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Comments on: ENVIRONMENTAL TOBACCO SMOKE: A GUIDE TO WORKPLACE SMOKING POLICIES [Draft] EPA 400/6-90/004 Response Addressing: Chapter 1: What Is ETS? Section: Hazardous Constituents in ETS Prepared by: David J. Doolittle, Ph.D. Senior Staff R&D Toxicologist Manager. Cell/Molecular Biology R.J. Reynolds Tobacco Company October 1990

SUMMARY: Based on the published, peer-reviewed data in the literature, the EPA document entitled "Environmental Tobacco Smoke: A Guide to Workplace Smoking Policies" (The "Guide") should list the following conclusions: 1) The relative biological activity reported for mainstream and sidestream smoke varies, depending on cigarette design. Sometimes mainstream smoke has greater activity, sometimes sidestream smoke has greater activity and sometimes the two have similar activities. Thus, it is not possible to make simplistic statements regarding the relative biological activity of mainstream and sidestream smoke. 2) Aged, diluted sidestream smoke is reported to be less mutagenic and less cytotoxic than fresh sidestream smoke. 3) There is absolutely no scientific evidence that ETS, or any of its constituents, are carcinogenic, mutagenic or cytotoxic at concentrations found in indoor air. Biological effects are only observable when ET S or its constituents are artificially highly concentrated. 4) There are no animal studies indicating that ETS, even at exaggerated concentrations, is carcinogenic. ~ 5) Claims regarding carcinogenicity, mutagenicity, and toxicity, are meaningless without reference to many factors such as specific animal species, dose, and many other factors.

COMMENTARY: The section entitled "Hazardous Constituents in ETS" in the Guide contains a pervasive, major conceptual error in that it neither mentions nor even considers the concept of delivered dose. The relationship between dose and effect is a cornerstone of pharmacology and toxicology, having been originally pointed out nearly 500 years ago by Paracelsus (1493-1541). Paracelsus wrote "All substances are poisons, there is none which is not a poison. The right dose differentiates a poison and a remedy." This concept has been shown to be so ubiquitous that it has become an axiom. Thus, it is remarkable that this could be overlooked by the EPA. For example, on page 8 of the Guide the following inflammatory statement is made: "Many of the chemicals in ETS are known carcinogens, mutagens........" Simply saying that ETS contains carcinogens and mutagens is a misleading, scientifically-irrelevant statement. The fact is that virtually every food (e.g., cooked meats, black pepper, table salt, fat, coffee, mustard, celery, parsley) and material (e.g., air, water, sunlight, our own bodies) on earth is either carcinogenic or mutagenic itself, or contains carcinogens or mutagens. Indeed, I do not know of a single material that has unequivocally been shown not to contain carcinogens and/or mutagens. The only substance on earth which has been certified by IARC as being non-carcinogenic to man, caprolactam, is mutagenic in several widely used test systems. The facts are that ETS has never been shown to be carcinogenic in any animal species. Furthermore, ETS has never been shown to be mutagenic in any animal or cell 0D culture system when tested at or near ambient concentrations. Also, none of the individual ~ .A CJ1 W 2 Ah

chemicals in ETS has ever been shown to be carcinogenic or mutagenic when tested at their concentrations in ETS. Another specific point to be addressed, on page 9 of the Guide, is the claim that "sidestream smoke has significantly higher concentrations of carcinogens and mutagens than mainstream smoke " This statement is extremely misleading and seriously flawed. Sidestream smoke has never been demonstrated to be carcinogenic in any species of animal under any experimental protocol. The only means available to compare the biological activities of mainstream and sidestream smoke are mutagenicity studies. The few studies in which mainstream and sidestream smoke mutagenicities were directly compared (Claxton et a1,1989; Monteith et aC,1987; Lofroth and Lazaridis, 1986; Ling et at:, 1987) have yielded conflicting results. These studies evaluated mainstream smoke and sidestream smoke collected directly from the burning end of the cigarette, or sidestream smoke generated by a smoking machine and introduced into a chamber to simulate a real room. In all cases, the greatest response in the Salmonella/microsome test occurs in strain TA98 in the presence of S9 metabolic activation. In the absence of metabolic activation or in studies conducted in strain TA100, mutagenicity is minimal. When compared on a revertant/cigarette basis in strain TA98 with metabolic activation Monteith et aL, (1987) found mainstream smoke to be more mutagenic than sidestream smoke, whereas Claxton et aL, (1989) found the opposite, i.et, sidestream smoke was more mutagenic than mainstream smoke. In the other two studies the results depended on the cigarette and air-dilution factors (Lofroth and Lazaridis, 1986; Ling et aL, 1987). 3

Ling et aL, (1987), compared the mutagenicities of mainstream and sidestream smokes from both 2R1 Kentucky reference cigarettes and a Swedish brand filter cigarette. Their results indicate that mainstream smoke from the 2R1 cigarettes was almost twice as mutagenic as sidestream smoke on a revertant/cigarette basis in the standard Ames test. Sidestream smoke from the Swedish brand was slightly more mutagenic than mainstream. These results show that mutagenicity is cigarette dependent, and simplistic statements regarding the relative biological activities of mainstream and sidestream smoke must be avoided. Some investigators have monitored ambient air, indoors and outdoors, and in the presence of tobacco smoking. These studies invariably involve the collection and concentration of the particulate matter from large volumes of air, usually onto filter pads. This concentrated particulate matter is then assayed in in vitro assays. The major conclusion one can draw from these studies is that virtually all air samples, both indoor and outdoor, both in the presence and absence of tobacco smoking, are mutagenic. Since the relative contnbutions of automobile exhaust, cooking fumes, ETS, and other airborne chemicals to the mutagenicity of air samples have not been characterized, one cannot draw any firm conclusions regarding the importance of ETS in the mutagenicity of air. Page 9 of the Guide contains the statement "N-nitrosamines are found in quantities up to 100 times greater in sidestream smoke " However, the EPA documents are concerned ~ ~ with ET'S, not sidestream smoke. An important fact ignored in the Guide is that ther.n ,Ah G't w 4 M

concentration of carcinogenic or mutagenic compounds in sidestream smoke, including N-nitrosamines, is lower than expected when the smoke is diluted in ambient air, probably due to chemical decomposition (Klus et al., 1987; Adlkofer et aL, 1989). To date, there is only one study in which the mutagenicity of fresh and air-diluted sidestream smoke was compared (L,ofroth and Lazaridis, 1986). In this study sidestream smoke was found to be slightly more mutagenic than mainstream smoke when the sidestream smoke was collected directly from the burning end of a cigarette. But when it was collected in a room, i.e., with mixing and air dilution (which more closely simulates ETS) sidestream smoke was much less mutagenic than mainstream smoke. It has also been shown (Sonnenfeld and Wilson, 1987) that sidestream smoke loses its toxicity toward cell cultures after only 30 seconds of "aging." Thus, statements on the chemical composition and biological activity of sidestream smoke relative to mainstream smoke are scientifically incorrect, as well as irrelevant to the present discussion on ETS. These statements should be deleted. Although highly concentrated mainstream and sidestream smoke is mutagenic, it cannot automatically be assumed that ETS is biologically active at ambient concentrations. In fact, as outlined below, results from several experiments suggest that it is not. Many studies have shown that nicotine is absorbed by nonsmokers exposed to ETS (Hoffmann et aL, 1984; Matsukura et aL, 1984; Mattson et al., 1989; Foliart et aL, 1983). While nicotine and its metabolite, cotinine, are themselves not mutagenic (Bishun et aL, 1972; McCann et aL, 1975; Florin et al, 1980; Riebe and Westphal, 1983; Brams et aL, 1987; Nakamura et aL, 1987), their presence in body fluids is often taken as an indication that a person has been 5

exposed to ETS (Eatough et aL, 1988). When nonsmokers are exposed to realistic amounts of ETS (by reference to nicotine and cotinine levels), it has been shown that their urinary mutagenicity (an indicator of genotoxic exposure) is not significantly different from that of non-exposed people (Husgafvel-Pursiainen et aL, 1987; Scherer et aL, 1987). These data are not surprising since elevated urinary mutagenicity in smokers is probably due to the uptake of particulate matter, which is minimal in individuals exposed to ETS (Adlkofer et aL, 1989). Q1 A G1 Gj 6 aD

REFERENCES Adlkofer, F.X., Scherer, G., Von Meyerinck, L., Von Maltzan, Ch. and Jarczyk, L (1989) Exposure to ETS and its biological effects: A review. In: Present and future of indoor air quality. Bieva, CJ., Courtois, Y., and Govaerts, M., (eds.) Elsevier Science Publishers, pp. 183-195. Bishun, N.P., I1oyd, N., Raven, R.W., and Williams, D.C. (1972) The in vitro and in vivo cytogenetic -effects of nicotine. Acta Biol. Acad. Sci. Hung. 23:175-180. Brams, A., Buchet, J.P., Crutzenfayt, M.C., De Meester, C., Lauwerys, R., and Leonard, A. (1987) A comparative study, with 40 chemicals, of the efficiency of the Salmonella assay and the SOS chromotest (kit procedure). Toxicol. Lett. 38:123-133. Claxton, LD., Morin, R.S., Hughes,T.J., and Lewtas, J. (1989) A genotoxic assessment of environmental tobacco smoke using bacterial assays. Mutat. Res. 222:81-99. Eatough, DJ., Hansen, LD., and Lewis, E.A. (1988) Methods for assessing exposure to environmental tobacco smoke. APCA Spec. Conf. Florin, I., Rutberg, L, Curvall, M., and Enzell, K. (1980) Screening of tobacco smoke constituents for mutagenicity using the Ames' test. Toxicology 18:219-232. Foliart, D., Benowitz, N.L. and Becker, C.E. (1983) Passive absorption of nicotine in airline flight attendants. N. Engl. J. Med. 308:1105. Hoffmann, D., Haley, NJ., Adams, J.D., and Brunnemann, K.D. (1984) Tobacco sidestream smoke: Uptake by nonsmokers. Preventive Medicine 13:608-617. Husgafvel-Pursiainen, K., Sorsa, M., EngstrSm, K., and EinistS, P. (1987) Passive smoking at work: biochemical and biological measures of exposure to environmental tobacco smoke. Int. Arch. Occup. Environ. Health 59:337-345. Klus, H., Begutter, H., Ball, M., and Intorp, M. (1987) Environmental tobacco smoke in real life situations. Proceedings of the 4th International Conference on Indoor Air Quality and Climate, 1987, p. 137-141. Ling, P.L, Lofroth, G. and Lewtas, J. (1987) Mutagenic determination of passive smoking. Toxicol. Lett. 35:147-151. LSfroth, G. and Lazaridis, G. (1986) Environmental tobacco smoke: Comparative characterization by mutagenicity assays of sidestream and mainstream cigarette smoke. Environ. Mutagen. 8:693-704. 7

Matsukura, S., Taminoto, T., Kitano, N., Seino, Y., Hamada, H., Uchihashi, M., Nakajima, H. and Hirata, Y. (1984) Effects of environmental tobacco smoke on urinary cotinine excretion in nonsmokers. New Engl. J. Med. 311:828-832. Mattson, M.E., Boyd, G., Byar, D., Brown, C., Callahan, J.F., Corle, D., Cullen, J.W., Greenbiatt, J., Haley, NJ., Hammond, S.K., Lewtas, J., and Reeves, W('989) Passive smoking on commercial airline flights. JAMA 261:867-872. McCann, J.E., Choi, E., Yamasaki, E., and Ames, B.N. (1975) Detection of carcinogens as mutagens in the Salmonella/microsome test: Assay of 300 chemicals. Proc. Natl. Acad. Sci. USA 72:5135-5139. Monteith, LG., Simmons, D.M., Myers, C.B., Hughes, TJ., and Claxton, LD. (1987) Comparative genotoxicity of sidestream and mainstream cigarette smoke. Environ. Mutagen. 9(Suppl 8):75. Nakamura, S., Oda, Y., Shimada, T., Oki, I., and Sugimoto, I{. (1987) SOS-inducing activity of chemical carcinogens and mutagens in Salmonella typhimurium TA1535/p/sk1002: examination with 151 chemicals. Mutat. Res. 192:239-246. Riebe, M. and Westphal K. (1983) Studies on the induction of sister-chromatid exchanges in Chinese hamster ovary cells by various tobacco alkaloids. Mutat. Res. 123:281-286. Scherer, G., Westphal, K., Biber, A., Hoepfner, I. and Adlkofer, F. (1987) Urinary mutagenicity after controlled exposure to environmental tobacco smoke (ETS). Toxicol. Lett. 35:135-140. Sonnenfeld, G. and Wilson, D.M. (1987) The effect of smoke age and dilution on the cytotoxicity of sidestream (passive) smoke. Toxicol. Lett. 35:89-94. 8