 909-915,1996. - 22. Ko, Y.C., et al., "Risk Factors for Primary Lung Cancer Among Non-Smoking Women in ro. Taiwan," International Journal ofEpidemiology 26: 24-31, 1997. 23. Koo, L.C., and Ho, J.H.C.,"Diet as a Confounder of the Association Between Air Pollution and Female Lung Cancer: Hong Kong Studies on Exposures to Environmental Tobacco Smoke, Incense, and Cooking Fumes as Examples," Lung Cancer 14(Suppl. 1): S47-S61, 1996. 24. Lee, P.N., and Forey, B.A, "Misclassification of Smoking Habits as a Source of Bias in the Study of Environmental Tobacco Smoke and Lung Cancer," Statistics in Medicine 15: 581- 605, 1996. - -2-
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75. Maclure, M., and Greenland, S., "Tests for Trend and Dose Response: Misinterpretations and Alternatives," Am JEpidemio1135: 96-104, 1992. 76. Martin, F., et al., "Urinary Excretion of Hydroxy-Phenanthrenes After Intake of Polycyclic Aromatic Hydrocarbons," Environ Int (15): 41-47, 1989. 77. Mayne, S.T., et al., "Dietary Beta Carotene and Lung Cancer Risk in U.S. Nonsmokers," Journal of the National Cancer Institute 86(1): 33-38, 1994. 78. McPhillips, J.B., et al., "Dietary Differences in Smokers and Nonsmokers from Two Southeastern New England Communities," JAm Diet Assoc 94: 287-292, 1994. 79. Mohtashamipur, E., et al., "Urinary Excretion of Mutagens in Passive Smokers," Toxicology Letters 35: 141-146, 1987. 80. Newell, G., "Re: Filing of Testimony and Evidence -- Hearing on Proposed Rule on Indoor Air Quality," OSHA Docket H-122, Ex. 10-206, 1994. 81. Nikula, K.J., et al., "Comparative Pulmonary Toxicities and Carcinogenicities of Chronically Inhaled Diesel Exhaust and Carbon Black in F344 Rats," Fund Appl Toxicol 25: 80-94, 1995. 82. Philip Morris, Submission on OSHA's Proposed Rule on Indoor Air Quality, OSHA Docket H-122, Ex. 9-47,500, 1994. 83. Phillips, K., "Assessment of Personal Exposures to Environmental Tobacco Smoke in British Nonsmokers," Env Int 20: 693-712, 1994. 84. Redhead, C.S., and Rowberg, R.E., "Environmental Tobacco Smoke and Lung Cancer Risk," CRS Report for Congress, Congressional Research Service, Library of Congress, November 85. 86. 14, 1995. 0 m Rodgman A "Environmental Tobacco Smoke " Reg Tox and Pharm 16: 223-244 1992 o+ , ~ , ., , , . W U1 , Rodgman, A., "The Chemical Composition of Environmental Tobacco Smoke: Some 10 ,- 10 Comments on the Occupational Safety and Health Administration's Notice on 'Indoor Air Quality' (OSHA, 1994)," OSHA Docket H-122, Ex. 9-47,532, 1994. 87. Roe, F.J.C., "Diet Is Important in Assessing Lung Cancer Risk Factors in Nonsmokers," Nutrition and Cancer 22: 203-206, 1994. 88. Roth, H.D., "Comments on OSHA's Analysis of the ETS Lung Cancer and Heart Disease Data," OSHA Docket H-122, Ex. 9-40,328, 1994. -7-
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Atmosphere: Emission Sources of BaP Osborne and Crosby (1987) cite the principal sources of BaP in the atmosphere as (1) coal- and oil-fired power stations, (2) domestic heating, (3) miscellaneous industrial processes, (4) vehicle exhausts, and (5) cigarette smoke, forest fires and volcanic activity. The yearly global emission of Bap is estimated to be about 5,000 tons, with the greatest contribution coming from coal combustion. BaP emissions in the U.S. have been estimated to be 1,260 tons/year, accounting for approximately 25% of the worldwide total (Grimmer, 1979). As can be seen in Table 1, the major emission sources in the U.S. are heating and refuse burning. The percentages in Table 1 are derived from a table presented by Grimmer (1979), reproduced herein as Table 2. Since Osborne and Crosby (1987) cited cigarettes as a principal source of BaP emissions, an estimate was calculated of the tons emitted in sidestream smoke/year. This estimate is based on cigarette consumption/year in the U.S. (Tobacco Manufacturers Association, 1997), and uses the value of 147 ng/cig BaP in sidestream smoke (SS) (based on values for the 1R4F reference cigarette cited in R.J. Reynolds, 1988). Figure 1 shows that for the years 1983-1996, the estimated emission ofBaP in sidestream smoke to the atmosphere is less than 0.099 tons/year, which calculates 0002235.01 3qN98 4:00pm -2-
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DRAf! T Benzo[a]pyrene: Environmental Distribution and Human Exposure Incomplete combustion of organic matter represents the major source of polynuclear aromatic compounds (PAH) in the environment. PAHs are found at detectable concentrations in air, water, and soil samples of all types. Concentrations are typically small, in the order of,ugJkg or ng/m3. Since PAHs are highly lipophilic, they accumulate in organic fatty material and therefore have the potential to concentrate in the food chain. Of the numerous PAHs, one compound that has perhaps received the most attention is benzo[a]pyrene (BaP). It is the focus of this discussion. The International Agency for Research on Cancer (IARC) has classified BaP as probably carcinogenic to humans -- IARC Overall Evaluation 2A (IARC, 1983, 1986a, 1986b). BaP has been identified in both mainstream and sidestream smoke from cigarettes, cigars, and pipes; marijuana smoke; and smoke-polluted environments (IARC, 1986b). Non-occupational inhalation exposure to BaP is primarily from tobacco smoke and urban air. However, Hattemer-Frey and Travis (1991) estimate that inhalation accounts for only 2% of the total daily intake of BaP. The focus of this paper therefore concerns the environmental distribution of BaP and human exposure to BaP. 0002235.01 3116/98 4:00 pm - t -
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Attachment A to Comments on Chapter 7-- Carcinogenic Effects Studies on Cancers Other than Lung Not Considered by OEHHA 1. Birch, J.M., et al., "The Inter-Regional Epidemiological Study of Childhood Cancer (Il2ESCC): Case-Control Study of Children with Central-Nervous-System Tumours," Brit JNeurosurg 4: 17-26, 1990. 2. Bosch, F.X., et al., "Prevalence ofHuman Papillomavirus in Cervical Cancer: A Worldwide Perspective," JNatl Cancer Inst 87: 796-802, 1995. 3. Bosch, F.X., et al., "Male Sexual Behavior and Human Papillomavirus DNA: Key Risk Factors for Cervical Cancer in Spain," JNatl Cancer Inst 88: 1060-1067, 1996. 4. Bunin, G.R., et al., "Risk Factors for Astrocytic Glioma and Primitive Neuroectodermal Tumor of the Brain in Young Children: A Report from the Children's Cancer Group," Cancer Epidemiol Biomarkers & Prev 3: 197-204, 1994. 5. Cordier, S., et al., "Incidence and Risk Factors for Childhood Brain Tumors in the Ile de France," Int J Cancer 59: 776-782, 1994. 6. Cress, R.D., "Characteristics of Women Nonsmokers Exposed to Passive Smoke," Prev Med 23: 40-47, 1994. 7. DeVita, V.T., et al., (eds.)., Cancer: Principles & Practice of Oncology, 4th Edition, Philadelphia, J.B. Lippincott Company, 1993. 8. Filippini, G., et aL, "Mothers' Active and Passive Smoking During Pregnancy and Risk of Brain Tumors in Children," Int J Cancer 57: 769-774, 1994. 9. Grufferman, S., et al., "Parents' Use of Cocaine and Marijuana and Increased Risk of Rhabdomyosarcoma in Their Children," Cancer Causes and Control 4: 217-224, 1993. 10. Gurney, J.G., et al., "Childhood Brain Tumor Occurrence in Relation to Residential Power Line Configurations, Electric Heating Sources, and Electric Appliance Use," Am JEpidemiol 143: 120-128, 1996. 11. Hellberg, D., et al., "Smoking and Cervical Intraepithelial Dysplasia: An Association Independent of Sexual and Other Risk Factors," Acta Obstet Gynecol Scand 65: 625-631, 1986.
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