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MICROBIOLOGICAL ASSOCIATES PROGRESS REPORT FOR CTR-0030 - SMOKE INHALATION STUDIES IN MICE SEPTEMBER 1, 1979 - AUGUST 31, 1980 . l. L ~-- a--.). EXHIBIT S. NELSOH CTR coNTRacTS 028259 11248009 a CTR HN 044. 337`

PROGRESS REPORT Contract CTR-0030 9/1/79 - 8/31/80 TA3LE 0P CONTENTS Page Abbreviations i Synopsis of the Progress Report ii I. Chronic Inhalation Studies A. Introduction 1 B. CTR-101A. Chronic exposure of BC3F1/Cum mice to 2R1 cigarette smoke. 1 C. CTR-101B. Chronic exposure of BC3F1/Cum mice to 2R1 and 3A1 cigarette smoke. 4 0. CTR-116. Comparative toxicity of 2R1 and 3A1 cigarette smoke. 5 F. CTR-117. Comparative toxicity of 2R] and 3A1 cigarette smoke when the rest period between exposures is eliminated. 5 11. Support Services A. Dosimetry 1. CTR-119. Deposition and distribution of 2R1 and'3A1 cigarette smoke using the continuous smoke exposure regimen. 6 2. CTR-105. Deposition, distribution and clearance of 3H-catechol (CAT) in BC3F1/Cum mice after exposure to 3H-CAT-2R1 cigarette smoke. 8 B. Distribution of cigarette smoke constituents 1. CTR-106. Autoradiographic analysis of lung tissues. 10 2. CTR-107. 3H-thymidine labeling (LI) index in lung, trachea, liver, bladder, spleen or kidney after exposure to smoke. 11 3. CTR-108. Effect of cigarette smoke on pulmonary mixed function oxidases. 11 C. Short-term assays relating to possible initiating events. 1. CTR-96. Inhibition of pulmonary DNA repair capacity after exposure to cigarette smoke (Dr. R. Rasmussen). 12 2. CTR-82A. Characterization of pulmonary cytochromes involved in smoke-associated MFO-induction (Or. I. uang). 15 3. CTR-109. Alteration in the immune response after exposure to cigarette smoke (Or. H. Herscowitz). 16 4. CTR-126.-Oocyte toxicity and atresia studies (Dr. 0. Mattison). ' 17 CTR caNTRRCTS 026260 11248010 C/ ! p /M! N 044'1.Yw• •WMM^C3

PROGRESS REPORT Contract CTR-0030 9/1/79 - 3/31/80 Page 5. CTR-127. Alkaline elution assay (CTR Grant !1?41). 19 6. CTR-128. Evaluation of chromosomal damaae or alteration after ex?osure to whole ciqarette smoke using a sister chromatid exchange (SCE) assay (Dr. W. Benedict). 21 0. Feasibility of short tesrrn assays relating to possible tumor-promoting events. CTR-111. Ornithine decarboxylase (ODC) induction as a marker of promotion in pulmonary tissues. 22 E. Aerosol studies 1. Introduction. 26 2. CTR-114. Deposition and retention of aerosolized TPA. 26. 3. CTR-115 and 123. Feasibility studies for aerosolization of selected chemicals and solvent vehicles. 26 4.' CTR.121. Aerosol dosimetry studies with 3H-catechol. 27 5. CTR-122. Studies with nicotine sulfate aerosols. 27 Tables 29 Figures 78 III. Appendices Publications 118 CTR CaNTRRCTS 028261 11248011 C/ / ! H/ f W' 1 I• 1r~•3.d" •

PROGRESS REPORT Contract CTR-0030 9/1/79 - 8/31/80 A83REVIATIOt:S L AHH BA BaP, BP BaP-7,8-diol CAT COHb CSC Co nA DMBA OTC, 14C-DTC EH ETR FA HA Hb HPLC 3H-TdR IT Igu IP LI LPS MCA MFO MMS NIC ODC ORNL PAG, PAGE PAH PFC PHA P&I RDS SCE SEM SRBC TCDO TPA TPM UDS UV WHSM aryl hydrocarbon hydroxylase benz(a)anthracene benzo(a)pyrene (s)trans 7,8-dihydroxy-l,8-dihydrobenzo(a)pyrene catecnoi carboxyhemoglobin cigarette smoke condensate concanavalinA 7,12-dimethylbenz(a)anthracene doctriacontane epoxide hydrase ethoxyresorufin flucir.olone acetonide hemagglutinating hemoglobin high performance liquid chromatography 3H-thymidine intratracheal irtmunoglobulin G intraperitoneal labeling index lipopolysaccharide 3-methylcholanthrene mixed function oxidase methylmethanesulfonate nicotine ornithine aecarboxylase Oak Ridge National Laboratory polyacrylamide gel electrophoresis polycyclic aromatic hydrocarbons plaque forming cells phytohemaggiutinin Process and Instruments replicative DNA synthesis sister chrocoatid exchange Smoke Exposure Machine sheep red blood cells tetrachlorodibenzodioxin tetradecanoyl phorbol acetate total particulate matter unscheduled ONA synthesis ultraviolet Walton Horizontal Smoking Machine i CTR caNTRRCTS 028262 11248012 VwM' l I w / MI I / lrr' /4' 4rrN' 4, .yr~'

PROGRESS REPORT Contract CTR-0030 9/1/79 - 8/31/80 SYNPOSIS OF PROGRESS .I. CiiRONIC INHALATION STUDIES .A. Eauioment. 1. A sham exposure machine was designed, developed, fabricated, delivered and tested. 2. The smoke exposure systems were upgraded by the addition of an additional animal containment unit, an improved air flow monitor- ing system with.temperature ccmpensated thermistors and direct reading air flow gauges. .3. Animal holding trays were evaluated, in terms of reducing neck abrasions., Inserts to cushion the neck edge and allow size changes were designed and developed. Fabrication was not completed due to the phase out of these inhalation studies in 1981. :4. An operation and service manual was drafted and 4 of 7 sections are complete. A fifth section is in final draft stage. B. Chronic Exposure of BC3F1/Cum Mice to 2R1 Cigarette Smoke. .1. As of October 24, 1980 (approximately 25 months of exposure), 389, 276 and 178 animals remain in the smoke-exposed, sham-exposed and shelf control, groups, respectively. :2. Smoke and sham exposures will continue for ti120 weeks (ti27 months). Exposures will be stopped at this time and remaining animals be allowed to die "naturally'. January 1981, February 1981 and June 1981 will be 020 weeks for mice on test since September 1978, October 1978, and February 1979. respectively. .3. All animals in CTR 101A will be removed from smoke and sham exposure by June 1981. 4. Histopathology of randomly selected groups of animals has sug- gested that the only smoke-associated lesion observed to date has been PAHA. Six lung carcinomas were observed in both the smoke and sham- exposed groups. ;5. The capacity of 2R1 cigarette smoke to alter BaP induced lung cancer is also on test. uata to date Indicate a higher incidence of lung carcinomas in the sham exposed animals compared to the smoke exposed animals. The only lesion which could be attributed to smoke exposure was the PAMA. ii CTR CaNTRRCTS 028263 11248013 CTR I I l 1 044341

PROGRESS REPORT Contract CTR-0030 9/1/79 - 8/31/80 ) C. Chronic Exoosure of BC3F1/Cum Mice to 2R1 and 3A1 Cigarette Smoke. l. A new exposure regimen was developed which resulted in higher TPM deposition and low associated toxicity for both 2R1 and 3A1 cigarette smoke. .2. A long term study employing this regimen was proposed, approved and initiated during this contract year, however, in July 1980 it was 'decided this study riould not be completed as proposed. Rather, smoke exposure using these newly defined conditions would be limited to the length of time during which animals in CTR 101A are to be exposed. Thus, all smoke exposure studies at MICROBIOLOGICAL ASSOCIATES will cease in June 1981. .3. The animals on test In this experiment will be used to evaluate the effects of 2R1 and 3A1 cigarette smoke on 6 short term markers at 3, 6, 9 and 12 months of smoke exposure. Both male and female mice will be evaluated. II. SUPPORT SERVICES A. Oecosition and Distribution of Whole Smoke in Model Animal Systems. .1. Analytical techniques h ve been utilized to monitor and quantitate the levels of TPM, NIC, ~4C-DTC and 3H-CAT in cigarette smoke. The procedures have been performed at MA. .2. Methods for reproducibly measuri.ng the radioactive content of tissues after exposure to radiolabeled cigarette smoke (or aerosols) have been determined using the Packard TRI-CARB Tissue Sample Oxidizer. .3. The deposition, distribution and clearance of 3H-CAT has been determined in mice after exposure to 3H-CAT-2R1 cigarette smoke. The half-life for 3H-CAT In the lung is <4 minutes. Over 90% of the radiolabel is found in the urine, conjugated as a glucuronide or sul- fate, within two hours after exposure. 4. Urine from smoke-exposed mice can be shown to contain several UV absorbing species compared to sham-exposed or shelf control mice. .5. Four smoke particulate constituents, DTC, NIC, 8aP and CAT, were shown to have widely different deposition and distribution patterns in mice exposed to whole cigarette smoke. B. Short-Term Effects of Exoosure to Cigarette Smoke. .1. AHH Pulmonary AHH,levelsare 7-8..fQ.ld higher in smoke exposed mice compared to controls after exposure to 2A1, 3A1 or 2R1 cigarette smoke. . iti CTR caNTRacTS 028264 11248014 L.I' 7 R l 1 N 'i./' 44ti..p 41~v+

PROGRESS REPORT Contract CTR-0030 9/1/79 - 3/31/80 2. DNA repair (Dr. R. Rasmussen) Unscheduled DNA synthesis was inhibited in pulmonary tissues from mice exposed to 2A1 ano 3A1 cigarette smoke, but not 2R1 cigarette smoke. Replicative DNA synthesis was stimulated after exposure to all three types of cigarette smoke. 3. Labeling Index The LI for pulmonary tissue was 2-3 fold higher in mice after exposure to 2A1 and 3A1 cigarette smoke, compared to pulmonary tissue from sham-exposed or 2Q1 cigarette smoke exposed mice. 4. ODC Exposure to smoke from 2A1, 3A1 or 2R1 cigarettes resulted in a 2-3 fold induction of ODC 6-9 hours after the final exposure. 5. Immune competence (Dr. H. Herscowitz) Acute or chronic exposure of BC3F1/Cum or BALB/c mice to 2R1 cigarette smoke failed to suppress the splenic PFC response. .6. Oocyte depletion (Dr. 0. Mattison) The rate of oocyte depletion was not influenced in ovaries of mice exposed to either 2A1 or 2R1 cigarette smoke compared to sham- exposed or shelf control animals. .7. DNA damage (Grant 11241) Exposure to 2A1 cigarette smoke resulted in minimal effects on pulmonary DNA compared to sham exposed animals. Enhanced damage to pulmonary DNA was observed in smoke exposed mice treated with BaP and BaP-7,8-diol compared to sham-exposed BaP treated mice. 8. Sister chromatid exchange (Dr. W. Benedict) Preliminary results indicate an. :SCE• rate which is 2 fold higher in mice exposed to 3A1 cigarette smoke, compared to sham-exposed mice. ,9. Lung weights _ Exposure to 2A1 and 3A1 cigarette smoke resulted in an increased lung iveight, compared to sham-exposed of 2R1 cigarette smoke exposed mice. iv CTR CONTRRCTS Q26265 11248015 CTR I-IN 044~~ 43

PROGRESS REPORT Contract CTR-0030 9/1/79 - 8/31/80 C. Aerosol Studies 1. Heasurements of aerosol concentration as a function of solution concentration have been completed for two chemicals. 2. Particle size distribution have been initiated using a 7 stage cascade impactor. 3. Preliminary calculations of a"1 ng deposition factor" have been made for 34-CAT after exposure to ~H-CAT-2R1 cigarette smoke. v CTR caNTRacTS 028266 11248016 CTR HN 044,`344

PROGRESS R:?ORT Contract CTR-0030 9/1/79 - 8/31/80 I. CHRONIC i`HALATION STUDIES A. Introduction The chronic cigarette smoke exposure studies were divided into three areas: a) chronic exposure of BC3F1/Cum female mice to 2R1 ciga- rette smoke (CTR-101A), b) comparison of effects of exposure to 2R1 or 3A1 cigarette smoke in BC3F1/Cum female mice (CTR-101B), and c) de- velopment of a new smoke exposure regimen* which eliminates the rest period (CTR-117). These studies were initiated as scheduled and are on- going at this time. Other studies were also completed during this funding year which were /n preparation for the studies comparing the effects of 2R1 and 3A1 cigarettes. :n March, 1980, a sham exposure machine was delivered to MA from Process and Instruments (P&I. Brooklyn, NY). The sham exposure machine duplicates the performance of the SEli II smoke exposure machines without generating whole cigarette smoke. Control animals can thus be treated exactly as the smoke exposed but without exposure to smoke ("sham- exposed°). The availability of this machine allowed the SEM II B and C machines to be scheduled full time for smo.ke generation, with the control animals sham-exposed on the sham machine. In this way, the capacity for Inhalation studies was greatly increased and the biological effects of both cigarette types could be analyzed simultaneously and with sham- exposed controls. The smoke exposure systems were upgraded by the addition of an additional animal containment unit .(a total of four are now available), improved air flow monitoring with temperature compensated thermistors, and direct reading air flow gauges. The servicing of the exposure systems were also upgraded by the development of the operation and service manual. The production of operation and service manual has been coordinated by Mr. Tom Gayle at ORNL, with assistance provided by Mr. Doyle Mullinax at MA and Mr. Leroy Florant at P&I. The manual provides schematic wiring diagrams and specifications for all components, detailed instructions for operation, ir.cluding trouble shooting sections. Four of the seven sections of the manual are complete (Table 1). :opi'es of the final drafts have recently been distributed to CTR. PSI, and MA. Section IV, Animal Handling Procedures and Techniques is in the final draft stage. The last two sections to be prepared are scheduled for completion in 1981. The tables of contents for Sections I-I'/ are given in Tables 2-5. The manual represents.the culmination of the ' development of not only the sophisticated smoke generation monitoring and exposure system, but the procedures and techniques for conducting long- term inhalation experiments with animals. B. CTR-:01A. Chronic exDosure of BC3F1/Cum mice to 2R1 cioarette smoke. This study was initiated in September, 1978, with a total of 2053 mice exposed to 2R1 smoke alone, a total of 1014 mice sham-exposed, and a total of 449 mice for shelf controls. -1- C1"R caHTRRCTS 028267 11248017 Md' /-R IMII / V• / I 3! Vu.'

PROGRESS REPORT Contract CTR-0030 9/1/79 - 6/31/80 An additional 710 mice were utilized to evaluate the cocarcinogenic effects of 2R] cigarette smoke and benzo(a)pyrene (BaP). As of October 24, 1980, a total of 389, 276, and 178 mice remain on test in the smoke, sham, and shelf control groups, respectively. Of the 389 smoke- exposed mice, 80 have been on test since September, 1978, 147 since October, 1978, and 122 since February, 1979. At monthly intervals, animals were weighed to ascertain their gen- eral health status. The average weights of smoke-exposea, sham-exposed and shelf control'animals at four weekly intervals over the first 80 weeks of the study are presented in Figure 1. There was a significant suppression of weight gain in both the sham and smoke-exposed'animals compared to shelf controls. 7here was no difference in weight gain between the sham and smoke exposed animals. As observed earlier in CTR-100, daily exposure and restraint on either the SEM II or sham exposure machines results in significantly slower weight gain. After 20-30 weeks of exposure, certain animals were observed to have reddened skin and worn away hair around the neck area which fits Into our "stock-type° holders. These- lesions progressed to open sores by 40-60 weeks of daily restraint. Approximately 90% of the animals express some level of neck abrasions by 80 weeks of exposure and 30-40% of these lesions are quite severe. Polycarbonate inserts to cushion the neck slot and of varying sizes were analyzed and found to be quite effective in limiting these neck-cuts, however, the scheduled fabrication and use of these inserts was not carried out because of the decision to discontinue any further life-time smoke-exposure studies. It is our opinion that these inserts must be used for any long term studies which may utilize animals of different ages and/or sizes. At monthly intervals, 3-5 mice per group were tested for carboxy- hemoglobin (COHb) levels immediately after smoke or sham exposure. The percent COHb at 4 weekly intervals is presented in Table 6. Every mouse ' which was exposed to 2RI cigarette smoke expressed high•levels of COHb with the average being %.18X COHb over the ti2 year observation period. No animal in the snam or in the shelf control groups expressed significant levels of COHb. These results indicate that theanimals were unable to avoid smoke exoo- sure. The level of smoke exposure has been documented throughout the experiment. Other studiei in our laboratory have shown that there is a high correlation between COFb levels in an animal and the level of total particulate matter (TPM) deposited in the respiratory•tract of the smoke exposed animals (r-.8, p<.01). Thus, these data confirm that this smoke exposure system generates smoke in a manner which results in the reproducible deposition of both particulate phase and gas phase constituents in the respiratory tract of the exposed animal. The disposition of all animals for CTR-101A is given in Table 7. A total of 1177 mice have been histopathologically examined and another 485 are being processed. Every individual animal has been accounted for. -2- CTR CONTRACTS 028268 11248018 CT~"~ PIN 04#4346

PROGRESS REPORT Contract CTR-0030 9/1/79 - 8/31/80 Analysis of the reasons for death in this study are given in Table 8. Virtually 80: of animals were,taken off test by the impact of the•exposure system itself. These animal deaths were randomly distributed over the 104 week exposure period and the incidence of any histopathologic lesion for any 4 week period should reflect the incidence of that lesion. in the population of animals on test at that time. The abbreviations used for the lung lesions observed are given in Table 9. A selected sumnary is given in Table 10 of all pulmonary histopathology for all six groups of CTR-101A animals. 1. Exposure to 2R1 cigarette smoke alone. A total of 415 animals have been diagnosed in the smoke alone groups, a total of 295 in the sham exposed group, and a total of 66 in the shelf controls. The only smoke-associated lesion observed to date has been the accumulation.of pigmented alveolar machrophages (PAMA). Six lung carcinomas have been observed and they were found.in both smoke- and sham-exposed animals. Leukemia, lymphosarcoma, osteogenic sarcoma and reticulum cell sarcoma have also been observed, and these tumors also seem to be evenly distributed over both smoke- and sham-exposed groups. The actual time of occurrence of these various lesions for the smoke, sham or shelf groups is presented in Tables 11 through 16, respectively. 2. Exposure to 2R3 smoke after intratracheal inoculation of BaP. Lung lesions observed following intratracheal treatment with 1.2 mg BaP given with and without smoke exposure are summarized In Table 10. Several lung carcinomas were induced by BaP, including AAC, SCC. and POC. The total incidence of lung carcinomas was higher in the sham exposed compared to the smoke-exposed animals (67/162 vs 43/159). The only lesion which could be attributed to smoke exposure was PAMA. The incidence of these lesions at 4 week intervals is presented in Tables 17-22 for the smoke + BaP, Sham + 9aP, and shelf + BaP groups. Preliminary analysis of the data from those animals which died during the experiment and which are known to have died with a random dis- tribution (125 smoke exposed and 106 sham exposed animals) has been performed- using the Mantel-Haenszel statistical procedure. The results suggest that there are significantly more malignant lung cancers (AAC, SCC and POC) in the sham-exposed compared to s.oke-exposed animals (p-.0386). There is no difference in the time-dependent incidence in premalignant lesions (SN, ANCN, ACN) (p-.5535). Incidence of premalignant and malignant lesions was also not different between the two groups (pa.8145). Since many animals remain to be analyzed it is premature to draw firm conclusions. However, it seems that as we observed in the C'R-100 study, whole cigarette smoke has very little activity as a cocarcirogen with BaP at the dose used in this study. -3 - CTR CONTRRCT6 029269 11248019 CTR `4 M/ / 04/' 34•! t

PROGRESS REPORT Contract CTR-0030 9/1/79 - 8/31/80 C. CTR-101B. Chronic exoosure of BC3F1/Cum mice to 2R1'and 3A1 Ciaarette smoke. ' 1. Background During the last year, an exposure regimen was developed which was estimated to result in high TPH deposition (ti1 mg TPM/day) and low associated toxicity for'both 2R1 and 3A1 cigarette smoke (see discussion CTR-117). These exposure conditions allowed the scheduling of a long term study whereby the biological effects of smoke from both cigarette types could be simultaneously evaluated. Such a long term study employing this new smoke exposure regimen was proposed, approved and initiated during the 1980 contract year (this study was referred to as CTR-118 in the Proposed Studies for 1980, which i's now termed CTR-101B). In July, 1980, however, it was decided that this study should not be complPted as proposed.and that smoke exposure under these newly defined conditions would be limited to the length of time during which animals in CTR-101A are to be exposed to smoke. As a result, all smoke exposure studies at Microbiological Associates will•c.ease in June, 1981. The animals in CTR-lO1B will be used to evaluate the effects of 2R1 and 3A1 cigarette smoke on 6 short term markers at 3, 6, 9, and 12. months of smoke exposure. The short term markers to be analyzed in male and female mice are: a) inhibition of pulmonary DNA repair, b) stimulation of pulmonary DNA synthesis, c) induction of pulmonary AHH activity, d) ip- duction of pulmonary ODC activity, e) augmentation of the DNA damaging effects of particular lung carcinogens, and f),alteration of.certain physical characteristics such as lung weight and incidence of PAMA. The smoke exposure'regimen for this study is significantly different from other exposure regimen carried out in this laboratory. • There are no rest periods between successive cigarettes. • The exposure period•for smoke exposure is :5 seconds per minute. • Smoke expasures are repeated "continuously" over 140 minutes. • Each "run" results in exposure to the ecuivalent of C 17 2R1 or 3A1 cigarettes (8 puffs/cigarette). The disposition of animals in this study are presented in Table 23. A total of 417 mice are presently be,ing exposed to 3A1 smoke, 322 mice are being exposed to 2R1 smoke, 401 mice are being sham exposed, and 156 mice are shelf controls. These animals will be removed from test at 3, 6, 9, and 12 months of smoke exposure and analyzed for the 6 markers previously described. -4- CTR C0NTFZRCTS5 026270 11248020 CTR NN 044-7;'3~• A-1

PROGRESS REPORT Contract CTR-0030 9/1/79 - 3131/80 The precent COHb in these mice irmediately following this exposure regimen is presented in Table 24. These values for the 2R1 smoke exposea animals are approximately twice that for the mice in CTR-101A. The COHb values for the 3A1 smoke-exposed animals are similar to that observed in CTR-100. These data suggest that. much more smoke is being taken up by these animals during this new smoke exposure regimen. A more complete discussion of the biological effects of this smoke exposure regimen is discussed in CTR-117. 0. CTR-116. Comoara:'.ve toxicity of 2R1 and 3AI cicarette smoke. This study was to be implemented if the animals could not survive the continuous exposure regimen. Data presented in CTR-117 show that this continuous exposure regimen worked very well and CTR-116 was, therefore, not required. A comparison of the toxicity of 2R1 and 3A1 cigarette smoke is given in the discussion of CTR-96. E. CTR-117. Comoarative toxicit of 2R1 and 3A1 ci arette smoke when the rest oeriod between exoosures is e iminated. As discussed in CTR-101B, a new exposure regimen. has been utilized where the rest periods between successive cigarettes has been eliminated. TPM deposition per mouse has been estimated to be twice that deposition observed in CTR-101A while requiring the same amount of machine time. The effect of this smoke exposure regimen on BC3FI/Cum female mice after exposure to 2P.1 cigarette smoke Is given In Table 25. The toxicity was 0.9% per week. The toxicity observed for the exposure regimen used in CTR-lOlA was ti1.6S per week, while TPM deposition and percent COHb were approximately one-half that in this regimen. Thus, this exposure regimen seems to result in a higher dose of smoke taken up by the animals with less toxicity. This exposure regimen was also used to evaluate the toxic effects of smoke exposure to male BC3F1/Cum mice. Table 26 shows the toxic effects of 2R1 cigarette smoke in male 8C3F1/Cum mice. Tnxicity was ti1.8S per weex, which is higher than that observed in female BC3FI/Cum mice suggesting that male mice are slightly more sensitive to the toxic effects of 2R1 cigarette smoke (see Figure 2). The percent of COHb was similar in both female and male mice. Histopathology on female BC3F1/Cum mice over the first 20 weeks of smoke exposure is presented in Table 27. Twenty-three of 24 animals which were examined•after 1? weeks of smoke exposure were observed to have PAMA. This is in direct contrast to CTR-10IA, where high levels of PAMA have still not been observed after approximately 2 years of daily smoke exposure. These data indicate that this exposure regimen results in a higher dose of smoke to the mice, with a higher T?M deposition In the respiratory tissue. -5- CTR COHTRRCTS 028271 11248021 . CTR HN 044,349

PROGRESS REPORT Contract CTR-0030 9/1/79 - 8/31/80 II. SUPPORT SERVICES A. Dosimetry 1. CTR-119. Deoosition and distribution of 2R1 and 3A1 cigarette smoke usina the continuous smoke exoosure regimen. The results frcm'CTR-117 have shown that the new exposure regimen results in fairly low toxicity to BC3F1/Cum fe.male and male mice. The actual distribution and dosimetry of TP?4 from radiolabeled smoke from the 2R1 and 3A1 cigarettes under these exposure conditions are scheduled for CTR-119. This study has not been completed due to scheduling difficulties on the SEM II B and C, which have not allowed shut down of the machines for the necessary 3-4 day period in order to generate the radiolabeled smoke. It is anticipated that machine time will be available in early December for this experiment. As a part of the monitoring of th smoke generated for the dosimetry experiments, T?M, nicotine and 14C-DTC content of radio- labeled cigarette smoke was determined in our laboratory at MA and the data compared and verified with previous experiments at ORHL.(see below). The tissues for this dosimetry experiment will be analyzed for radioactive content using the new Packard 306 TRI-CARB tissue oxidizer. In order to insure that techniques and procedures have been adequately implemented with this instrument, dosimetry studies using the small Walton Horizontal Smoking Machine (WHSM) were scheduled. Data could then be compared with that of previous dosimetry experiments analyzed by tissue solubilization procedures. Results from both the smoke monitoring and the WHSM dosimetry studies are presented in the following sections. a. Analysis of smoke constituents. Nicotine (NIC) is the principal alkaloid present in cigarette smoke. It is associated with the particulate phase of smoke and is found with the total particulate matter (T?M) collected on a Cambridge filter pad. NIC was quantitated by extractions with various organic solvents to compare the extraction efficiency of nicotine from Cambridge filter pads. Table 28 shows that hot chloroform extraction from Cambridge filter pads gives 100% recovery, whereas 95%, 89% and 33% of recovery was found by using pyridine, ethanol, and p-dioxane, respectively. NIC in these solvent extracts was quantitated by utilized gas liquid chromatography (GLC,PERKIN ELMER, Model 3920 B). The extracts of Cambridge filter pads were dissolved in chloroform, and 1 ul and 2 ul were infected into the GLC equipped with a flame ionization detector. The column was a silanized 3 ft x 4 irm ID coiled glass tube packed with ~ 3% OV-17 on gas chrome Q (80-100 mesh, Supelco). The column and in,iec- tion port temperature were 1400C and 2000C, respectively, and detector oven temperature was 3000C. -6- CTR CCNTRRCTS Q28272 11248022 C T R I ~~N 0 - -1• 4 3 1: V' 0

PROGRESS REPORT Contract CTR-0030 9/1/79 - 8/31/80 L.. Helium gas was used as a carrier at a flow rate of approximately 40 ml/min and attenuator setting of 8 x 10. The NIC elution profiles shown in Figures 3 and 4 were compared with NIC standards (Eastman, Rochester, N.Y.) dissolved in chloroform. Figure 5 demonstrates the linearity response of a flame ionization detector against the various nicotine concentrations using an external standard method. Figure 6 indicates that it is feasible to use 3-methylindole as an internal standard for GLC analysis to quantitate the amount of NIC on the pads as well as in biological samples. NIC samples generated from reference cigarettes on a Phipps and Bird-Smoking Machine were analyzed both at ORNL and MA, to compare the analytical methods used at both laboratories. The results from both laboratories are shown In Table 29. Chloroform extraction and the 0V-17 chromatography column were highly efficient in determining the amount of NIC in these samples. The 20% recovery difference observed resulted from differences in inf ection techniques. In the laboratory at MA, the dead space volume of the injection syringe is accounted for, yielding higher values than if it is not accounted for. A correlation coefficient of 0.99 was obtained for the relationship between NIC and TPM for NIC determined at MA, while a correlation coefficient of 0.97 was obtained for the data obtained by ORNL. These analytical techniques have therefore been utilized to reproducibly quantitate levels of NIC generated in the smoke exposure• stud es at MA. Oata are presented in Table 30 for analysis of TPM, NIC and 4C-DTC levels generated from 14C-DTC-2R1 cigarettes on the WHSM. A11 of these experiments and subsequent analyses were performed at MA. These data demonstrate the direct relationship between increase In TPM, NIC and 14C-OTC-dpm with increasing smoke exposure (puffs per cigarette). The correlation between these three constituents is also demonstrated, indicating that any two measurements of TPH, NIC, or 14C-DTC-dpm can be used to monitor the levels of the third parameter. These data also point out the difference in NIC yield when cigarettes are burned on the WHSM, compared to cigarettes burned on the Phipps and Bird analytical sroking machine. On the WHSM cigarette butts are "free" to the atmosphere between puffs, while in the Phipps and Bird, the cigarette butts are "restricted" between puffs and are not exposed to the atmosphere. NIC yields are significantly higher from the WHSM, ccrpared to the yield from the Phipps and Bird (Stokely, J.R., Woneyhum, J.H., Guerin, M.R., Florant, G., and Greenspan, J. In Tobacco Smoke Inhalation Bioassay Chemistry, edited by M.R. Guerin, 3:R. Stokely and C.E. Higgins, ORNL-5424, 1979, pp 1-17). These data point to the need for moni.toring of the smoke generated under actual experimental conditions rather than extrapolating from the analytical data. It should be noted that the NIC levels in the smoke generated on the wHSH from 2R1 cigarettes are. approximately 1.5-2.0 times higher than when the smoke is generated from the Phipps and Bird. Since the cigarette butts are also "free" in the SEH II smoking machine, the levels of NIC were determined for smoke generated on the SEM U. -7- CTR CaHTRRCTS5 026273 11248023 C T R VIN 0 4 4,3 =5 I

PROGRESS REPORT Contract CTR-0030 9/1/79 - 8/31/80 The monitoring system for the SEM II has the capacity to instanteously and simultaneously determine TPM, COZ and CO levels in smoke. For determinations of nicotine or radioactivity present in smoke, the same Cambridge filter is analyzed which is used to filter the smoke for analysis of the gas phase constituents. The data in Table 31 summarize the measurements of TPM, C02, CO and nicotine in 2R1 cigarette smoke generated on the SEM II. The NIC/TPM ratio from 2R1 cigarette smoke is lower than that observed on the WHSM. The effect of the "free" vs. "restricted" configuration for the cigarette butt does not appear to be as great in the SEM II as in the WHSM, but again points to the necessity of monitoring the smoke actually generated for inhalation_experiments. b. 14C-DTC dosimetry on the WHSM. The previous section showed that techniques re now available for the rapid quantitation of TPM, nicotine and t4C-DTC in cigarette smoke. No preliminary 14C-DTC-dosimetry experiments were performed using the WHSM so as to become familiar with the tissue sample oxidizer and to be able to compare the data in terms of previous experi- ments andlyzed by tissue. solubilization. Data are presented in Tables 32 & 33 for TPM deposition and distribution in BC3F1/Cum female mice after exposure to 10S-2R1 cigarette smoke•using the 15/45 second (smoke/ air) exposure cycle. Mice were sacrificed by C02 asphyxiation immed- iately after smoke exposure. Tissues (lungs, trachea, larynx and turbinates) were removed and placed directly into combusto cones In scintillation vials, stored at -60oC until analyzed with TRI-CARB Sample Oxidizer (Packard, Downers Grove, Iil.). The procedures for sample combustion were those recommended by Packard Instruction Manual 2130/1. Combusted samples were analyzed for radioactivity using a Beckman LS-330 Liquid Scintillation System. All samples were corrected for background and quenching as determined by the External Standard Method. When mice were used which had not been previously exposed to smoke before the dosimetry experiment, a large coefficient of variation (CV) for lung deposition was observed (Table 32). However, when smoke "adapted" mice were used, the CY was reduced (Table 33) and a linear dose response for TPM deposition in the lung was observed as a function of total smoke exposure time (Table 33). The fifteen second smoke exposure interval on the WHSM results in a lower deposition than that estimated after exposure on the S-7M II. Techniques and procedures have now been established at MA to perform the large dosimetry experiment using the SEM fI. This experiment will quantitate.the TPM deposition and distribution in BC3F1/Cum mice after exposure to the "continuous" regimen discussed previously. This experiment will be completed by December, 1980. 2. CTR-105. Ceoosition, distribution and clearance of 3H- catechol (CAT) in BC3F1/Cum mice after exposure to 3H-CAT-2R1 cigar- ette smoke. Previous studies have examined the pharmacokinetics of quantities of BaP, MCA, UIC and DTC in mice after exposure to these aerosolized chemicals in cigarette smoke. -8- CTR CoNTRRCTS 028274 11248024 C T R N V 1 0 4 4'-~~.', -a

PROGRESS REPORT Contract CTR-0030 9/1/79 - 3/31/80 BaP, HIC and OTC are constituents of smoke found in the particulate phase and represent, respectively, the polycyclic aromatic hydrocaroons, the tobacco alkaloids and a straight chain hydrocarbons. BaP, MCA, and NIC were shown in previous studies to be rapidly cleared (th<2 hours) from the mouse lung, while OTC was retained at significant levels up to a week post exposure. CAT, 1,2-dihydroxybenzene, is of interest for these studies iri that it represents a fourth class of chemicals (diphenols), is present in moderately high concentration in cigaret:e smoke 220-550µg/cigarette, (Mold, J.D., Pepton, M.P., Means, R.E., and Walker, T.B., Analyst 91, 189, 1966), and has been reported to be a weak carcinogen. (van Ourren, B.C., Katz, C., and Goldschmidt, J. Naa . Cancer Inst. 51, 703, 1973). We report h re the pharmacokinetics of inhaled 3H-CAT in mice after exposure to ~H-CAT aerosolized in cigar- ette smoke. BC3F1/Cum female mice were exposed to 10% (v/v) smoke from 3H-CAT-2R1 cigarettes under standard conditions on the WHSM for a total of 300 seconds. Mice were sacrificed by C02 asphyxiation at the following times after exposure: 1:25, 2, 4, 4:25, 5:25, 6:30, 7, 7:60, 8:30, 9:25, 10, and 10:9 minutes. Levels of radioactivity in Individual tissues was determined by tissue oxidation (using a Packard TRI-CARB Tissue Oxidizer). 3H-CAT was rapidly cleared from the respiratory tract as shown in Figure 7. The half-life (t4) of H-CAT In the lung was less than 4 minutes. 3H-CAT was rapidly absorbed in the blood and rapidly removed (tk<4 minutes, Figure 8). Significant levels of radioactivity were found in the liver and kidney immediately after exposure (Figures 9 and 11). These levels decreased rapidly (th-. 4 minutes, Figures 9 and 11). Small amounts of radioactivity were observed In the spleen and pancreas (Figure 9) and brain and heart (Figure 10). Two hours after exposure, approximately 90% of the 3H label was excreted in the urine (Table 34). Less then 10% of the labeled compound remaine~ in the lung, turbinates, liver, or kidney (Table 34). Over 90% of the H label in the urine was not extracted by chloroforn- methanol (3:1 , v/v, Figure 12) or an ethyl acetate (Figure 13), suggesting that CAT was metabolized to another form before excretion. High performance liquid chromatoqraphy (HPLC) was used to analyze both the ultraviolet (UV) absorbing and radioTabeled metabolites in u-rine. As shown in Figure 13, free CAT Is not detected in urine from smoke-exposed mice. Following g-glucuronidase and arylsulfatase treatment of the urine, both UV and radiolabeled CAT were detected (Figure 14) in HPLC profiles. These data indicate that CAT is detoxified through conjugation as a glucuronide and/or a sulfate before excretion ine the urine. Enzymatic digestion of the urine from smoke exposed mice resulted in additional UV absorbing peaks in the HPLC elution profiles (compare Figures 138 and 148). Urine from mice on test in CTR 1018 was analyzed ` to determine whether these additional peaks were smoke-associated. :n Figures 15, 16 and 17 are presented thg. HPLC elution profiles of. enzyme treated urine from shelf controls, sham-exposed and smoke-exposed mice from CTR 101B. -9- CTR caKTRacTS a2-8275 11248025 rM I R ! I N •-/' , I43M5V/."

PROGRESS REPORT Contract CTR-0030 9/1/79 - 8/31/80 > The HPLC profiles of urine from shelf controls and sham-exposed mice are almost identical (Figures 15 and 16). The HPLC profile of urine from smoke exposed mice reveals many additional peaks, suggesting that many constituents inhaled in smoke are excreted in the urine in con- jugated form. Longer exposure of BC3F1/Cum mice to smoke aerosols containing 3H-CAT resulted in'deposition and distribution of label similar to that seen after shorter exposures. Data in Table 35 indicate that TPM deposition does not accumulate in the respiratory tract after 1200 seconds of exposure. TPH deposition as represented by 3H- CAT is observed in the liver and kidney immediately after this 1200 second exposure (Table 35). Data are summarized in Table 36 for the percent distribution of the four smoke constituents, DTC, NIC, BaP and CAT, in the internal tissues of the mouse after exposure to whole cigarette smoke. Deposition in the lung of the four particulate constituent ranges from 2.5 to 69%. TPM can be represented by all four of these chemicals but different results are observed for the deposition and'distribution of each, thus indicating the complex nature of cigarette smoke and the potential difficulties in ascribing biological activity to cigarette smoke. B. Distribution of Cigarette Smoke Constituents 1. CTR-T06. Autoradiograohic analysis of lung tissues. Autoradiography of lung tissue after appropriate exposure to radioactively labeled chemicals allows determination of the deposition and distribution pattern within the lung. After exposure of the mice to 14C-DTC aerosolized in 2A1 or 2R1 cigarette smoke, we demonstrated that the tracer could be seen to have reached all portions of the lung and was somewhat concentrated along the bronchi and bronchioles (Progress Report, 1979). Since the technique of using frozen cut sections without organic solvents has been reproducible and routine in our laboratory, we wished to extend these studies to determine the irrmediate localiza- tion of either 3H-tetradeconyl phorbol acetate (TPA) generated as a chemical aerosol or 3H-CAT, generated both in a smoke aerosol and as a single chemical aerosol. Data from CTR-105 demonstrated that the half-life of 3H-CAT in the lung was less than 4 minutes. Due to rapid clearance from the lung wnich occurs with 3H-CAT and low quantities of radioactivity found in pulmonary tissue, autoradiographic analysis was not attempted. Should hi gher quantities of radioactivity be observed after aerosolization of 3H-TPA or 3H-CAT from ethanol, lungs from the aerosol exposed mice will be quick-frozen and autoradlograpnic analysis performed. L -10- CTR CoNTRRCTS o2e276 11248026 CTT%'> I-IN 0443 -4

PROGRESS REPORT Contract CTR-0030 9/1/79 - 3/31/80 2. CTR-107. 3H-Thymidine labelina index (LI) in luno, trachea, liver, biadder, saleen or kidney after exoosure to srroKe. Results obtained in collaboration with Dr. R. Ramussen (University of California at Irvine, Irvine, CA) suggested that one of the effects of exposure of mice to 2A1 cigarette smoke is an increase In the fraction of cells in the lung in which 3H-Thymidine (3H-TdR) is incorporated (Progress Report, 1979). These data are presented as a labeling index (LI), defined as the number of cells labeled with 3H-TdR divided by the total number of cells examined x100Y. The LI is deter- mined from autoradiographic analysis of sections of fixed tissue. Since in most eukaryotic cells the 3H-TdR-LI is proportional growth rate, measurement of LI was undertaken to further investigate the effect which cigarette smoke might have on cellular turnover rate in the lung and other tissues. Experiments performed in collaboration with Dr. Rasmussen and reported previously have indicated that pulmonary replicative DNA synthesis (RDS) was stimulated after exposure of mice to smoke from 2A1 cigarettes but not 2R1 cigarettes (Progress Report, 1979). To Investigate these observations, the LI was determined in BC3F1/Cuar female mica after exposure to three different cigarette types, 2A1, 3A1 and 2R1, as part of a rather large comprehensive experiment with Dr. Rasmussen. Results and details of this experiment, Including the LI data, are discussed under CTR-96, however, the data suggest that the LI is 2-3 fold higher in lung tissue from mice exposed to 2A1 or 3A1 cigarette smoke, compared to lung tissue from mice exposed to 2R1 cigarette smoke or sham exposed. These effects were observed after 9 and 13 weeks of exposure. Tissues from mice exposed for 24 and 41 weeks are under investigation. 3. CTR-108. Effect of cigarette smoke on pulmonary mixed function oxidases. Measurement of MFO activity in lung tissue is a rapid, sensitive and reproducible method to biochemically assess the effect of exposure of various chemicals to pulmonary tissue. The substrates used for these assays are either BaP or ethoxyresorufin. An advantage to the latter chemical is that unexposed or control tissues have little capa- city to metabolize this chemical and, therefore, low levels of induced MFO can be detected. MFO levels have been determined in pulmonary tissues of BC3F1/Cum mice after exposure to smoke from 2A1, 3A1 and 2R1 cigarettes. Results and details of this experiment, including the Induction of MFO, ,• are discussed under CTR-96, however the data suggest that the AHH levels in the smoke exposed lung tissues are 7-8 fold higher than the lung tissue from sham-exposed mice. Further, the level of induction was constant over 24 hours after exposure to smoke from all three cigarette ~ types. CTE2 CONTRRCTS 0282 77 11248027 CTR NN 0443' r,."~ 5

PROGRESS REPORT Contract CTR-0030 9/1/79 - 8/31/80 As discussed under CTR 101B, the levels of pulmonary and hepatic MFO will be determined in BC3F1/Cum female mice after exposure to 3A1-and 2R1 cigarette smoke for 3, 6, 9 and 12 months using the "continuous" exposure regimen. Data will thus be available concerning whether differences exist in HFO-induction kinetics or levels following exposure to different cigarettes using different smoke exposure regimens. C. Short-term Assays Relating to Possible Initiating Events. 1. CTR-96. Inhibitton of oulmonar DNA reoair caoacity after exposure to cicarette smoke (collaboration with Dr. R. Rasmussen). a. 'Introduction Collaborative studies with Dr. R. Rasmussen (University of California at Irvine, Irvine. CA) continue to further characterize the effects of whole cigarette smoke on the capacity of pulmonary tissue to repair DNA damaged in an in vitro assay. Previous results indicated that chronic exposure of BC3F1/Cum mice to 2A1 cigarette smoke resulted in an inhibition of DNA repair as determined by unscheduled DNA synthesis (UDS) and that this inhibition persisted for at least 6 months after exposure was terminated (Progress Report, 1979 and Appendix A, Rasmussen, R.E., Boyd, C.H., Dansie, D.R., Kourt, R.E. and Henry, C.J., DNA Repli- cation and Unscheduled DNA Synthesis in Lungs of Mice Exposed to Cigarette Smoke, submitted to Cancer Research, 1980). When these studies were extended to include exposures using 2R1 cigarettes and C3H/Anf Cum mice, in addition to BC3F1/Cum mice, no inhibition of UDS was observed up to 27 weeks exposure (Progress Report, 1979). To investigate these observations a rather large comprehensive study was proposed last year and the results are presented here. b. Experimental Protocol The SEM II 8 and C were used to generate smoke from 2A1, 3A1 and 2R1 cigarettes. Groups of BC3F1/Cum mice, 8-10 weeks old, were adapted to smoke from all three cigarette types. In the following table the exposure conditions are summarized for each cigarette, all utilizing 10% (v/v) smoke concentration and standard exposure ccnditions of 2 second puff duration, 35 ml average puff volume: Cigarette Type Exposure Cycle Smoke/Air (sec) Total Smoke Exposure/ Day (sec) Number of Cigarettes 2A1 30/30 2400 10 3A1 30/30 2400 10 2Rl 20/40 1600 10 Sham 0 0 0 Shelf 0 0 0 -12- CTR CaNTRRCTS 02E3278 11248028 C.; T R N t 4 0 4, 4, °~G

PROGRESS REPORT Contract CTR-0030 9/1/79 - 8/21/80 Eight puffs per cigarette were used to reduce the toxicity of high nicotin,e containing 2R1 cigarette, yet maximize the dose. The exposure regimen was given in two sessions per day (5 cigarettes in the morning, 5 cigarettes in the afternoon). Ten minutes of air were given between each cigarette. Mice were shipped to Or. Rasmussen after 9, 13, 24 and 41 weeks of exposure for analysis of pulmonary DNA repair capacity (Progress Report, 1979 and Appendix A). Mice from each grcup and time were retained at MA for analysis of ODC, MFO and 3H-TdR-LI. Smoke exoo- sures were documented and entered on computer so that the TPM dose for each cigarette type cociid :e determined. c. Results (1) Toxicity It was proposed that this experiment be on test for 6 months, however, with results obtained during the study, it was extended to as long as possible, approximately 8~ months. Sroke expo- sures for CTR-96 stopped in June, 1980, due to Initiation of the CTR-1018, which required the use of both the SEM II B and C and utilized a completely different exposure regimen (see CTR-1018). The exposure regimen used in CTR-96 was similar to that used in CTR-100. Intermittent smoke exposures (e.g.. 30 seconds smoke followed by 30 seconds air per minute over 8 minutes) were followed by 10 minutes of air before the next smoke exposure. Five exposure sessions requiring 90 minutes actual machine time were given in the morning and another five in the afternoon (equivalent to '10 cigarettes/ day"). The disposition of the animals for this study is shown in Table 37. The toxicity observed using this exposure regimen was 1.8%, 2.4%, 2.3%, and 0.9% per week for 2A1, 3A1, 2R1 smoke and sham-exposed mice, respectively. As a point of comparison, the toxicity observed in CTR-117, which requires one-half the machine time to deliver equivalent doses, was 0.9% per week. (2) UDS and RDS Data are presented in Table 38 for the UDS and RDS levels observed in vitro in lung tissue from BC3F1/Cum mice exposed to smoke from 2A1, 3A1 or 2Rl cigarettes. The cumulative TPM deposition has been estimated from the daily smoke exposure documentation, resulting in approximately 1 mg TPH/day/mouse for 2A1 and 3A1 cigarettes and ti.8 mg TPM/day/mouse for 2R1 cigarettes. Results for UDS and RDS in tissues from smoke exposed mice are given relative to the common sham-exposed controls. UDS appears to be depressed after 13 weeks exposure to 2A1 and 3A1 cigarette smoke, but not after exposure to 2R1 cigarette smoke. RDS appears to be stimulated approximately 2-4 fold after 9 weeks of exposure to 2A1 and 3A1 cigarette smoke, while no more than 2 fold after exposure to 2R1 cigarette smoke. The lack of effect with the 2R1 cig- arette may be a consequence of the lower dose required to maintain equivalent toxicity with the ZAl and 3A1 cigarettes. -13- CTR CaNTRRCTS 028279 11248029 CT'~'~ I-IN C~~'4•.~..-~.~ -`

PROGRESS REPORT Contract CTR-0030 9/1/19 - 8/31/80 However, by 24 weeks of exoosure, a TPM dose from 2R1 cigarette smoke had been attained which was equivalent to the TPM dose from 2A1 or 3A1 cigarette smoke which resulted in depression of UDS. Thus, there appears to be a significant difference between the effects of UDS and RDS from the high nicotine containing 2R1 cigarettes compared to the low nicotine containing 2A1 and 3A1 cigarettes. These differences will continue to be investigated during the remaining contract period through use of the "continuous' exposure regimen with both 3A1 and 2R1 cigarettes, described under CTR-1018. (3) Labeling Index (LI) As described under CTR-107, the LI was determined in BC3Fl/Cum mice exposed to smoke from 2A1, 3A1 and 2R1 cigarettes as part of CTR-96. 3H-TdR was administered IP (100µCt/ mouse) for 1 hour, after which the mice were sacrificed by-pentobarbital overdose, tissues removed, fixed in forQ+alin and processed for autoradiography as pre- viously described (Progress Report, 1979). To date, data have been analyzed for lung tissue from mice exposed for 9 and 13 weeks. Data are presented in Table 39. Exposure to 2A1 and 3A1 cigarette smoke for 9 or 13 weeks resulted in approximately a 3 fold higher LI than exposure to 2R1 cigarette smoke or sham exposure. These increases in the LI in vivo, determined immediately after the last 2A1 or 3A1 smoke exposure, agree well with the stimulation observed in the RDS determined in lung tissue in vitro, 24-48 hours after exposure (Tables 38 and 39). Enhancement of ROS was smaller following exposure to 2R1 cigarette smoke as was the LI. The evaluation of lung_tissu.q!t _fl•om.lnic.e,exposed to smoke for 24 and 41 weeks is in progress.s. A,qu,alttative assessment of the LI in other tissues from mice exposed to smoke for 13 weeks is given in Table 40. Similar analyses after exposure to 3Al and 2R1 cigarette smoke using the 'continuous" exposure regimen described under CTR-1013 is also underway. (4) Induction of Mixed Function Oxidase (MFO). l.. The induction of MFO by the exposure regimen utilized in CTR-96 was ssonitored after 41 weeks exposure (Figure 18). BaP was used as the substrate and AHH data are presented as nmoles 3-OH-8aP/minute/mg microsomal protein for 3, 6 and 9 hours post exposure. Exposure to smoke from all three cigarettes resulted in approximately a 10 fold induction of AHH compared to the sham exposed controls, at 3, 6 and 9 hours post the final exposure. While other assays have suggested a lack of effect for 2R1 cigarette smoke, AHH is efficiently induced by smoke from all three cigarettes. MFO will continue to be monitored after exposure to 3A1 and 2R1 cigarette smoke using the "continuous" regimen described under CTR-101B. (5) Induction of Ornithine Carboxylase (OOC). The induction of OOC was also monitored in this experiment. The data will be presented under CTR-111. Exposure to smoke from all three cigarette types resulted in a 2-3 fold induction of OOC 6-9 hours after the final smoke exposure. -14- cTR Caf~TRRCTS a2aleo 11248030 CTTZ NN 04•4:3=BR_

PROGRESS REPORT Contract CTR-0030 9/1/79 - 8/31/80 2. CTR-82A. Characterization of oulmonar' c tochromes involved in smoke-associated MFO-ir.auction Co labora:ive studies with Or. I. wanal. In collaboration with Dr. I. Wang (University of South Carolina, Medical School, Charleston, SC), the nature of the P-450 cyto- chrome involved in pulmonary MFO activity has been investigated by ultra- violet and visible spectroscopy and polyacrylamide gel electrophoresis (PAGE) patterns. As reported last year.(Progress Report, 1979), frozen microsomal preparations were shipped from MA to the University of South Carolina with very little loss of protein integrity. The various hepatic cytochrome P-450's from these frozen preparations could be ident- ified by PAGE. The hepatic microsomal preparations served as controls for the pulmonary tissue since they are more fully characterized and because specific changes in these hepatic cytochromes following AHH induction have been detected. As reported previously, treatment of the AHH inducible C5761/6 (86) and C3H/Anf stains of mice with MCA or g-napthoflavone resulted in high hepatic AHH activity and also in the appearance of a 53.000 dalton molecular weight protein band on PAGE. Similar treatment of the AHN non-responsive OBA/2 (02) strain resulted in no hepatic AHH induction and no new protein band on PAGE. Tetra- chlorodibenzodioxin (TCOD) treatuent caused induction of hepatic AHH activity in all three stains and the appearance in all three strains of this 53,000 dalton molecular weigh protein band. Preliminary attempts at finding this same PAGE protein band in pulmonary tissue from these same mice were unsuccessful. Either the pulmonary microsomal prepara- tions had few new cytochromes or the concentration of these was too low to be detected by PAGE. Aicrosomal preparations of pulmonary and hepatic tissues were sent to Dr. Wang from 86 and 02 mice treated either IT or IP with high doses of TCDD (ti20 ng). The hepatic microsomes from TCDD treated mice demonstrated high AHH activity (5-10 nmoles 3-OH-BaP mg microsomal protein/min) for both 86 and 02 mice. The 53,000 dalton molecular weight protein band was also visible on PAGE. At equivalent microsomal protein concentrations (^.2 mg/ml), the AHH activity in lung microsomes was 0.5 nmoles 3-OH-BaP/mg microsomal protein/min. No new protein band was detected in lung microsomes from these TCDD treated 86 or 02 mice, when the TCDO was given either IT or IP. Further studies on the pul- monary cytochromes must await completion of studies In Dr. Wang's laboratory. When requires, mice will be treated at MA and frozen micro- somes sent to Dr. Wang for analysis. -15- cTR coHTRRCTIES a282e1 11248031 , Cf R f f f'"f 04,432159

PROGRESS REPORT Contract CTR-0030 9/1/79 - 8/31/80 > 3. C;R-109. Alteration in the Immune Resvonse after Exoosure to Ciaarette Smoke (Collaborative studies with Or. H. Herscowi t], a. Introduction Collaborative studies with Dr. H. Herscowitz (George- town University, Washington, D.C.) have investigated the immunosuppres- sive effects of cigarette smoke in BC3F1/Cum female mice. The following criteria have been used to determine the immune response: 1) antibody production at the cellular level as reflected by the spleen plaque assay (PFC assay),2) antibody production in vivo as determined by cir- culating hemagglutinating antibody (HA)•, and 3) mitogen induced prolif- eration of spleen cells. Previously Dr. Herscowitz has demonstrated that all 3 responses were significantly suppressed in BALB/c mice after exposure to 1R1 cigarette smoke but not to lAl cigarette smoke using the WHSN (Progress Report, 1979). Exposure of BC3F1/Cum mice to 2R1 cigarette smoke did not result In a consistent depression of the anti- body production (PFC assay) when compared to sham-exposed controls. In order to determine whether the lack of depression L of the PFC response in BC3F1/Cum mice was due to differences in strains, experiments were designed to expose both BC3F1/Cum and BALB/c Cum mice under the same conditions and at the same time. The BALB/c mice would serve as positive controls of inhibition and the results from the PFC assay for the two strains could be directly compared. b. Background experiments to optimize the system The system employed to measure systemic immunologic suppression was the Jerne plaque technique. Animals were injected IP with 0.2 ml of a 10% suspension of Sheep Red Blood Cells (SRBC). Three to seven days later animals were killed, their spleens were removed and teased apart, and the resulting spleen cells were assayed for their ability to produce antibody against SRBC (PFC assay). Previous experi- ments had observed a raximum response 5 days after SRBC injection. The kinetics of the PFC response are given in Table 41 and confirm the previous observations. 3C3F1/Cum and BALB/c Cum mice demonstrated a maximal response at 5 days post Injection. It was therefore decided to assay the PFC response on day 5 in all later studies. Cyclophosphamide, a widely used chemotherapeutic agent and bifunctional alkylating agent with well characterized immunosuppres- sive qualities was used as a positive control for the PFC assay. The results in Tabie 42 show that cyclophosphamide causes a dose dependent decrease in the PFC response both In old (59 weeks) and younger (22 weeks) BC3F1/Cum mice. Cyclophosphamide treatment was used as a positive control for the PFC assay in the following experiments. -16- CTR CaNTRRCTS 02820022 11248032 C TI V J// W/ 0 4 !, 3' 6- 0

PROGRESS REPORT Contract CTR-0030 9/1/79 - 8/31/80 Q c. Effects of acute cigarette exposure on the systemic immune response of mice Our initial attempt was to repeat the previous observa- tion that acute exposures to high nicotine containing cigarette smoke (1R1 cigarettes) for 5 to 15 days could substantially suopress systemic immunity. BALB/c Cum or BC3F1/Cum mice were exposed to 10% (v/v) smoke from 2R1 cigarettes on the SEM II. Exposure conditions were 20/40 seconds of smoke/air per minute over 8 minutes• (8 puffs/cigarette). Ten minutes of air followed each smoke exposure. The exposures were repeated for a total of 4 sessions per day (equivalent to 4 cigarettes/day). Animals were exposed for 5 days prior to the administration of SRBC antigen and for 5 days post SRBC in,iection for a total of 10 days. Results are shown in Table 43. No significant immunosuppression resulted from exposure to 2R1 cigarette smoke under these conditions. A repeat of these experiments using a 30/30 seconds of smoke/air per minute over 8-10 minutes similarly failed to cause any signifi wnt decrease in the splenic PFC response. Thus it would appear that acute exposure to smoke from 2R1 cigarettes did not cause any significant immuno- suppression. immunity d. Effects of chronic cigarette smoke exposure on systemic . In a further attempt to confirm the previous observation by Dr. Herscowitz that substantial immunosuppression was caused by chronic exposure to high nicotine containing cigaretta smoke, the follow- ing experiment was performed. BC3F1/Cum mice were exposed to 2R1 cigarette smoke for 52 weeks under the exposure regiment used in CTR-101A (10% (v/v) smoke, 20/40 seconds smoke/air per minute over 8 minutes, followed by 8 minutes of air, 5 repeated exposures/day). Five days prior to the termination of the experiment, the mice were ir,munized with SRBC. Smoke exposure was continued for 5 days following im:nun- ization. The results in Table 44 show that no: significant irmunosup- pression occurred after exposure to 2R1 cigarette smoke. In conclusion, neither acute nor chronic exposure to 2R1 cigarette smoke caused significant immunosuppression in either BALB/c C::m or BC3F1/Cum mice under the conditions employed in these experiments. Further experiments are in progress in Dr. Herscowitz's laboratory using the 1R1 cigarette and the WHSM in an attempt •o con- firm his previous observations. ~ 4. CTR-126. Oocvte Toxicity and Atresia Studies (Collaborative studies with Or. D. Mattison . a. Introduction Collaborative studies with Dr. 0. Mattison (National Institue of Child Health and Human Development, Bethesda, MD) have ex- amined the effects of various treatments, Including exposure to cig- arette smoke, on the rate of depletion of oocytes in the mouse. -17- CTR CaNTRRCTS Q282Q3 11248033 C1 I_ Vl I I •-I' 441,pr+' imrl' W.

PROGRESS REPORT Contract CTR-0030 9/1/79 - 8/31/80 A relationship has been suggested to exist between smoking and onset of menopause in humans, indicating that women between the ages of 44 and 53 who smoke one or more packs of cigarettes per day are more likely to be postmenopausal than non-smokers (Jick, H., Porter, J., and hlorrison, A.S., Lancet, 1354, 1. 1976). Menopause occurs when the ovaries are depleted of oocytes. The number of oocytes decreases from a maximum before birth, to zero at about SO years of age in humans and at about 1 year in mice. Since ovulation accounts for only about 0.06% of the oocytes in the ovary, factors other than ovulation are clearly important in determining the age of menopause (Mattison, 0. and Thorgeirsson, S.S., Lancet, 187, 1978). The major factor is thought t3 be a poorly understood process called atresia, whereby oocytes are destroyed in the ovary (Ingram, D.L., in The Ovary, edited by S. Zuckerman, Vol 1, p 247, New York, 1962). The question raised by these observations was whether oocytes in mice exposed to whole cigarette smoke were destroyed at an accelerated rate. Since the smoke exposure studies utilized lifetime exposure of animals to smoke; the oocyte depletion was determined as a function of age for BC3F1/Cum, C57B1/6 Cum (86 Cum), C3H/Anf Cum (C3 Cum) and DBA/2J (02J) mice. The oocyte depletion was also determined as a function of exposure to 2R1 cigarette smoke. b. Genetics of murine atresia For the studies of atresia as a function of age in the 4 strains of mice, 3 mice per strain were sacrificed by cervical dis- location every 2 months for a total of 18 animals per strain. Abdominal ovariotomies were performed immediately after sacrifice. Both ovaries were removed with the fat pad Intact, fixed for 24 hours in Bouin's solution. Ovaries were then transferred to 70% ethanol for storage until delivered to Dr. Mattison for sectioning and analyses. The entire ovary was sectioned and the oocytes in•every 20th section determined. Data are presented as a mean number of oocytes per ovary as a function of the age of the mouse. Data are presented in Figure 19 for the four strains held at MA (C3 Cum, BC3F1/Cum, 86 Cum and D2J) and two strains held at VICHO (DBA/2N (OZN) and C57BL/6 (86N)). The C3 Cum strain appears to have the most rapid rates of atresia. There may be differ- ences between the 86 Cum and 86N strains, while the 02J and D2N appear to have similar rates of atresia. The rate of atresia from BC3F1/Cum mice appears to be intermediate between C3 Cum and 86 Cum. c. Effects of cigarette smoke exposure on atresia The ovaries from BC3F1/Cum mice on test for smoke and sham exposure under CTR IOIA were utilized to determine what effects, if any, exposure to 2R1 cigarette smoke had on the rate of oocyte depletion. Ovaries were taken from age matched smoke and sham exposed mice at selected intervals, fixed,sectioned and the number of oocytes counted as described above. Data are presented in Figure 20 for the number of oocytes/ovary from BC3F1/Cum mice from 6 to 39 weeks of age. -18- CTR CaNTRRCTS Q26284 11248034 C T R H N 4~.0 4 43 G2

PROGRESS REPORT Contract CTR-0030 9/1/19 - 8/31/80 L.• Mice were first exposed to 2R1 cigarette smoke or sham treated at 8-10 weeks of ace. Smoke exposure conditions were described previously (see CTR 101A). The data indicate that the rate of oocyte depletion is not influenced by exposure to 2R1 cigarette smoke compared to sham exposure or untreated controls (compare Figures 19 and 20). d. Effect of BaP given IT on atresia' Dr. t4attison has previously demonstrated a dose response for oocyte destruction by BaP given IP. These results were compared to BaP given IT to determine whether chemicals given via the lung could exert any effect on the ovary and be toxic to oocytes. Four weekly doses of 1.2 mg BaP were given IT to groups of BC3F1/Cum mice. Mice were sacrificed at weekly intervals, one week post BaP treatment. Data are not yet available for the results of this study. S. CTR-127. Alkaline Elution Assay (Collaborative studies with Grant 11241, Ors. Henry, Lubet and Kouri . a. Introduction Methods have been developed under Grant 01241 for the utilization of an alkaline elution assay to detect daaw ge to DNA in vivo• (Progress Report, Grant /1241, 1980). Animals exposed to smoke under CTR-96 have been used to determine whether exposure to smoke alone results in damage to pulmonary DNA or whether exposure to smoke con- currently with chemical treatment results in damage to pulmonary DW1. A brief summary of the results are given here. b. Description of the assay The alkaline elution assay measures the rate of elution of DNA from a polyvinylchloride filter in the presence of an alkaline buffer system. The rate at which DNA elutes from such a filter apparatus is dependent of the molecular weight of the DNA. Large strands of DNA from many chemically or physically (x-ray) damaged cells elute from the filter at a faster rate than DNA from untreated control cells. In the case of lung cells from methylmethanesulfonate (MMS) treated BC3F1/Cum mice (Table 45), the rate of elution is 8-10 fold greater in h41S treated animals than in lung cells of control animals. Recent experiments have shown the increased rate of elution in damaged lung cells to be dose dependent with a dose of 150 ug/kg MMS resulting in a K value of 1.64. K, the elution rate constant, is defined by one equation: WHERE, Qh a Qoe-kh Qh - amount of DNA remaining on filter after time h Qo = amount of DNA on the filter at zero time k - elution rate constant h  time in hours -19- CTR CQNTRRCTS 028285 11248035 CTR HVI 04436"1

PROGRESS REPORT Contract CTR-0030 9/1/79 - 8/3T/80 A K value of 0.80 is obtained for 50 ug/kg WS and a K value of 0.20 for control lung cells. Thus this rethodology is capable of quantitating the extent of DNA damage resulting from in vivo exposure to a variety of chemicals. This approach will be used to determine both the•direct effects of exposure to cigarette smoke on lung DNA, and the possible interactions between cigarette smoke and other chemical agents whith result In pulmonary DNA damage. c. Damage to pulmonary DNA induced by smoke exposure BC3F1/Cum mice were exposed to 10% 2A1 cigaratte smoke using the SEt4 I: smoking machine. The exposure conditions were 30 seconds smoke alternating with 30 seconds air, over 10 minutes (10 puffs/cigarette), followed by a 10 minute rest period. This regimen was used 5 times in two sessions each day (10 cigarettes/day) with a pulmonary deposition of ti 1 mg TPM/day/mouse for 40 weeks. Sham exposed mice were treated the same but without smoke. Animals were exposed to 5 cigarettes/day on the day of the assay, sacrificed by cervical dislocation 7 hours after initiation of smoke exposure. Two mice per condition were used, pulmonary cells were pooled and duplicate samples were analyzed for elution of DNA from the filters. t4MS was used as a positive control, given intraperitoneally at 120 mg/kg body weight approximately 4 hours prior to sacrifice. Data are presented in Figure 21. Exposure to cigarette smoke resulted in minimal effects on pulmonary ONA relative to that. found in sham-exposed animals. d. Damage to pulmonary DNA induced by chemical treatment and smoke exposure. Our preliminary results studying the effects of smoke exposure with intratracheal treatment with BaP or BaP-7,8-diol demon- strated that smoke exposure augmented the DNA damaging effects of these chemicals. Conditions for smoke exposure are described above. BC3F1/Cum female mice, 48 weeks old, exposed to 2A1 cigarette smoke for 40 weeks were treated intratracheally with 180 ug BaP-7,8-diol In DM50 (60 µg/0.02 ml) or 1.2 mg BaP in gelatin saline (1'.2 mg/0.02 ml) approximately 3 hours prior to sacrifice. Two mice per condition were used, pulmonary cells pooled and duplicate samples were analyzed for elution of DNA from the filters. Data are presented in Figure 22 and show the enhanced damage to pulmonary DNA from BaP and BaP-1,8-diol smoke exposed mice but not BaP or BaP-7,8-diol sham exposed animals. The mechanism for this aug- mentation is not known but may include effects on carcinogen metabolism and/or effects on DNA repair. Experiments are in progress to more fully detail the specifics of this interaction (see Grant 11241 Renewal Appli- cation, 1980) These studies have been designed to answer questions.. tncluding,l) how lo.ng an exposure to cigarette smoke is necessary to see this interaction?, 2) is the effect specific to cigarette type?. L -20- CTIR CONTRACTS 028286 11248036 • C) T "M I ) ) I 044,2364

PROGRESS REPORT Contract CTR-0030 9/1/79 - 8/31/80 6. CTR-128. Evaluation of chromosomal damaae or alteration after exoosure to whole ci arette smoke ustn a sister cnromatid exchange SCE assay (Collaborative studies with Or. W. 8eneaictl. a. Introduction The sister chromatid exchange (SCE) assay measures the number of times the genetic material of the chromosome is exchanged or recombined per metaphase chromosome. It has been suygested that human smokers have an increased number of SCE's in their blood lymphocytes compared to non-smokers (Hopkin. J.M. and Evans, H.J., Nature 283, 388, 1980). Thus it was of interest to determine whether mice exposed to various doses of whole cigarette smoke would also display an increase in SCE's in either lymphocytes or bone marrow cells. In collaboration with Dr. W. Benedict (Children's Hospital of Los Angeles, Los Angeles, CA), bone marrow cells from mice exposed to cigarette smoke are under evalua- tion for SCE. b. Experimental protocol Cellular DNA Is labeled with bromodeoxyuridine (BUdR) . during chemical treatment. Femural bone marrow is separated, cells are suspended in colchicine to obtain metaphase cells, harvested, spreaa on glass s.lides, fixed and stained. Generally 20-30 netaphase chromo- somes are scored for the number of SCE's per chromosome. •Two groups of animals were utilized in this study. In one group, the BudR pellet was implanted subcutaneously at MA 2 hours prior to further treatment. In the second group the pellet was implanted in Dr. Benedict's laboratory %24 hours after the last treatment at NA. Smoke exposed, sham-exposed and shelf control BC3F1/Cum female mice were included in each of these groups for shipment to Los Angeles. BC3F1/Cum female mice were exposed to 3A1 cigarette smqke for 4 weeks using the "continuous" exposure regimen described under CTR 1018 (10% (v/v) smoke concentration, 15/45 seconds smoke/air cycle per minute 8 minutes, 8 puffs/c.igarette, repeated over ^.130 minutes). Cyclophosphamide (IP, 5^sg/kg body weight) was given 2 hours post pellet implantation and served as a positive control. c. Results In the initial experiment in which the BUdR pellet was Implanted 2 hours before the last smoke exposure, metaphase chromo- somes were obtained but no differential straining of these chromosomes was observed. This section of the protocol will be repeated, using minimal light exposure (to prevent inactivation of the BUdR) during both the implantation procedure and actual housing of the animals during ~, shipment. -21- CTR CONTRRCT5 028287 11248037 CTFZ MN 044..~ 6=5'

PROGRESS REPORT Contract CTR-0030 9l1/79 - 8/31/80 The initial results are given in Table 46 for the second section of the protocol where the BudR pellets were implanted ti24 hours after the last smoke exposure. While these data are preliminary and require confirmation, the results suggest that the SCE rate is two fold higher in animals exposed to smoke compared to sham exposed animals. Experiments are in progress to confirm these results and to determine the effects of cigarette type or length of exposure to smoke. D. Feasibility of Short Term Assays Relating to Possible Tumor - Promoting Events. CTR-111. Ornithine decarboxylase (ODC) induction as a marker of promotion in oulmonary tissue. 1. Introduction This study was des.igned to determine the conditions and parameters of 00C induction in pulmonary tissue. Results presented previously (Progress Report, 1979) demonstrated that ODC activity could be induced in pulmonary tissue after treatment with tetradeconyl- phorbol acetate (TPA) and that TPA administered by aerosol provided a useful model system for the analysis of induction of 0DC in pulmonary tissue. In addition, preliminary results were presented which indicated that chronic exposure to whole cigarette smoke resulted in slightly elevated 00C activity above sham-exposed ODC levels. 2. Whole cigarette smoke as an inducer of 00C activity in pulmonary tissue. Initial studies on the effect of exposure to cigarette smoke on lung OOC levels were performed using smoke from 2R1 cigarettes under the exposure regimen utilized for CTR 101A (the experiment to determine the effects of lifetime exposure to whole cigarette smoke in BC3F1/Cum mice). The exposure conditions resulted in ti200 ug TPM/ mouse/day and have been previously described (see CTR 101A). Mice were exposed to smoke for nine to 150 days and were sacrificed and lung 00C activity was measured as previously described. At nine days, ttie_earliest time at which lung,00C levels were measured, lung ODC activity was 1.2 to 1.5 times higher in smoke-exposed mice (Figure 22). However, at days 24, 44 and 88, 00C levels of 2-5-fold higher than control levels were observed (Figure 22). On day 150, the highest activity was only 1.7-fold higher than in sham-exposed con- trols. The reason for this lower level of induction is not known at this time. Slightly higher liver ODC levels were obtained in smoke exposed mice than in sham exposed controls. On day 9, liver ODC activity was approximately 30-40% higher in the smoke exposed than in sham exposed mice. Liver 00C levels were 5-17% higher in smoke exposed mice assayed on day 150, a time at which lung 0DC activity from smoke exposed mice was 1.7 fold higher than sham exposed controls. -22- CTR CaNTRRCTS 026286 11248038 C TR NN 04• 43-GG

PROGRESS REPORT Contract CTR-0030 9/1/79 - 8/31/80 As previously described, mice exposed to 2R1 cigarette smoke for up to 44 days had lung AHH activity which was elevated up to 8-fold or more above sham exposed control levels. These data strongly indicated that exposure of mice to cigarette smoke elevated pulmonary OOC levels. The following experiments were designed to verify and expand these observations. The effect of exposure to smoke for 2A1, 2R1 and 3A1 cigar- ettes (at high doses) on induction of ODC and AHH in lungs of 8C3F1/Cum female mice was determined after various periods of smoke exposure. The smoke exposure regimen was that described under CTR-96. Mice were exposed to 10% (v/v) smoke for either 1600 seconds (2R1 cigarettes) or 2400 seconds (2A1 and 3A1 cigarettes) total smoke exposure/day for up to nine months. Mice were sacrificed at 3, 6 and 9 hours after cigarette smoke exposure, or sham exposure. Lungs were removed, weighed and processed for OOC and AHH assays. Mice tested 9 weeks after start of smoke exposure exhibited the highest ODC levels in their lungs at nine hours after exposure, which were 1.8-3.4 times higher than sham exposed controls (Figure 24). Smoke from 3A1 cigarettes induced the highest pulmonary OOC leve7s, followed by 2R1 and 2A1 cigarette smoke. After 13 weeks of smoke exposure, OOC levels were again highest at nine hours after smoke exposure and ranged from 1.6-2.3 times that of sham exposed mice (Figure 25). Mice exposed to smoke for 41 weeks (ti9 months) were examined to determine whether smoke continued to induce elevated ODC levels during chronic smoke exposure. Figure 26 presents the results of the ODC assays after 41 weeks of smoke exposure, corresponding to an accum- ulated TPM deposition of %214, 232 and 134 mg TPM/mouse. ODC levels at 3 hours ranged from 1.8 to'S times that of sham-exposed mice. The results confirm those obtained in our earlier, prelim- inary studies that exposure to cigarette smoke results In elevated ODC activity. In addition. the results demonstrate that very long (41 weeks) exposure to cigarette smoke does not result in a reduction of pulmonary ODC levels and that ODC activity continues to be induced following cig- arette smoke exposure over an extended period. In order to monitor the direct effect of smoke exposure on the lung physiology and its relationship to altered enzyme activities, lung wet weights were obtained at the time of sacrifice. The ratios of lung weights from smoke exposed mice relative to sham exposed mice are presented in Table 47. The greatest effect was observed in lungs of mice exposed to 2A1 and 3A1 cigarette smoke where an increase in lung to body weight ratio of 26-41% ratio was seen after 9 weeks of smoke exposure. Exposure to 2R1 cigarette smoke had the least effect (<10%). Similar results were observed after 13 weeks (Table 47) and 41 weeks. -23- CTR C0NTRRCTS 028289 11248039 CTR MN 04'4_-3F_.x '.-r"4

PROGRESS REPORT Contract CTR-0030 9/1/79 - 8/31/80 The results on altered lung weights suggest that the increase in lung weights is not directly linked to the increase in ODC activity. This can be seen in Table 47 and Figure 24. Exposure 2A1 and 3A1 cigar- ette smoke had the highest increase in lung weight ratio (26-41% above sham exposed controls) after 9 weeks (Table 24) however, 2R1 cigarette smoke induced a higher leve.l of ODC by 9 hours than did 2A1 cigarettes (Figure 24). Experiments confirming the results are in progress using the "continuous~' exposure regimen for both 3A1 and 2R1 cigarettes described under CTR 101B. 3. Level of OOC activity induced in lungs by other chemicals. The purpose of these studies was to investigate the possible effects of exposure to solvents utilized in our aerosol studies such as ethanol, water and dimethyl sulfoxide (OMSO). These studies have been deferred due to the problems of coating the modules with materials to protect the modules against the effects of these solvents. 4. Relationship between the genetic control of AHH and ODC activities. ODC Induction occurs as a result of exposure to chemicals which cause gene(s) activation. TPA is an example of a chemical which causes non-specific gene activation (and induction of OOC), but does not induce AHH activity. MCA, however, causes specific gene activation (e.g. the Ah locus) and treatment results in the. induction of both OOC and AHH activity. OOC induction by MCA, therefore, could result from either specific activation of AHH genes or activation of genes responsible for the "promotion-activity" of this chemical. Thus the mechanism of OOC induction by TPA may be quite different from the mechanism of OOC induction of MCA. An approach was suggested last year in that specific recom- binant inbred (RI) lines would be analyzed for the kinetics of OOC induc- tion following TPA or MCA treatment. These RI lines were derived from C57BC/6J and OBA/2J strains (B602 RI lines) were selected because of the availability of AHH responsive and AHH non resposive animals. These animals were to be treated with either MCA by intratracheal inocul- ation or aerosol generated TPA. If OOC were linked to AHH, then it would be expected that in the AHH inducible animals, ODC would be in- duced after treatment with.MCA and TPA, while in the AHH non-inducible animals, ODC would be induced only by TPA. Sufficient numbers of animals needed for these studies were not commercially available and it was necessary to breed these strains at MA. Low fertility has been a problem with many of these RI lines and In some, breeding has been completely unsuccessful. The status of these lines is given in Table 48. When sufficient numbers (50 from each RI line) are attained, the mice will be treated as previously des- cribed and assayed for AHH and ODC activity. -24- CTR coNTRRCTS 02B290 11248040 CJ/ R 1 I I / ld' 4I /, 1or GG

PROGRESS REPORT Contract CTR-0030 9/1/79 - 8/31/80 5. Characterization of pulmonary 0DC induction in the inbred strains of mice. Characterization of pulmonary 0DC in the inbred strains of mice was proposed to establish an initiation and promotion (two. stage) carcinogenesis model system where pulmonary carcinomas are the tumor end- point. This model system would be analogous to the two stage system historically used for epidermal tissue. We have shown previously that pulmonary ODC is induced after exposure to a known epidermal 0DC inducer (TPA) and by exposure to whole cigarette smoke (see CTR-111). TPA induced epidermal 0DC has been shown to be inhibited by retinoic acid (Verma, et al, Can. Research 39, 419, 1979). Thus we wish to test whether pulmonary ODC is regulated in a similar manner as epidermal 00C. Pulmonary ODC induction by aerosolized TPA and/or cigarette smoke will be evaluated after exposure to retinoic acid, an inhibitor of TPA- induced 00C in epidermal tissue. A series of retinoic acid derivatives are now in the lab- oratory and will be tested for toxicity after intratracheal treatment. Those available are 13-trans-retinoic acid, 4-2-hydroxy-ethylretinamide, trans-retinol , retintol acetate and 13-cis-retinoic acid. BC3F1/Cum mice will be treated IT with these chemi'Ma s in gelatin saline at 10-100 and 1000 nanogram doses. The retinoid which results in the least toxicity will be used to inhibit TPA-induced 00C activity. These experiments will be completed by the end of 1980. -25- cTR caNTRacTS 028291 11248041 CTR HN 044'36.9.

PROGRESS REPORT Contract CTR-0030 9/1/79 - 8/31/80 E. Aerosol Studies L l. Introduction Our aerosol inhalation studies have been predicated on the idea. that certain of these studies could serve as positive or negative controls for the cigarette smoke inhalation studies. In the case of ODC Induction (CTR-111), this has proven to be extremely useful 1n that, depending upon dose, T?A delivered as an aerosol to the lungs of mice results in a 20-100 fold increase in ODC induction, whereas intratracheal inoculation was much less efficient or reproducible (Progress Report, 1979). Aerosolized TPA has thus served as a positive control for OOC induction studies using whole cigarette smoke, which also Induces ODC (CTR-111). Lung deposition of a chemical is a function of a) concentra- tion of chemical in the aerosol, b) particle size distribution of the aerosol, c) time of exposure, d) respiratory rate, and e) collection efficiency of animal. Our previous dosimetry studies have allowed calculation of a "lung deposition factor°, defined as the product of respiratory rate and collection effi.ciency of the animal. From TPA aerosol dosimetry studies, the lung deposition factor was found to be 0.00054 liter/minute. This value is about 4.5 times lower than the value of 0.0025 liter/minute calculated for smoke deposition studies. Confirmation of this difference in "lung deposition factor° has required measurement of the particle size of the aerosol as a function of aerosol concentratton. We have used catechol as a model chemical since 1t can be aerosolized from ethanol with the aerosol generator and can also be aerosolized in tobacco smoke. The status of: the aerosol studies is given in the following section. 2. CTR-114. Deposition and retention of aerosolized TPA. 3H-TPA required for the aerosol dosimetry study to be per- formed to determine the "lung deposition factor" for TPA aerosols gen- erated from 0.2: ethanol solution was not available from the former source (Dr. P. Borchert, University of Minnesota, Minneapolis, Minn.). It has recently been made available from another source (New England Nuclear, Boston, Mass.) which will now allow us to perform the study as proposed, by December 1980. 3. CTR-115 and 123. Feasibility studies for aerosolization of selected chem ca s ano solvent venicles. We have reported earlier that BaP or MCA aerosols may be generated from their solutions of OMSO: ethanol (1:1, v/v) (Semi- Annual Progress Report, 1979). Such solutions can not be used with the polycarbonate exposure modules since the OMSO dissolves the module. To date, we have not found a suitable method of protecting the modules from the effects of the solvents. Fabrication of modules from another material has been explored, but will not be pursued at this time due to the phase out of the inhalation studies at MA. -26- CTR CCHTRRCTS Q2G'2~32 11248042 C TR t1H 0 4,4~~ r"` CO

PROGRESS REPORT Contract CTR-0030 9/1/79 - 8/31/80 3. CTR-121. Aerosol dosimetry studies with 3H-catechol. Smoke dosimetry experiments have enabled: the determination of a"lung deposition factor", the product of ventilation and collection efficiency. Such a factor allows estimation of the amount of chemical deposited into the.lung after completion of exposure if the aerosol concentration and exposure time are known. Smoke dosimetry studies indicated a lung deposition factor of 0.0025 liter/minute, compared to 0.00054 liter/minute indicated by TPA aerosol dosimetry experiments. 3H-CAT aerosol may be generated by nebulization of an ethanolic solution of catechol. Thus aerosol dosimetry studies with 3H-CAT will enable us to determine the "lung deposition factor" and to determine the effect of chemical or type of aerosol on this factor. 3H-CAT-2R1 cigarette smoke dosimetry experiments have been completed (CTR-105) and demonstrate a rapid clearance from lung tissue. Preliminary estimates of the "lung deposition factor" from these ex- periments suggest values of 0.00088 liter/minute, or about the same order of magnitude as determined from the TPA experiments. The results must be evaluated with the following points In mind: a) the smoke dosimetry studies utilized dotriacontane, a chemical whish is not meta- bolized and is not rapidly cleared from lung tissue, b) H-CAT in smoke is r4pidly cleared from lung tissue, c) if estimates of total amount of H-CAT in the body are made and corrected to initial amount of TP!i deposition in the lung, the factor more closely agrees With that obtained in the smoke dosimetry studies, and d) the °iung deposition factor" may be different for a chemical aerosol compared to a smoke aerosol since it involves the respiratory rate of the animals. The• 3H-CAT aerosol studies should aid in addressin~ these points and will permit direct comparison of the deposition of H-CAT from a smoke aerosol and as a single chemical aerosol. The measurement of the aerosol concentration and determination of particle size distribution is, therefore, extremely important for these aerosol experiments. Aerosol concentration as a function of solution concentration was determined for phenolphthalein, a model chemical in the development of these aerosol studies, and catechol (Table 49). Aerosol concentrations from 60 to 260 µg/liter were observed for phenoiphthalein and from 20 to 320 ug/liter for CAT. Preliminary measurements of the particle size distribution using a 7 stage cascade impactor have been attempted. ( A diagram of the cascade impactor used is shown in Figure 27.) Interaction was observed between the CAT and the silicone spray used to coat the glass cover slips mounted in each stage so that these measurements will be repeated without this coating. It has been suggested that for non-mineral aerosols the coating of the cover slips may not be necessary. 11ith the completion of these aspects of the CAT-aerosol experi- ments, the 3H-CAT aerosol dosimetry experiment will be scheduled for December 1980. BC3F1/Cum mice will be exposed to 3H-CAT from a 0.2% and a 1.0% solution, sacrificed by C02 asphyxiation at zero, 0.25, 0.5, 1.0,.2.0 and 24 hours post exposure. -27- CTR CQNTRRCTS 028293 11248043 ~ ~ CTR HN 0443

PROGRESS REPORT Contract CTR-0030 9/1/79 - 8/31/80 The following tissues will be removed from each animal: head, larynx, trachea, lung, liver, stomach, kidney and bladder. Urine will be collected for 2 hours after exposure. Tissues will be analyzed for radioactivity as described under CTR-105. ,4. CTR-122. Studies with nicottne sulfate aerosols. Aerosols of nicotine sulfate or tartrate could be generated from aqueous solutions using the generator presently in use for CAT or TPA aerosols. However, based on the conplexity of these aerosol studies, it was decided to emphasize the CAT studies and if time permitted, fur- ther investigate nicotine aerosols, since it was determined that nicotine sulfate or tartrate would have to be synthesized and purified at either ORNL or MA. -28- CTR CaNTRRcTS 028294 11248044 CTFZ MN 04437 df-.2

TABLE 1 OPERATION AND SERVICE MANUAL Title Status Introduction Rough Draft I. The SEM II Completed I I. Smoke and Fl ovi Moni tori ng and Safety Systems Completed III. Animal Containnent and Exposure System Completed IV. Animal Handling Procedures and Techniques Final Draft V. Gas and Aerosol Sampling and Measurement Completed VI. Sham-Exposure Machine Proposed Completion in 1981 VII. Computer Interface for Smoke Monitoring and Automatic Docuroentation Proposed Completion in 1981 l -29- CTR COHTRRCTS 028295 11248045 CI R i 1N 04431 `LsF

TABLE 2 OPERATI;IG AtrO SERVICE MANUAL for MICROBIOLOGICAL ASSOCIATES ---- COUNCIL FOR TOBACCO RESEARCit-US.1, INC. SMOKE INHALATIOtt FACILITY r„. ' Bethesda, 6?a r; l and SECTION I SEM SERIES II A. GENEP.AL DESCRIPTICU 8. SPECIFICi1TI0it AND CONTROL' SETTIItGS C. OPERATION 1. Pre-start up proc:dure 2. Start up procedure . 0. ~1AINTE?IANCE . 1. Dai ly 2. Periodic E. CQ`4P0HENT DETAILS AN'D SERVICING 1. Air regulator, filter and gage assembly 2. Spent cigarette (butt) containment systeui 3. Cigarette drurn drive systen 4. Dilution air and purge atr controls 5. Done and hood asseably 6. Machine electrical circuit 7. Ejecting function 8. Drum prograi.u;:er 9. Hopper 10. Humidifier 11. Liyhting function 12. Loading function 13. Puff counter and runs counter 14. Puff pressure and dame pressure controls 15.. Slider block and smoke lines 16. Recycle shutdown and mode suitch functions 17. S:ioke distribution valve 18. Snoke distribution valve control circuit 19. Relative humidity and temperature indicator F. CALIBRATION l. Puff volu.;e 2. Dilution air G. TROUBLE-SIt00TING -30- cTR caHTRRCTS 028296 11248046 C! R l wI I I 0I` 1/ Sq+^ 7`I

TABLE 3 OPERATING AND SE3VICE MANUAL _ for MICROBIOLOGICAL ASSOCIATES and THE COUNCIL FOR TOBACCO RESEARCH - USA, St40KE INHALATION FACILITY Bethesda, Maryland SECTION II St40KE r,40 FLOW MONITORING AND SAFETY SYSTEM G. A. GENERAL OESCRIPTION . . . . . . . . . . . . . . . . . . . . . . . 2-1 B. SPECIFICATIONS AND CONTROL SETTIt1GS . . . . . . . . . . . . . . . . . 2-4 C. OPERATION . . . . . . . . . . . . . .' . . . . . . . . . . . . . . 2-4 0. MAINT~';fANCE . . . . . . . . . . . . . . . . . . . . . . . ... . . . . 2-7 1. Dai ly . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-7 ' 2.. Periodic . . . . . . . . . . . . . . . . . . . . . . . . . . 2-7 E. COMPONENT OETAILS ANO S:.R`IICING . . . . . . . . . . . . . . . . . . . 2-8 1. Auxil i ary Ai r System . . . . . . . . . . . . . . . . . . . . ... 2-8 2. Flow Safety Valve . . . . . . . . . . . . . . . . . . 2-10 3. FTotir Sensor and Alarm Circuit . . . . . . . . . . . . . . . . . . 2-12 4. Smoke Sensor . . . . . . . . . . . . . .. . . . . . . . . . . . 2-16 S. Smoke t4onftoring.System . . . . . . . . . . . . . . . . . . . . . 2-17 6. Smoke Al arm System . . . . . . . . . . . . . . . . . . . 2-21 7. Recorder Power and Integrater Control Yetwork . . . . . . . . . . 2-23 8. Smoke Monitoring Recorders . . . . . . . . . . . . . . . . . . . 2-24 F. CALIBRATION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-27 1. Flow Sensor Calibration . . . . . . . . . . . . . . . . . . . . . 2-27 .2. Smoke Sensors and Readout . . . . . . . . . . . . . . . . . . . . 2-29 TROUBLE-Sii00TI NG . . . . . . . . . . . . . . . . . . . . . . . . . . 2-34 1. Containment and Air/Smoke Handling . . . . . . . . . . . . . . 2-34 2. Recorder and Smoke Monitoring . . . . : . . . . . . . . . . . . . 2-36 1. Initial Instrument Settings for Particulate Sensors .... . 2-39 2•. Amplifier-Integrator Checkout P rocedure . . . . . . . . . . ... • 2-43 3. Abrevfations Used in Manual . . . . . . . . . . . . . . . . . . . 2-45 4. Operators tianual, for Recorders . . . . . . . . . . . . . . . . . 2-46 H. REFERENCES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-38 CTR CoNTRacTS 028297 11248047 CTR MN 044• :-475

TABLE 4 OPE?AT I N G AKO SE?YIC: :!CNUAL for YICROBIOl.OGICAL ArSOCIATES and T1E COUNCIL FCR TOSACCO RES'utRCH-USA, INC. SMOKE IMR1.LATiON FaC ILITY Bethesda,.Haryl and SECTION III ANIMAL CONTAI?Q1EXT ANO EXPOSLRE SYSTEM A. S. C. 0. E. G. GEilERAL OESCRIPTION . . . . . . . . . . . . ... . . . . . . . . . . . 3-1 SPEC IFICATIONS . . . . . . . . . . . . ... . . . . . . . . . . . . . 3-S 0 PERAT IOIl . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-5 MAIYCE`NX E . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-7 1. bSouse Trays . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-7 • 2. . 'Racf:s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-7 3. 'tdodul es . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-8 COMPONEhT OETAILS AtrO SERVICING . . . . . . . . . . . . . . . . . . . 3-17 1. 2. 3. 4. 4. 6. Mouse T rays and Supports . . . . . . . . . . . . . . . . . . . . 3-17 hbuse Modul es . . . . . . . . . . . . . . . . . . . . • . . . . . 3-20 Module Racks . . . • . . . . . . . . . . . . . . ,. . . . . . . 3-29 uodule Magnets . . • . . . . . . . . . . . . . . . . • • . • • . 3-32 Oashpocs . . . . . . . . . . . . . . . . . . . . . 3-34 Smoke Connectors and Window Fittings ... . . . . . . . . . . . . 3-37 F. TROU8L:-Srtv"OTING . . . . . . . . . . . . . . . . . . . . . . . . . . 3-40 REFERENCES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-42 1. Abbreviations used in the Nanual . . . . . . . . . . . . . . . . 3-42 2. List of Illustrations and Captions . . . . . . . . . . . • . . . 3-43 -32- CTR caNTRRCTS 026298 11248048 CT~"~ I-IN 04'43?"G

TABLE 5 . OPEBAiL`tG AND SnVICE 2SANUAL FOR 2SICROBIOLOCIC.SL .1SSOCLlTES bND THE COUNCII. FOR TOBACCO RES?11i2CR-iJSA, i::C. S2i0n INHALATIOY FACILITY SECTION IQ ANIMAL SANDLL'IG PROCEDIIRES AND TEC'@iIQVES A. GrZ. iERAI. CARE AND liAIllTF2IANCE OF ANIMALS FOR A LARGE LMaT-aTION FACILITY. 1. Restricted Access Facility; Traffic Flow Patters 2. Acqui•stion of 6nimals 3. Quarantine of /lnimals 4. Preparations for the Experiment S. Initiation of Experimental Trastment 6. Observations during the Experiment B. DAILY PROCEDURE.S FOR E3y8RItENT 1. Loading onto Animal Trays 2. Loading of Trays Onto Exposure Hodules 3. Paperwork 4. Monitoring of an{-l A During Exposure S. Treatmmc and Handling of Dead or KorYbund Aaimals 6. Unloading of Animals and Return to Cages 7. Transportation and Bold3ng Carts 8. Observation Program C. HONTFII.Y PRDCgDL'RES 1. Weighing Schedules• - Automatic. Systea 2. Carboxyhenoglobin Determinations 3. Urine Collection and Analysis D. NECROPSY PROCEDLiRES 1. Necropsy facility 2. Equipment 3. Clinical Fxamiaation of Animals (Yrior to Necropsy) 4. House Necropsy Procedures 5. Positive Pressure Apparatus 6. Processing of Specimeas for Fiistology and Pathology E. REHERENCES 1. Selected Standard Operating Procedures 2. Selected Reporting Foras and Record Sheets -33- CTR CC1htTRRCTS 028299 11248049 CTR NN 044,'3f' " r

i TABLE 6 Percent Carboxyhemoglobin (xCOHb) 1n BC3F1/Cum Female Mice After Chronlc Exposure to 2R1 Cigarette Smoke or Chronic Sham Exposurea 2R1 Smoke Exposure Sham Exposure Untreated Mean + S.D.b Mean + S.D. Mcan + S.D. September 1978 9.2 + 1.2 1.5 + 1.0 2.5 + 0.4 October 1978 7.1 + 1.2 1.8 t 0.3 1.3 + 0.3 November 1978 26.1 + 3.9 2.0 + 0.6 1.8 + 0.7 December 1978 15.0 + 5.4 1.6 + 0.3 2.1 + 0.7 January 1979 24.2 + 2.2 1.8 + 0.1 1.9 + 0.5 February 1979 17.7 + 1.3 0.9 + 0.2 1.4 + 0.1 March 1979 15.1 + 1.0 4.8 t 0.4 1.7 + 0.8 April 1979 17.9 + 0.9 1.2 * 0.2 1.3 + 1.5 May 1979 10.8 + 1.1 1.2 + 0.3 1.2 + 0.6 ~. June 1979 22.2 + 1.8 1.2 + 0.1 1.4 + 0.3. o, n A July 1979 5 21 7 + 1 2 + 0.3 1 1.5 + 0.4 ~ ~ August 1979 . . 18.9 + 0.6 . 1.5 + 0.3 1.3 + 0.2 ~ September 1979 16.1 + 0.5 2.2 + 0.4 1.9 + 0.5 October 1979 19.7 + 1.0 1.5 + 0.4 2.2 + 0.3 ~ November 1979 not done not done not Aone 0 December 1979 14.84 f 1.36 1.25 + 0.40 2.04 + 0.70 January 1980 14.60 + 3.37 1.52 + 0.45 2.22 + 0.36 ~ February 1980 18.13 + 2.40 1.13 + 0.74 2.17 + 0.46 ~ March 1980 16.20 + 3.15 1.06 + 0.38 1.92 + 0.40 ~ April 1980 16.62 + 2.20 1.92 + 0.48 2.10 + 0.20 M May 1980 16.42 + 1.68 1.08 + 0.53 1.68 + 0.48 ~ June 1980 17.00 + 2.71 1.32 + 0.29 1.80 + 0.37 "..~ July 1980 17.86 + 1.45 1.82 + 0.19 1.86 + 0.45 l.n August 1980 16.20 + 0.54 2.12 + 0.74 2.28 + 0.28 °Conditions for smoke exposure: 10% 2R1 smoke; 20 seconds of smoke alternating with 40 seconds of air for a period of 6-8 minutes (equals one cigarette); five cigarettes per day with approximately 10 minutes rest period between cigarettes. * bYalue given represents the mean (3-5 animals) plus or minus the standard deviatlon.

TABLE 7 CINtONIC INMLATION OF 2R1 CIWRETTE SMDKE IN l0FI/Cu. FEMAIE N1CE DISPOSITION OF Iq11NALSa ~ J A 00 0 "'4 CA ~ J s,~ ~„~ n ti..i_-T 70 ~_ 1) ~ .w.. r"~ Ln h e Muober first Te.rd Second Ye.r Nu.h.r Chnical [xoosur on Test Dtagrased InFrocess .g No NecropsY Dlagnos.d I. rocess ng No Necropsy Allvee None 221 Snot. 2063 157 59 97] 2S8 193 24 389 None Sham 1014 111 14 320 21/ 90 19 276 None None 449 4 0 le 62 147 40 178 !aP 2111 Sook. 320 109 2 01 50 22 4 98 LP Sham 260 62 2 31 99 21 11 28 8aP None 130 6 0 A )S 12 IS 14 aSlnce September 1978. hl.tratrache.l Installations once per week for three weeks prior to first SEN II esposur.t 1.2 .g senio(.)pyrene (!ap) In 0.02 wl of a 0.2% g.latln-s.line solutton was gIven per lesttllatton. cCondlttons for swke exposure conststed of 101 2R1 elgarette snokei 20 seconds of s.oke alternating with 40 seconds of air for a period of 6-6 minutes. flre such esposeres were given each day. Sham e.posure was t1u saoe but witifout smoke. dDuring the flrst six months of the experiment approximately 20% of th. anls+.ls (animal nuwb.rs eud/ng tn 3 or 0) had their tissues saved at n.cropsy. Only 10% (a.l.al nu.hers end/q lo 3) wero processeJ for histopalholuglc diagnosis. eNwher alive as .f • 10/24/80. •

TAOLE NUMOERS OF OC3FT/Cus FEMALE MICE 011114 OF TARIOUS CAUSES AFTER TWO YEARS Of EXPOSURE TO 2R1 CIGARETTE SMOtE OR SIIRM EIPOSIRIE ON THE SEM Ila'h first Tear Weeks After Flrst Exposura Second Description of Oeith Exposure Totals 56 60 64 68 72 76 80 84 08 92 96 100 104 Tear Totals Cueulatlve Totals Iblder Related 2R1 Smoke 198 10 4 0 0 S S 1• 2 1 1 3 0 0 32 230 Deathse Shee 101 7 2, 2 3 3 3 0 1 S 0 0 1 0 27 134 Modules Opened T o ~ 2R1 Smoke 55 2 0 0 1 1 0 0 0 0 0 0 0 0 4 59 Rapldly/Oropped Sham 23 0 0 1 I 1 0 0 0 0 0 0 0 0 3 26 Documented Improper 2R1 Smoke 90 0 0 0 0 0 0 0 0 .0 0 0 0 0 0 98 Seoke/A/r flow Sham 13 12 0 0 0 0 0 1 0 0 0 0 0 0 13 26 Exposure Related ZRI Smoke 695 20 14 11 iS 16 23 22 12 10 17 26 10 10 206 901 r OeathsR Sbaw 197 18 8 14 16 16 10 8 14 9 12 7 1 1 134 331 Cage Related 2RI Smoke 34 ' 0 3 S 0' 0 0 0 0 0 0 0 0 0 8 42 t s Deaths Slu. Ro exposure 18 10 0 6 1 0 0 0 0 0 0 0 1 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 3 S 21 iS l Found Dead In 2R1 S.oke 56 5 6 3 2 4 8 S lz 13 7 13 l0 1 00 144 the CeRe Shar 31 6 3 S 2 3 7 6 S 10 9 10 4 4 73 104 No e.posure 4 0 6 2 4• 7 3 10 13 13 10 ' 22 17 4 119 123 J IV ~ Moribund. Klllcd 2R1 Smoke 23 3 2 1 2 2 3 2 3 6 4 4 6 2 42 65 s Shaw 7 0 0 1 1 2 1 1 3 4 2 4 0 1 20• 27 O ~ ~ ' at Ro E.posure 3 0 0 2 2 6 0 2 3 0 1 1 2 2 21 24 CYI 70 : Undocumented. 2R1 Smoke 30 1 1 3 2 0 1 1 1 2 0 1 1* 0 14 44 Collaboration Sbe. 19 2 1 I 0 0 1 2 1 0 1 3 I 0 17 32 ond/or Other Causes No exposure S 0 0 1 0 1 1 0 2 0 0 0 0 1 6 11 _ 0 ~ :z ~ Totals 2R1 Smoke Sham 1189 415 41 44 29 15 23 24 22 23 28 26 40 23 31 16 30 24 34 29 29 24 47 24 27 1 13 6 394 206 1583 101 ~ ~ No exposure 22 S 6 S 6 14 4 12 iR 13 19 23 19 1 1S1 173 7a . eCondttlons for smoke exposure consisted of l0i 211 clgarette sooke; 20 seconds smoke alternatln0 with 40 seconds of air for a period of "'y„' ~ . 6-8 .Inutes. F1ve such exposures were 91vee each day, s.1 k ~ h Total number of animals: smoke exposed • 2053; shan exposed • 1014; no exposure - 449 ( n cAs of 0/31/80: Series 1• 2 have 6een on test for 102 weekst Series 3 6 4 for 9/1 weeks; Series S for 82 weeks. . dRnt.als were not swoke or shara exposed during the time 4/12/79 to 4/17/79. For Series I R 2 this period was during week 31; for Series 3 6 4 ~ 1~.m 'k~ 4V~ ~Y N 0 this period was during week 26; for Series S this period wao during week 10. eCauses*of death lnclude .1ce stru9911n9 against the holder resulting lo a hroken neck. and excessive head rove.ent resulting In non-a119nrent of the nese with the module. Oeaths are due to asphyxiation or broken necks. f0ashpot .alfunctlon. 9Causes of death Include wtce dying on the module (before. durlnk. after esposure), or In the holder or cage within one hour after exposure.

TABLE 9 ABBREVIATIONS USED PAMA Pigmented Alveolar Macroohage Accumulation AH Alveolar Hyperplasia ANCN Alveolar Non-Cornpressing Nodule ACN Alveolar Compressing Nodule AC Adenocarcinoma AAC Alveolar Adenocarcinoma ASC Adenosquamous Carcinoma SM Squamous Mecaplasia SN Squamous Neoplasm SCC Squamous Cell Carcinoma p.d. poorly differentiated -37- CTR CQNTRRCTS 028303 11248053 CTR HN 044381

.. AE 10 m t 1 SEItCI(0 SIMNRT Or f1NJqRMT NISTOtAl/0L00T fRON KXI/CIM IWLLE MIC( Afi(R (VOSORE T0 2R1 CICAR(fT( S/qtL. SNIW ESfOSOR(. OR N0 EIf01YNE A/p YITN p YITIRMIT fR10R 1R(AINENT NiTN /EN10(.)1tRENE TIfATM NTa CRt111CA1t 'R.n. N... Noee (It010R(l 2R1 S.ote SA.. N.ne e e.ocarc a..a alve.lar adawcarclnert A1v..l.r Ad..oc.rc/ao.a, p.rly dllf.r..tla.d 3/i1S 1/29S t/66 (ACI A6tt Mt. r.d.) +beasma uus 0/29s 0/6s Ad....atslt 0//15 0/295 0/66 AdeM/1W..et c.rcl...a (ASC 01415 01295 0/66 Ai...lar c..lreul.6 .od.l. IACN) 2//1S 1/29S 0/66 Al.e.l.r e/ter'Le16 (AN) 2//1S 6/29S 0/66 Al..dar rcrqAap .cctaul.tlen 2//IS )/29S 0/66 Al.eel.r Re.-c.yratl.6 md.l. (AIKN) 2/I1S . S/29S 1166 ere.c6lal .de..r. 0//1S 0/295 0/66 er.ec6als 0/416 0/295 0/66 Ir.ncMMt.o.l. 0/61S 0/295 0/66 Coq.stl.n 122/415 110/295 1/66 fl6ros.re...; fl6r.s.rcer, .et..t.tlc 1//1S 1/29S 0/66 Cra'rlo'+ 0/11S 2/29S 0/66 Ne.orrAy. 11//16 1/29S 1/66 Nept.c.ll.lar e.rclao.e. ..tyctals 1/11S 0/29S 0/66 f.f.rctl.. .f ACL MCI MC, e.d. • 0//IS 0/29S 0/66 t laf.rctl.a .f POC 01416 0/29S 0/66 lar.rcll.n .r scC 0/4ls 0/295 0/66 f.6.ted 61..d 11416 0/29s 0/66 l.l.rttltlal l.ew.l. 1/IIS . 0/29S 0/66 (..1t.la1 I~yllc lotw/. 6//lS 11/29S 11/66 l2yA.ld latl tr.ll..t <+er16r..cMt.1 Il.fMld l.fllt.r.tl.e; 9/I1S 9/29S 1)K L terlr.ec.l.r 11.fA.id /Mftltr.tl.. tl.r/atracar 11//1S 6/t9S 12/66 Maclr.c .d..... 0/I1S 0/29S 0/66 1 Fac•sw•»•••6•n... 0/61s 6v29S 0/a NeMt/.. (.ne+.t. wt) 224/(1S 12e/29s 30/66 Neell.s.; tlfe Md.t.r.l.ed 0/(1S tl/29S 0/66 Oue.ss .ct.p1a1. 21(1S 1/29S 0/K 0et..qeMe ..rc...; .at.qe.le ..rc.... n.tatatte 0//1S 0/29S 1/66 P.r..I.nc6..lI1/.( p.r16r..c6L111L; r.rlM.acNl.llll. 0/I11 S/29S 0/66 nq...t det.ettl.. • 0/t16 0/29S 0/66 11pat.d .l...lar rcq6.ee accwlal.. (NIMR) 20/e1S 0/295 0/66 f Iwr.l Invasion .f ACI MCt MC, e.d. ' 2//1S e/29S 0/66 Pleural I..u/...f ASC 0/us 0/295 0/66 Ilewal t...sl...l lOC 0//ll 0/29S tl/K Pleural l.eal...r ue o/us o/29S 0/66 L.rll dlfferatl6led t.reln... (tOC) 0//1S 0/29S 0/66 Post ..rle ..wllelt 3/p1S 0/29S 7/66 R.tlc.l... c.11 .arc... 0//1s 0/29s 1/66 S.rcw, rt.st.slUl urca.a wndlffer..tl.ttl/ t.re.r 11416 1/196 1/66 Ss...wt eell c.rcl.«. (SCC); es.....6 c.n e.re/...., r..rll dlff«.nu.t.d 01415 6/r9s 0/66 .aa. I../. (sn) 0//16 0/295 o/66 sh..~.. ...r~.e. (16) 0/e16 e/29S o/66 Ilpe 11 Lsl.. ' 0//11 11/29S tl/66 T.scrl.r Invasion .f AC; MC; AM. r.e. 0/41s 0/29S 0/66 tascul.r Invasion .f tOC 0/11S 0/295 0/66 t.sc.l.r Invasion .f SCC 0/I1S 0/29S 0/66 fasc.lllls; lerl..ic.l/llt 0/(1f 1/29S 1/66 6a/ 6ef e.f M1 S..Ie SA.. Ibnt 31/le9 69/162 se/e1 0/1s9 6/.let o/el 1/159 11162 0/el 0/15f 0/162 3/01 26/ls9 u/162 44/e1 )1/1S9 60/162 12/01 1l/169 15/162 20/e1 21/1S9 $1/162 29/O1 1/IS9 1/162 0/01 2/159 1/162 0/el 0/159 0/162 7/el 29/1S9 71/162 1/t11 0/IS9 6/162 0/01 1/1s9 t/162 0/el 2n59 1/162 I/el 0/1S9 t/!62 0/el 1/IS9 S/162 1/el 1u1s9 1/161 vel 0/1s9 1/16( 1/el o/1s/ 1/162 lvel 3/1sf 2n62 2/el t/ls9 e/162 2/et 2/159 . e/!62 vel 1/1S9 0/162 0/01 2/1S9 0/162 0/01 I/ISO 6/162 wel 21v1s1 141162 1/el 0/1s9 1/162 o/el 0/t69 6/16! 0/et 1/lsl 6/162 0/el 4/a9 3/ul 0/e1 0/1s9 6n61 I/el 1e/IS9 u/lu o/el 1s/1s9 u/162 le/el 0/fs9 e/ts2 uel 6nS1 1/162 6/e1 o/1S9 uu2 0/e1 6/lsl 1/162 e/el o/1S9 e/162 S/el e/IS9 en62 o/el 0/1s1 6/1a2 2/el 0/1s9 S/162 1/el t/IS9 1/161 a/el 0/169 6/162 0/el 0/1s9 1/162 0/el 0/169 21162 2/e1 1/1s9 0/162 2/el o/1S9 2/162 vel 0/1s9 2/162 0/el tMlce nert treated .e16 6a/ .ece per r.a f.r 3 neae rrl.r t. ..y.ar.( 1.2 M e.r I. 0.02 .1 .1 a 0.2% 9.L11.-u1Ine s.lutl.n per LtraracReal Lsl/ll.ll.e. C.Mllluat f.r srte eeps.r.c.eelated .f 10%. 'llt cipr.tle srlel 20 sec..Js .t s..te .IternallnM +Ilb 40 aec.ads of o/r f.r a eerl.d .f 6-0 .lawtes. fl.e sucA ..pswes vere gIv.. ..c6 day. L.w eur.sw. ..s t6e s..e 6ut .Itlat sa6..

f TABLE 11 HISTOPATHOLOGY SUMMARY IN BC3F1/Cuaa FEMALE MICE AFTER CHRONIC EXPOSURE TO 2R1 CIGARETTE SMOKE Weeks After First Smoke Exposure 4 8'12 16 20 24 28 32 36 40 44 '48 52 lst.yr. TOTALS iUMBER OF ANIMALS no necropsy 166 195 108 52 96 66 69 59 55 51 40 15 1 973 with necropsy 33 33 21 15 11 14 15 11 11 11 6, 18 17 216 diagnosed 16 17 10 10 6 11 14 11 11 11 6 17 17 157 )IAGNOSIS negative 10 10 4 7 2 9 10 8 8 7 4 8 9 96 PAMA 0 0 0 0 0 0 0 0 0 0 1 1 1 3 ~ AN 0 0 0 1 0 0 0 0 0 0 0 0 0 1 -' ANCN 0 0 0 0 0 0 0 0 0 0 0 0 0 0 ACN 0 0 0 0 0 0 0 0 0 0 0 0 0 0 o --i AC; AAC; AAC,p.d. 0 0 0 0 0 0 0 0 0 0 0 0 0 0 ~ ~j ASC 0 0 0 0 0 0 0 0 0 0 0 0 0 0 SM 0 0 0 0 0 0 0 0 0 0 0 0 0 0 ~ SN 0 0 0 0 0 0 0 0 0 0 0 0 G 0 n 0 Scc 0 0 0 0 0 0 0 0 0 0 0 0 0 0 ~ ~ Congestion 3 7 5 2' 4 2 2 3 3 3 2 9 6 51 Fibrosarcoma 0 0 0 0 0 0 0 0 0 1 0 0 0 1 70 Hemorrhage 3 1 0 0 0 0 2 0 0 0 0 0 1 7 ~ 33 Interstitial pneumonia 0 0 1 0 0 0 0 0 0 0 0 0 0 1 Perivascular lymphold Infiltration 0 1 0 0 0 0 0 0 0 0 0 0 1 2 C-101 8/26/80

i TABLE 12 HISTOPATHOLOGY SUMMARY IN BC3F1/Cum FEMALE MICE AFTER CHRONIC EXPOSURE TO 2R1 CIGARETTE SMOKE FIRST xeeks After First Smoke Exposure SECOND YEAR YEAR CUMULATIVE TOTALS 56 60 64 68 72 76 80 84 88 92 96 100 104 TOTALS TOTALS ur•18 ANIMALS ~ no necropsy 973 2 3 4 2 0 2 2 1 3 1 2 2 0 24 997 with necropsy 216 39 26 19 20 28 38 29 29 31 28 45 25 13 370 586 diagnosed 157 39 26 19 18 28 36 23 25 31 12 1 258 415 DIAGNOSIS negative 96 27 18 10 10 15 16 3 8 16 5 0 128 224 PANA 3 2 0 2 0 0 4 5 4 7 1 0 25 211 All 1 0 1 0 0 0 0 0 0 0 0 0 1 2 ~ ANCN 0 0 0 0. 1 0 0 0 0 0 1 0 2 2 ACN 0 0 0 0 0 0 1 0 0 1 0 0 2 2 N A AC; AAC; AAC,p.d. 0 0 0 0 0 0 0 1 U 1 0 1 • 3 . 3 00 0 ~ 0 ~ ASC 0 0 0 0 0 0 0 0 0 0 0 0 0 0 SM 0 0 0 0 0. 0 0 0 0 0 0 0 0 0 rn ~ SN 0 0 0 0 0 0 0 0 0 0 0 0 0 0 SCC 0 0 0 0 0 0 0 0 0 0 0 0 0 0 4 J n 0 Adenoma - 0 0 0 0 0 1 0 0 0 0 0 1 1 Alveolar Macrophage accumulation 0 0 0 0 0 0 1 1 0 0 0 2 2 Congestion 51 10 7 8 6 6 7 14 5 7 1 0 71 122 73 Fibrosarcoma 1 0 0 0 0 0 0 0 0 0 0 0 0 1 e Hemorrha 7 0 0 0 0 4 2 0 1 0 0 0 7 14 ~ 3:) g Hepatocellular carcinoma, metastatic - 0 0 0 0 0 0, 0 0 1 0 0 1 1 Inhaled blood _ 0 0 0 0 0 0 0 0 1 0 0 1 1 Interstitial pneumonia 1 0 0 0 0 0 0 0 0 0 0 0 0 1 Lfl Lymphocytlc leukemia - 0 0 0 1 0 2 2 2 0 1 0 8 8 Lymphoid Infiltration - 0 0 0 0 0 0 0 0 0 1 0 1 1 ~.. (Q Lymphosarcoma - 1 0 0 0 0 1 1 4 1 3 0 11 11 •~ N Osseous metaplasia - 0 0 0 0 1 1 0 0 0 0 0 2 2 Peribronchial lymphoid Infiltration - 0 0 0 0 0 1 0 0 0 0 0 1 1 ~ Perivascular lymphoid infiltration 2 0 0 0 0 1 1 0 1 2 0 0 5 7 w d 0• 0 0 0 0 0 0 0 1 0 1 2 2 ~ 0 .) Pleural invasion (AC; AAC; AAC.p. - * Post mortem autolysis - 0 0 0 1 0. 1 0 1 0 0 0 3 3 ~ ~ (J k Sarcoma - 0 0 0 0'1 0 0 0 0 0 0 C-101 1 1 9/11/80

TABLE 13 HISTOPATHOLOGY SUMMARY IN BC3F1/Cum FEMALE MICE AFTER CHRONIC SHAM EXPOSURE NUMBER OF ANIMALS no necropsy with necropsy diagnosed DIAGNOSIS negative PAMA AH ~ ANCN ~ ACN AC; AAC; AAC,p.d. ASC SM SN SCC Congestion Hemorrhage Lymphosarcoma Pertvascular lymphold infiltration Weeks After First Sham Exposure 1st yr. 4 8 12 16 20 24 28 32 36 40 44 48 52 TOTALS 65 37 31 22 26 20 20 13 14 31 29 11 1 320 c+ ~ ;a o 13 8 2 3 6 3 2 3 4 6 4 11 30 95 0~ 9 6 1 1 3 2 2 3 3 6 4 11 30 81 3 UI a&A " 0 4 4 1 1 1 2 0 1 3 3 1 3 14 38 w~ 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 5 2* 0 0 2 0 1 0 0 3 3 8 14 38 1 0 0 0 0 0 0 1 0 0 0 0 1 3 0 0 0 0 0 0 1 0 0 0 0 0 0 1 0 0 0 0 0 0 0 1 0 0 0 0 1 2 C-101 8/26/80

TABLE 14 IIISTOPATIIOLOGT SUWMtRY IN OC3F1/Cu. FEM/LLE MICE AFTER CIIRONIC SHAM EXPOSURE NUMBER OF ANIM/ILS no n.crepsy rlth necropsy dl.qnosed OIAGMOSIS ~ neO.t v. PAMA All ANCN ACN AC; AACI AAC,p.d. ASC SM SN N ~. SCC ~ Alveolar .rcroph.0e accu.olat/on 00 N Coagesttoe 0 ~ Flbrosarcom.. .xt.st.ttc [n 00 7U Granule.r ~ -4, n 0 Hemorrhage Ly.phocytlc leulewla lywplbld /otlltr.tl.n ly.phot.rco.w Osscous w.l.yl.sl. ~ P.rlbronehlalltls, Peribronchi.lltls PerlbroncAl.1 lymphold Inflltr.ttoe ~ P.rlvscular 1y.phold tnf/ltratl.n S ~ ~ ~IV .i.J Perlv.seulltls Pest .orte. .utolysts S.rc.w., .etast.tle ~ .w.~. ~ L ~ fl ~ ~ ~ W ~ 0 CD Flrst fear Y..tc Aft.r First Sha. Exposure Second Tot.ls 56 60 6/ 68 72 76 80 84 88 92 96 100 104 Year Totals Cu.ul.tiw Tota s 320 5 2 1 0 0 1 2 1 0 2 4 1 0 19 • 339 95 39 13 23 23 25 22 16 23 29 22 20 6 6 267 362 81 39 13 23 22 24 20 16 ' 22 29 6 214 29S 38 23 7 10 8 12 11 1 5 7 0. 90 128 0 0 0 0 0 0 0 0 0 0 0 0 0 1 • 0 0 0 2 2 1 0 0 0 0 5 6 0 0 0 0 1 0 1 1 0 1 1 6 S 0 0 0 0 0 0 0 0 1 0 0 1 1 0 0 0 1 0 O 0 0 0 0 0 1 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0. 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 2 1 0 0 a 3 38 1S S 0 10 10 4 3• 13 10 2 e0 118 - 0 0 0 0 0 0 0 0 0 1 1 1 2 0 0 0 0 0 0 0 O 0 ' 2 2 3 0 0 1 0 0 0 0 0 3 0 4 7 - 0 1 0 1 0 2 3 0 2 2 II 11 0 0 0 0 0 1 0 0 0 0 1 I 1 0 0 I 1 0 0 0 0 S 0 7 6 - 0 0 0 0 0 0 1 0 0 0 1 I - 0 0.0 0 0 0 1 2 2 0 S S 0 0 0 0 1 0 0 0 0 0 1 1 1 2 0 0 0 0 2 0 0 2 0 6 7 - 0 0 0 0 0 0 0 1 0 0 I I - 0 0 2 0 0 0 0 0 0 0 2 2 - 0 0 0 0 0 0 0 0 1 0 1 1 4

i i TABLE 15 ~ HISTOPATHOLOGY SUMMARY IN UNTREATED BC3FI/Cum FEMALE MICE ~ WEEKS ON TEST 16t yr 4 8 12 16 20 24_28_32 36 40 44_48 . 52 TOTALS_ ' NUMBER OF AN ( MALS no necropsy 1 1 0 1 0 5 3 0 6 0 1 0 0 18 :0 0 ` with necropsy 0 0 0 0 0 0 0 0 0 0' 0 2 2 4 c,`~ o ce diagnosed. 0 0 0 0 0 0 0 0 0 0 0 2 2 11 rt^ ~ ~ D I AGNOS I S nn ~ p negat ve 0 0 0 0 0 0 0 0 0 0 0 0 2 2 00 PAMA 0 0 0 0 0 0 0 0 0 0 0 0 0 0 o.t 0 ~ AH 0 0 0 0 0 0 0 0 0 0 0 0 0 0 ~ ~ ~ ANCN ACN 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0. 0 0 0 o ""1 AC; AAC; AAC,p.d. 0 0 0 0 0 0 0 0 0 0 0 0 0 0 ~ ~ ASC 0 0 0 0 0 0 0 0 0 0 0 0 0 0 SM 0 0 0 0 0 0 0 0 0 0 0 0 0 0 ~ () SN 0 0 0 0 0 0 0 0 0 0 0 0 0 0 •-4 [D SCC 0 0 0 0 0 0 0 0 0 0 0 0 0 0 ~ -7 ~ ,.,.j congestion 0 0 0 0 0 0 0 0 0 0 0 1 0 1 70 lymphosarcoma 0 0 0 0 0 0 0 0 0 0 0 1 0 1 ..~. .M.. ~ 1.~1 ~ c-101 N 8/27/80 ~ p ~~

TABLE 16 "-r M ".rZ. l ,J LF) LJ 0 .~ CD ~ ~ o C) C~J i NtNMER OF ANllY1Lf no n.creysy ulth .ecroP:Y diagnosed DIAGNOSIS negatlve Conyestlon Iknorrheye Lywphocyttc leut..l. tY.phosarco.. Osleogenlc sarcoma. ..tastetlc Perlhrenchl.l 1y.phold I.//ltretlon Post .orten autolys/s Betlculua cell sarco.e Sarcoma. .etastattc (origin undetermined) vasculltls IIISTOPATIIOL06T SIN9NIT IN UNTREATED 8C]F1/Cu. FEMALE MiCE Ftrst Tear Meeks on T.st S.eond Totals 66 60 64 68 72 76 80 84 88 92 96 100 104 Tear Totals Cu.ulatlve Totals - le S 4 2 1 1 2 5 3 4 4 ' 5 2 0 40 se 4 0 2 3 6 11 2 7 15 9 15 18 17 7 111 115 4 0 2 3 6 11 2 6 15 8 10 62 66 2 0 1 1 3 5 0 4 7 7 0 28 30. 0 0 0 0 0 0 D 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 1 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 I 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0. 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 2 0 1 3 4 - 0 0 1 0 0' 0 0 0 0 0 I 1 - 0 0 1 0 2 1 2 2 1 2 I1 11 1 0 1 0 0 0 1 0 4 0 5 11 12 - 0 0 1 0 0 0 0 0 0 0 1 1 • 0 0 0 0 0 0 0 1 0 0 1 1 - 0 0 0 0 3 0 0 0 0 0 3 3 - 0 0 0 0 0 0 0 0 0 1 1 1 - 0 0 0 0 1 0 0 0 0 0 I 1 - 0 0 0 1 0 0 0 0 0 0 1 1 C-101 9112180

"c r TABLE 17 4 CD W ~ a-+ !„o HISTOPATHOLOGY SUMMARY IN BC3F1/Cum FEMALE MICE AFTER INTRATRACHEAL INSTILLATION OF BENZO(a)PYRENE AND CHRONIC EXPOSURE TO 2R1 CIGARETTE SMOKE NUMBER OF ANIMALS no necropsy with necropsy diagnosed ; DIAGNOSIS negative .~ ~ P n PAMA AH ANCN ACN AC; AAC; AAC, p.d. ASC SM SN SCC Alveolar macrophage accumulation Bronchial adenoma Bronchitis Congestion Grenuloma Hemorrhage Infarction (AC, AAC) Interstitial pneumonia Lymphocytic leukemia Parabronchiolitis, Peribronchiolitis Perivascular lymphoid infiltration Pleural invasion (AC; AAC; AAC, p.d.) Weeks After First Smoke Exposure 4 8 12 16 20 24 28 32 36 40 44 48 52 lst yr. TOTALS c,v -~ ~ 23 4 48 0 2 2 1 0 1 1 1 1 0 84 A 0 18 38 10 4 3 9 6 2 6 5 3•4 3 111 o m 18 36 10 4 3 9 6 2 6 5 3 4 3 109 ~y N a ~o n M 9. v 0 12 6 0 0 2 0 1 0 0 0 0 0 0 21 o11 or+ W 0 0 24 10 3 1 3 2 2 5 5 2 1 1 59 0 1 1 2 0 2 1 1 2 3 1 3 1 18 0 0 0 0 0 1 1 1 2 2 1 2 1 ,11 0 0 0 0 0 0 0 0 1 1 1 2 2 7 0 0 0 0 0 0 1 1 0 0 1 2 1 6 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0-0 0 0 1 0 0 0 0 0 0 0 2 0 0 0 0 0 0 0 0 0 1 0 0 0 1 2 0 0 0 0 0 0 0 0 0 0 0 0 2 010 3 2 0 6 0 0 0 4 1 0 0 26 1 0 0 0 0' 0 0 0 0 0 0 0 0 1 0 1 0 0 0 0 0 0 0 0 0 0 1 2 0 0 0 0 0 0 0 0 0 0 0 1 0 1 0 0 0 0 0 0 0 1 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0'0 1 1 2 2 0 0 0 0 0 0 0 0 0 0 0 4 1 0 0 0 0 0 0 0 0 0 0 0 1 2 0 0 0 0 0 0 1 0 0 0 0 2 0 3 C-101 8/26/BO I

(^ TABLE 18 NISTONITNOLOGT SUMOIRT IN OC3f1/Cum FENOLE NICE Af(ER INTRATRACNEAL INSTILLATION OF OEN20(.)PTRENE AND CNRONIC EttOSURE TO 2R1 CIGARETTE SMOKE Flrnt Yeeks Aftor ftrst Smoke E.posure Second Tear Cuwulatlve Tear lotall 56 60 61 6e 72 76 00 e4 ee 92 96 100 104 , Totals Totals R no necropsy 84 0 1 2 0 0 1 0 0 0 0 0 0 0 4 6B with necropsy 111 11 4 4 0 9 13 0 5 1 3 3 2 1 72 1e3 dlayeosed DIAGNOSIS 109 11 4 4 6 2 7 7 5 1 3 50 IS9 e0,c e 21 . 0 0 0 0 0 0 0 0 0 0 0 21 PAµ 59 e 3 1 3 1 3 3 6 0 2 29 Oe AN le 6 3 1 z 1 2 3 1 0 0 19 37 ANCN 11 3 1 2 2 1 2 2 1 1 1 16 21 ACN 7 1 1 1 2 0 3 4 4 1 2 21 20 AC; AAC; AAC,p.d. 6 3 1 4 4 2 6 7 3 1 0 31 ~7 ~~.. ASC 0 0 0 0 0 0 0 0 0 0 0 0 3 0 4 N SN 1 1 0 0 0 0 0 1 1 0 0 0 0 A ~ SN 0 0 0 0 0 0 0 0 0 0 0 00 0 SCC 0 0 0 0 0 0 0 1 0 0 0 I I O ~ s 0) ~ Adeno.atosls - 0 0 0 1 0 0 0 0 0 0 • 1 1 N Alveol.r .acrophaye accumulation 2 0 0 1 0 1 2 3 2 1 1 11 13 1 ~ Orenchlal adenor 1 0 0 0 0 0 0 0 0 0 0 0 2 \ J Oronch/tls 2 0 0 0 0 0 0 0 0 0 0 0 0 3 29 ~ 1 Coeyestlon 26 1 0 1 1 0 0 0 0 0 1 O Granuloer 1 0 0 0 0 0 0 0 0 0 0 0 2 Ne.orrha9e 2 0 .0 0 0 0 0 0 0 0 0 0 3 Inf.rctton (AC; AACt AACIp.d.) 1 0 0 0 0 1 1 0 0 0 0 2 3 3 Infarctlon (P0C) • 1. 0 0 1 0 0 0 1 0 0 3 Interstitial pnau.onla 1 0 0 0 0 0 I 1 0 0 0 2 0 1 ty.phoc,tie lewkeda 1 0 0 0 0 0 0 0 0 0 0 1 1 lr.phosarco.a - 0 0 0 0 0 0 0 0 0 1 2 Nuctnors adenoa, - 0 1 0 0 0 0 1 0 0 0 0 0 0 2 Nveosrrs.ous adenow, - • 0 0 1 0 0 0 0 0 0 0 0 0 0 0 ; ; -1 OsteoOe.le sareor - 1 0 0 0 4 ~~ Vr ParaOroscAlellelsl Perlt+ronchlolltls ' 4 2 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 2 Perlvase.l.r ly.psw/d /ntlltral.a 2 3 1 0 0 0 12 IS tleeral Invaslo. ACI AAC; MC.p.d.) ~ 3 2 1 1 2 1 0 0 0 1 0 0 4 4 POC) Pieural Invesloa - 1 1 0 6 6 .~ N Poorlydlfferentlated carclnoea-'PDC' - 1 1 0 2 0 1 0 1 0 0 0 3 3 Y.sc.lar Invasion (POC) - 0 0 0 1 0 1 0 1 0 ~. rn ~W~~ ~hs t+o 0-A N 0 C.101

C TABLE 19 ~ HISTOPATNOLOGY SUMMARY IN BC3F1/Cum FEMALE MICE AFTER INTRATACHEAL INSTILLATION OF BEN20(a)PYRENE AND CHRONIC SHAM EXPOSURE Weeks After First Sham Exposure lst yr. 4 8 12 16 20 24 28 32 36 40 44 .48 52 TOTALS NUMBER 0 ANIMALS no necropsy 13, 6 4 0 3 3 1 2 0 1 2 2 0 37 with necropsy 7 0 1 1 6-5 5 2 5 7 9 9 7 64 diagnosed DIAGNOSIS 1 0 1 1 6 5 5 2 5 7 9 9 6 63 ..~'.. N negative 4 0 1 0 2 2 2 1 1 0 0 0 0 13 A co n PAMA 0 0 0 0 1 0 0 0 3 2 1 1 0 •8 0 AH 0 0 0 0 0 1 1 0 2 4 5 5 3 21 0) w 70 ANCN 0 0 0 0 1 0 1 1 1 1 3 3 1 12 ACN 0 0 0 0 1 0 0 0 0 2 2 1 2 8 AC; AAC; AAC, p.d. 0 0 0 0 0 0 0 0 1 0 2 1 2 6 • ASC 0 0 0 0 0 0 0 0 0 0 0 0 0 0 SM 0 0 0 0 0 0 0 0 0 0 0 0 0 0 SN 0 0 0 0 0 0 0 0 0 0 0 0 0 0 SCC 0 0 0 0 0 0 0 1 0 0 1 0 1 3 ~ M Alveolar macrophage accumulation 0 0 0 0 0 0 1 0 0 0 0 1 0 2 Bronchitis 0 0 0 0 0 0 0 0 1 0 0 0 0 1 Congestion 0 0 0 1 3 3 0 0 0 6 2 6 2 23 ~ U) Infarction AC; AAC ; AAC, p.d.) 0 0 0 0 0 0 0 0 0 0 1 0 1 2 Infarction ISCC) 0 0 0 0 0 0 0 1 0 0 1 0 1 3 0 0 0 0 0 0 0 0 0 3 0 Parabronchiolitis 0 Pleural tnvasion A p C; AAC; AAC d ) 3 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 1 4 N Pleural tnvasion iS , . . CC) 0 0 0 0 0 0 0 1 0 0 0 0 0 1 Vascular tnvaston .p.d.) AAC 0 0 0 0 0 0 0 0 0 0 0 0 1 1 .~ W Vascular invasion , SCC) 0 0 0 0 0 0 0 0 0 0 1 0 1 2 C-101 9/4/80 ~~ M 0 ~ o m = IA C? N w ;o 0 m , r. v 0

e- TABLE 20 NISTOPATINILOGY SUMSART IN 1C]F1/Ca. fENILE NICE AFTER IMTRATRACIIEAL IMSTILLATION OF OEN20(a)PTRENE AND CBRONIC SIIAM EXPOSURE ffrst Neeks After flrst Sh.. Exposure Second Year Tear Cualative MO R 1 S Totals S6 60 64 60 72 76 tl0 e1 00 92 96 100 104 Totals Totals p"Cropsy 77 2 3 2 1 0 1 0' 0 1 0 1 0 0 11 90 rith necrepsy 6/ 12 13 10 16 9 1/ 12 9 7 5 5 7 2 120 101 dlagnostd 6l 12 13 10 1) 7 12 12 0 7 5 99 162 DIAGIIOSIS aegat va 17 1 0 0 0 0 0 0 0 0 0 I 14 rA,u a 0 All 21 5 ANCN 12 7 ACN 0 5 AC; AAC; AAC,p,d. 6 2 AsC 0 0 SM 0 0 SM 0 0 SCC; SCC.p.d. 3 0 Adeno.rtosls - 0 Alveolar oaceophage accu.ulatlon 2 0 Bronchial ade.o.a - 1 Oronch/tls 1 0 Congestlon 23 4 Ile.orrluga - 0 Infarctlon ACt AAC; MC,p.d.) 2 0 ln(.rctlon pOC) - 0 Infarct/on SCC) 3 0 Interstitial pneueonla - 0 Neoplas., type undertermtned - 0 Para6nnchlolltls 3 0 Pleural Lvasloa AC; MC; MC,p.d.) 1 0 Pleural lavasloa MC) - 0 Pleural Invaslo. SCC) 1 0 Poorly dlfferentlated carcinoma - 0 Type 11 leslon - 0 Vascular Iavaslon AC; MC; MC, p.d.) 1 0 Vascular Invaslon SCC) 2 0 3. 0 0 0 0 0 0 0 0 7 7. S 6 S 2 / 1 1 6 7 S 2 S 4 2 5 2 0 ] 0 3 7 0 7 3 3 5 S 6 4 9 7 6 6 4 0 0 0 0 0 0 0 0 0 0 1 0 0 0 1 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 1 0 0 0 0 1 0 0 0 0 0 0 1 0 7 1 1 1 4 1 1 0 0 0 0 0 0 0 0 0 0 01 0 0 0 0 0 0 1 0 1 1 0 0 2 0 I 0 0 0 0 1 0 0 0 0 0 0 0 0 2 1 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1. 1 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 1 0 1 1 S 2 2 1 3 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 1 0 0 1 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 C-10t 9/12/00

~ ~ ~. TABLE 21 NISTOPATHOLOGY SUMMARY IN BC3F1/Cum FEMALE MICE AFTER INTRATRACHEAL INSTILLATION OF BENZO(A)PYRENE Weeks on Test lst yr. 4 8 12 16 20 24 28 32 36 40 44 48 52 TOTALS NUMBER OF ANIMALS -~-+ no necropsy 3 0 0 0 0 0 0 2 0 0 0 0 3 8 ~ 0 xith necropsy 0 0 0 0 0 0 0 0 0 0 2 0 4 6 ~~ diagnosed 0 0 0 0 0 0 0 0 0 0 2 0 4 6 n m ~.~ DIAGNOSIS o -°+ negative 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0" PAMA 0 0 0 0 0 0 0 0 0 0 0 0 0 0 AH 0 0 0 0 0 0 0 0 0 0 1 0 3 4 -~ ANCN 0 0 0 0 0 0 0 0 0 0 1 0 2 3 '' ACN 0 0 0 0 0 0 0 0 0 0 1 0 0 1 ~ (7) AC; AAC; AAC, p.d. 0 0 0 0 0 0 0 0 0 0 0 0 1 1 o --~ ASC 0 0 0 0 0 0 0 0 0 0 0 0 1 1 0) 7U SM 0 0 0 0 0 0 0 0 0 0 0 0 0 0 . SN 0 0 0 0 0 0 0 0 0 0 0 0 0 0 4~ SCC 0 0 0 0 0 0 0 0 0 0 0 0 0 0 ~ 0 Leukemia 0 0 0 0 0 0 0 0 0 0 1 0 0 1 ~ ~ Pleural Invasion (PDC) • 0 0 0 0 0 0 0 0 0 0 0 0 1 1 Poorly differentiated carcinonw-"PDC" 0 0 0 0 0 0 0 0 0 0 0 0 1 1 7U ~ M C-101 Ln 9/4/80 S..f ~ Q .~ N ~

TABLE 22 t IIISTOPATNOIOGf SUNN/lRT IN eC7f1/Caio FEMALE NICE AFTER tNTRATRACIIEAI INSTILLATION Of 0EN20(a)PIRENE flrst Tear iotols 01 M N OF A 1 BER _ IINLS 00 necrop.r 8 rlth necrapsy 6 dleynosed 6 1 GNOS t noya ve 0 PANA 0 AN 4 ANCN 3 ACN 1 AC; MC; MC,p.d. • I ASC I SN 0 SN o SCC 0 Alveolar mcrophaye accu.ulatlon Rronchop.eu.onla ConReetlen Ne.orrhaye seforctlen lnlorctlen POC Infarctlon SCC I nterstitlol pneueonta - leute.le; lyp hocytle laate.la 1 ly.phold Infiltration . - PlO.ent depositloe - Pleurel tnvaslon AC; MC; MC, p.d.) - Plearal lnvaslo. ASC - Pleural Invasion POC 1 Poorly dlfferentlated corclnoeu-•PDC' I Post wrtem autelysls - Sarcoor<,rndlff. (origin undeter.) - Vascular lensloa AC;MC;MC,pd) tascular levoslon POC Vascular Invdsfon SCC Weeks on Test Second Year Cuaulative 56 60 64 66 72 76 80 04 88 92 96 100 104 Totals Totals 1 0 4 3 1 2 0 1 1 2 0 0 0 is 23 2 4 7 11 16 16 10 6 4 5 2 2 0 87 93 2 4 7 17 12 13 9 6 4 5 75 e1 0 0 0 0 0 0 1 0 0 0 ' 1 1 9 0 0 0 0 0 0 0 0 0 0 0 0 2 0 2 0 2 2 0' 0 0 e 12 1 1 1 6 4 4 1 3 0 3 26 29 0 3 3 / S 8 3 6 2 S 43 44 2 4 S 11 / 10 7 4 3 3 S) Se 0 0 0 0 2 0 0 0 0 0 2 3 1 0 1 0 0 0 0 1 1 0 4 4 0 0 0 e 0 0 0 0 0 0 0 0 1 1 1 0 0 0 0 0 0 0 > > 0 3 1 2 3 S 2 1 1 2 20 • 20 0 0 0 1 0 0 2 0 0 0 3 3 0 0 0 1 0 0 0 0 0 0 . 1 I 0 0 0 0 1 0 0 0 0 0 1 1 0 0 0 0 1 0 0 0 0 0 1 1 0 0 1 0 0 0 0 0 0 0 1 I 0 1 0 0 0 0 0 0 0 0 1 1 0 0 1 0 0 0 0 1 0 0 2 2 0 0 0 0 0 1 0 0 0 0 1 2 0 0 0" 0 0 1 0 0 0 0 1 1 0 0 0 0 1 0 0 0 0 0 1 I 2 1 3 2 1 2 3 •2 1 1 le le 0 0 0 0 1 0 0 0 0 0 1 1 0 0 0 1 2 0 1 1 0 0 S 6 0 0 1 1 3 1 0 1 0 0 1 e 0 0 0 0 2 0 0 0 1 0 3 3 0 0 0 0 0 1 0 1 0 0 2 2 0 0 0 0 1 1 0 0 0 0 , 2 2 0 0 0 0 1 1 0 0 0 0 2 2 0 0 1 0 0 0 0 0 0 0 1 1

~ C) ~ 0 ~ ~ 3Y Ln TABLE c3 DISPOSITION OF ANIMALSa CHRONIC "CONTINUOUS" EXPOSURE TO 3A1 OR 2R1 CIGARETTE SMOKE Number Complete Respiratory No Dosimetry/ Numbef Exposure on Teste Necropsyd Tract° Diagnosed Necropsy Collaboration Alive 3A1 Smoke 480 5 28 0 5 25 417 2R1 Smoke 360 4 15 0 2 17 322 Sham 440 2 14 0 8 15 401 None 156 0 0 0 0 0 156 aSince June, 1980. Experiment CTR- 101-B bCondltlons for smoke exposure: 10% 3A1 cigarette smoke or 10% 2R1 cigarette smoke; 17 clgarettes.per day; 8-10 puffs per cigarette; 15 seconds of smoke alternating with 45 seconds of air; continuous exposure (no rest periods); program 1 on the SEM (1. Sham exposure was the same but without Smoke. `Serles I on test 6-2-80 ~ 180? 3A1; 1804 2R1; 180 4 Sham; 56 Y no exposure. Series 2 on test 7-14-80 ~ 120? 3A1; 80? Sham. Series 3 on test B-11-80 ~ 1809 3A1; 1802 2R1; 1809 Sham; 1004 no exposure. dAll tissues taken at necropsy and submitted for dlagnosls. °Resplratory.tract " those animals from which the lung complex, larynx, trachea and head were taken at necropsy. These tissues were stored for possible use In the future. fNumber allve as of 8/26/80.

TABLE 24 PERCENT CARBOXYHEMOGLOBIN (% COHb) IN BC3F1/Cum FEMALE MICE AFT'eR CHRONIC EXPOSURE TO 3A1 OR 2R1 CIGARETTE SMOKEa Date 3A1 Smoke 2R1 Smoke June 1980 49.7 + 6.2b'c 32.4 + 6.7 August 1980 48.2 + 2.8 28.7 + 4.0 aConditions for smoke exposure consisted of: 10% 3A1 cigarette smoke or lOZ 2R1 cigarette smoke; 17 cigarettes per day; 8-10 puffs per cigarette; 15 seconds of smoke alternating with 45 seconds of air; continuous exposure (no rest periods); program 1 on the SEM 11. Sham exposure was the same but without smoke. bYalues given represent the mean (3-5 animals) + the standard deviation. C% COHb values for sham exposed and untreated animals were equal t0 or less than 2.4%. -52- CTR CONTRRCTS 028316 11248068 CTR NIV`71 0,443~`~G

TABLE 25 CHRONIC TOXICITY AFTER CONTINUOUS EXPOSURE OF BC3F1/Cum FEMALE MICE TO 2R1 CIGARETTE SMOKEa Weeks on Test Average Daily Inte rated TPM ,?gtSO)b Number of Mice Al ivec Percent Collb (+SO)d 0 -- 180 -- 4 2.8+0.3 178 34.5+ 5.0 8 2.5+0.5 178 26.0+ 4.9 J 12 2.4iU.2 175 26.4+ 8.9 16 2.1+0.2 157 30.9+ 5.0 N ~ ~ ~ 20 2.0+0.5 138 23.1+ 6.6 co ~ 24 2 1+0 4 137 38 8+ 4 1 0 ~ . . . . 0) 28 2.0+0.4 125 33.7+12.6 co 32 2.4+0.1 123 33.67 3.3 36 2.4 108 33.6+ 7.9 °ConAitions for continuous exposure on the SEN II were 15 seconds smoke/ 45 seconds air for 8 consecutive minutes (8 puffs/cigarette), repeated without rest for 17 runs over 136 minutes. bintegrated TPM values were recorded with the digital integrators, once per week for 4 consecutive weeks. cThe number of mice alive at the end of the time Interval. dCarboxyhemoglobin values were determined once per week, averages 33.5+9.1% over 36 weeks. to %D IQ

TABLE 26 CHRONIC TOXICITY AFTER CONTINUOUS EXPOSURE OF BC3F1/Cum MALE MICE TO 2R1 CIGARETTE SMOKE° Weeks on Average Daily Number of Percent Test Inte rated TPM M1ce A11vec CoHb ?9;S0)b• (+SD)d J -' 150 -- A 4 1.9+0.5 136 31.2+14.0 co ?• 8 2.1+0.4 123 31.1t 4.4 ~ 12 2.640.4 120 29.279.8 o 16 2.4+0.1 112 32.6+ 4.5 , aConditlons for continuous exposure on the SEta 11 were 15 seconds smoke/ 45 •seconds air for 8 consecuttve minutes (8 puffs/cigarette), repeated without rest for 17 runs over 136 mtnutes. bIntegrated TPM values were recorded with the digital lntegrators, once per week for 4 consecutive weeks. ~. cThe number of mice alive at the end of the time interval. dCarboxyhemoglobin values were determined once per week. ~ QO (A) E.~ 0 a)

i - ~ .. ( ~ TABLE ~. HISTOPATHOLOGY SUMMARY IN BC3F1/Cum FEMALE MICE AFTER CHRONIC I "CONTINUOUS" EXPOSURE TO 2R1 CIGARETTE SMOKE° r~ o 0 o n n -.m ~ Weeks After First Exposureb Flrst ~~ ' Year p ! 4 8 12 16 20 24 28 32 36 40 44 48 52 Totals A o ~ No necropsy 0 0 0 0 15 0 10 0 25 0-A4 With necropsy 2 0 3 18 4 1 2 2 32 0 W 0 i diagnosed 2 0 3 18 3 26 negatlve 0 0 0 1 0 1 PAMA 0 0 3 17 3 23 AH 0 0 0 0 0 0 ! J ANCN 0 0 0 0 0 0 ~ ACN 0 0 0 0 0 0 A Ln AAC 0 0 0 0 0 0 o ~ ~ ASC 0 0 0 0 0 0 y SM 0 0 0 0 0 0 L ~ SN 0 0 0 0 0 0 SCC 0 0 0 0 0 0 Q Congestion 2 0 0 0 0 2 7a aCondltlons for smoke exposure: 10% 2R1 cigarette smoke; 17 cigarettes per day; 8-10 puffs per cigarette. ~ :D 15 seconds of smoke alternating with 45 seconds of air; continuous exposure - no rest periods; program I on the SEM 11. Ln Females were put on test 1-7-80 (CTR-117-4) ~' 0

'TABL `8 14 . ..... , , COMPARISON OF C-NICOTINE EXTRACTION EFFICIENCY BY VARIOUS ORGANIC SOLVENTS x CPM % EFF OPM D.F. Recovery J~gd 89.2 .930 95.9 C-Nicotine „1 381,983.9 .938 407,232.3 Corrected Stock (0.1 ml) 2 386,797.2 .936 413,244.87 AV. DPM 410, x3 - 1,230,428 OPM/filter 142.68/0.1 ml Pad (0.3 ml) Bkgd a 37.0 .942 39.28 Chloroform lA 6198 .928 6678.88 1B 6262.5 .931 6726.64 Corrected 2A 5447.5 .933 5838.69 AV. DPM - 6335.35 x200 (Dilution Factor)- 2u 5829 .932 6254.29 1,267,070 102.9% b Ethanol 1 A 5625.5 .943 5965.53 18 5597.5 .942 5142.14 Corrected 2A 5484.5 .942 5822.19 AV. DPM - 5867.73 x200 D.F. - 1,173,546 95.38% 2B 5556.0 .942 5898.09 Pyridineb lA 5150.5 .939 5485.09 Corrected ~ 0 ~ 1B 5225.0 .940 5558.51 AV. OPM - 5458.33 x200 D.F. = 1,091,666 88.7% "'i 2A 5113.0 .940 5439.36 70 28 5166.0 .938 5507.46 n b Dioxane 1A 1952.5 .943 2070.52 Corrected 0 1B 1956.5 .942 2076.96 AY. DPM - 2019.09 x200 D.F. - 403.,818 32.8% z 2A 1908.5 .942 2026.01 20 1940.5 .942 2059.98 "i Chlorofor~ lA 5714.0 .933 6124.33 Corrected 33 1B 5903.0 .931 6340.49 AV. OPM - 6207.53 x200 D.F. = 1,241.506 100.9% ~ (Overnight) 2A 5720.5 .932 6137.88 ~ 28 5944.0 .931 6384.53 a14C-nicotine was extracted from the Cambridge pad by hot chloroform. The extraction was completed within one hour. • bEthanol, pyridine and 1.4 diuxane were used as extracting solvents by ORNL (24 hrs soaking technique) c14C-nlcotine Cambridge filter pad was soaked with chloroform overnight prior lo counting. I :. •

TABLE 2 9 DETERMINATION OF NICOTINE (NIC) AT MA AND ORNLa MA ORNL TPM (mg) Nicotine (mg) NIC/ TPM Nicotine (mg) NIC/ TPM 49.5 3.6 0.073 45.8 2.6 0.057 49.1 3.4 0.069 45.1 2.4 0.053 38.4 2.6 0.068 41.4 2.3 0.056 30.9 2.0 0.065 32.5 2.0 0.061 52.7 .4.0 0.076 51.3 2.8 0.055 45.7 3.3 0.072 47.1 2.7 0.057 45.1 3.3 0.073 48.3 2.7 0.057 Mean 0.071 (0.05)b 0.056 (0.04) a. 2R1 cigarettes were burned on a Phipps and Bird analytical smok- ing machine under standard conditions and TPM was collected on Cambridge filters. The filters analyzed at ORNL used ethanol extraction to elute the TPM and a Bendix Model 2600 gas chroma- tograph with a 10% Castorwaxcolumn to determine NIC. The filters analyzed at MA used chloroform extraction to elute the TPM and Perkin-Elmer 3920B gas chromatograph with a 3% OV-17 column to determine NIC. Matched samples were analyzed at MA and ORNL. b. Coefficient of variation. L- -57- CTR CONTRRCTS 028323 11248073 CTR HN 044,401

TABLE 30 ' ANALYSIS OF TPM, NICOTINE AND 14C-DTC FROM 14C-DTC-2R1 CIGARETTE SMOKE GENERATED ON THE WHSMa Number of puffs/ cigarette TPMb (mg) Nicotiqec (mg) 14C-DTCd (dpm) Calculated Calcula Nicotine/TPM speci Ratio activity (1 (mg/mg) dpm/mg ted fic 4C-DTC- TPM) 6 22.5±1.4 2.06±0.28 8.8x105 0.09 39.1 8 35.3 3.60±0.51 1.1x106 0.10 31.3 10 43.913.7 4.900.22 1.6x106 0.11 36.1 a. Exposure conditions were a 2 second puff duration, 35m1 puff volume, one puff per • minute, nominal lOx (v/v) smoke concentration, with an exposure cycle of 15 seconds smoke alternating with 45 seconds of air per minute over 6.8 or 10 minutes. b. TPM was collected on a Cambridge filter pad and weight determined after indicated number of puffs. Data result from duplicated samples. c. Nicotine was determined after chloroforms elution of TPM from Cambridge filter. Values are result of duplicate chromatographic analysis of two Cambridge filters. d. 14C-DTC (dpm) content was determined from liquid scintillation spectrimetry of duplicate aligicots of the chloroform solution of TPM from two Cambridge filters.. »

TABLE 31 k2tALYSIS OF TPH, NICOTI:fE, C02 AND CO FROH 2R1 CIGdAETTE SMOICE GIIIERATED IN TE1: SEH IIa OPTICA L SEiSOR READOD Tb CA2ffiBIDGE FILTEBc SAlPLES tzumber of "ruas" TPM (mst) C02 X CO (Z) TP`1 (mx) Nicotine (mA) Nicotine/TPK ratio 1 192 0.8 0.28 - - - 5 953 4.0 1.4 63.6 2.85 .045 5 928 3.7 1.4 59.5 3.05 .051 10 2132 8.7 3.2 138.6 6.80 .049 a. Exposure conditions were 10x (v/v) smoka concentration, 15/45 second (smoke/air) exposure cycle'per minute over 8 minutes (8 puffs/cigarette) for each "run". b. Data read directly from digital intestrators and converted -" to mg T?a or percent C02 and C0. c. Smoke was drawn through a Cambridge filter at approximately 100 m.l/minute. TP%1 vas determined gravimetrically, and nicotine determined after chloroform extraction of the fil- ter and analysis by gas chromatography. -59- CTR CQNTRACTS 428325 11248075 C TR VM4 044`4~`~ .--Z-13

C TABLE 32 DEPOSITION AND DISTRIBUTION OF TOTAL PARTICULATE MATTER (TPM) IN BC3F1/Cum FEMALE MICE AFTER EXPOSURE TO 2R1 CIGARETTE SMOKE USING 15/45 SECOND EXPOSURE CYCLE, 8 PUFFS/CIGARETTEa : I Number of Total Smoke Total Particulate Matter • ..14C-DTC Exposure (Ug TPM) labeled time claarettes (seconds) Turbinate • (Head) Larynx Trachea Lung .! 1 120 3 1 2 3 1 0 3 t 1 29 = 20 (.70)b 3 360 3 t 1 3 ± 0.5 5 s 0.5 37 t 7 (.19) 5 600 4 ± 0.6 5 1 3 6*_ 3 90 t 31 (.34) 0 ...~ t ~ '0 . , 14 ' a 0 to 10% 2R1 cigarette smoke containing C-OTC for 120 seconds (8x15 Four mice per time point were exposed 0 . ~ seconds) per ctgarette,•with no rest between. Mice were not previously exposed to smoke before the day of the dosimetry experiment. f ....~ bCoefflclent•of variation. Ln

TABLE 33 DEPOSITION AkD,DISTRIBUTION OF.T07AL PARTICULATE NATTeR (TPM) IN BC3F1/Cum FEMALE MICE AFTER EXPOSURE TO 2R1 SMOKE USING 15/45 SECOND EXPOSURE CYCLE, 10 PUFFS/CIGARETTE° Total Total Particulate Matter Ner of Smoke (pg TPM) M-DTC Exposure Labeled Time Turbinate Stomach Cigarettes (Seconds) (Nead) Larynx Trachea Lung Contents External Noseb 2 300 12±0.4 0.54.2 14. 7 39±8 8t3 28±6 (0.21 )c J ~ ... N .~ 5 750 4:1 1:0.3 1±0.5 61t19 11±2 53t41 v+ (0.31) ' CD O .i v ..-~ 8 1200 5t1 14.4 2±0.8 102t28 18t4 99.01 (0.28) aFour mice per time point were exposed to 10% 2R1 cigarette smoke containing 14C-DTC for 150 seconds (10 x 15 seconds)•per cigarette, with no rest between. Mice were previously exposed to smoke be- fore the day of the dosimetry experiment. bThe portion of the nose which protrudes through the rubber dam into the smoke channel was removed from the head and the deposition determined. cCoefficient of variation. ~'.~' .

TABLE 34 _____. _. _.....1?IS~3ISII~ION .Qr' .$AAIOAC~IVITY jN TISSUES AND ' ERCFtETA OF :YECE AFTER 'EM0StTRE TO 34i-CAT-2R1 CICARETTE SYOKE Percent External Eeadb Larynx Trachea Lung Stoaach Liver Ridney Otherc Urined Nosea' 0.17 -0- -0- 0.51 0.12 , 0.50 0.23 0.95 91.4 6.0 . a. 2welve SC3F1./Cua female "adapted" mice vere exposed to l0x (v/v) amoice generated on a WHSH from 38-CAT-281 cigarettes under standard conditions for a total of 300 sec- onds total smoke exposure time (30 seconds of smoke alternating vith 30 seconds of ~ air~er miaute o_ver- 10 miautes~. .lftar erposure,,niee vere,immediatelY olaCed.A:x._... - __metabolie ca$es to collect urine. After 2 hours._mice ver_e_¢a,qrift,cad by. ~02' _. asphyxiation and tissuea vera analyzed as previously described. b. 2urbinates and brain make up tha composite head sample. c. Includes blood and spleen. d. Urine is a pooled sasple, collected over 2 hours from 12 mice. e. Represents the area of the nose vhich protrudes into the smoke chamber. C• -62- CTR caNTRRCTs 026328 11248078 C T R ~ I N ~".-4, 4 -4_ 0 E- ~

TABLE 3S DEPOSi:-C:I AND DISTRIBUTION OF TPa IN BC3F1/CCM :QCE AleTER B.E?FATED F7SPOSURES TO 3H-Cl.T-2Rl CICARETTE SXOICra TPH,(ug) 2±0.6 1*0.3 External Zrachea Lung Stomach Liver eidney Otherc Nosed 3.1 0.2 -0- -0- 1t4 1-+0.8 2219 Percent of Total 9t5 3±0.5 41t17 8.7 .1.4 20.8 10.7 4.3 50.8 a. Twelve BC3F1/Cum f-le "adapted" nice were exposed to lOZ (v/v) smoke gen- erated on a WUSH from 3H-CAT-2R1 cigarettes. Staadard exposure eonditions were used to geaerate 1.5 seconds of smoke alternating with 45 seconds of air per ainute over 10 minutes. Eight such expo.ures were given for a total of 1200 seconds total smoke exposure time. Hice were not given a rest be- tween exposure sessions. After the last exposure, nice were sacrificed by C02 asphyaxiation. b. Turbinates and bu in make up the composite head sample. c. Includes pancreas, spleen, stomach contents and uterus. d. Represents the area of the nose which protrudes into the smoke chamber. -63- 9 CTR C(]NTRRCTS 02832 11248079 CTR HN 04440" ~` .

(1 TABLE 36 The Percent Distribution of Four Smoke Constitueqts in the Internal Tissues of the Mouse after Exposure to Whole Cigarette Smokea CHEMICAL , TISSUEb 1 Lung Larynx Head Stomach Others 14C-Dotriacontane 69.0 0.4 6.6 19.6 5.0 14C-Nicotine 2.5 1.2 15.8 3.5 77.1 14C-Benzo(a)pyrene 24.0 4.0 ~ 13.3 13.7 45.0 3N-Catechol 15.6 3.4 29.9c 2.6 48.4d aBC3F1/Cum mice were exposed to smoke from either 2A1 or 2R1 cigarettes using the Walton Hori- zontal Smoking Machine. Exposure conditions were 10% smoke concentration, 30 seconds•smoke alternating with 30 seconds of air per minute, separated over a period of 10 minutes. Mice were sacrificed immediately by C02 asphyxiation. 'bTissues were analyzed by solubilization.procedures at ORNL for dotriacontane, nicotine and benzo.{a)pyrene, and with a Tissue OxidizeratMA for catechol. cIncludes turbinates, nose and brain. dIncludes all other tissues except for the gastrointestinal tract.

TABLE 37 DISPOSITION OF BC3F1/CUM MICE AFTER EXPOSURE TO HIGH LEVELS OF 2A1, 3A1, OR 2R1 CIGARETTE SMOKEa Number of Mice Original iti Di Weecs on est Cigarette Number of Type Mice on _ . , _ spos 4 8 12 16 20 24 28 32 36 40 426. 2A1 120 Scheduled Sacrifices 0 21 21 0 0 5 0 0 0 0 5 Other Deaths 7 5 6 3 2 3 3 1 5 1 0 Alive 113 87 60 57 55 47 44 43 38 37 32 J J 3A1 120 Scheduled Sacrifices 0 21 21 0 0 5 0 0 0 0 5 co sm Other Deaths 2 6 1 8 6 11 5 1 •2 1 0 ~ 0 Alive 118 91 69 61. 55 39 34 33 31 30 25 oD ~. ..J, 2R1 120 Scheduled Sacrifices 0 21 21 0 0 5 0 0 0 0 5 0 , Other Deaths. Alive 4 116 5 90 1 68 4 64 12 52 7 40 3 37 3 34 3 31 0 31 0 26 Sham 120 - Scheduled Sacrifices 0 .21 21 0 0 0 5 0 0 0 5 Other Deaths 4 3 2 0 5 0 2 2 1 1 2 Alive 116 92 69 69 64 59 57 55 54 53 46 , M .~ ~ ` . Shelf 50 Scheduled Sacrifices 0 8 8 0 0 2 0 0 0 0 5 Other Deaths 0 0 0 0 0 0 0 0- 0 0 0 ~ Ln : Alive 50 42 34 34 34 32 32 32 32 32 27 ~ a. Exposure conditions on the SEM II were 10% smoke concentration, 8 puffs cigarette, either 30/30 seconds 0 N smoke/air for 8 minutes (2A1 and 3A1 cigarettes or 20/40 seconds smoke/air for 8 minutes. Five such expo- sures with an 10 minutes of air between exposures, were given In the morning and five in the afternoon. ~ OD ~ (A) W 0 6-6 Sham exposed animals were treated the same but without smoke. b. Smoke and sham exposure stopped due to the initiation of CTR101B(CTR)18) which required all SEN II B and C machine time. Wr

TABLE 38 i Unscheduled (UDS) and Replicative (RDS) DNA Synthesis Induced In vitro In Lung Slices Obtained from BC3FI/Cum Mice Exposed to Smoke from 3 Reference Cigarettesa UDS ~ RDS 3N-DPM g DNAc ! 3-D M g DNA Cigarette type Weeks of Exposure Cumulative TPM depositedb (mg) Smoked- Sham- Smoked/Sham Smoked- Sham- Smoked-/Sham- 2A1 9 47•8 425 ± 65 412 t'~47 1.03 7907 1 811 1775 1 114 4.58 13 64.4 236 a 22 430 t 42 0.55 3284 3 132 1721 1 241 1.91 24 105.2 46o ± 32 620 ±103 0.74 8763 1 482 2433 1 259 3.60 41 214.4 438 ± 69 539 = 59 0.81 8688 11278 2618 : 343 3.32 ~ oh I 3A1 9 51.3 386 t 67 412 t 47 0.94 7216 : 550 1725 t 114 4.18 13 71.2 221 s 27 430 s 42 0.51 4747 t 134 1721 ± 241 2.76 24 116.2 571 ± 58 620 t103 0.92 8650 t 500 2433 t 259 3.56 41 231.6 397 1117 539 s 59 0.74 7192 11018 2618 : 343 2.75 2R1 9 .25.8 531 t 62 '412 s 47 1.29 3370 ± 276 1725 ± 114 1.95 13 36.4 435 t 33 430 t 42 1.01 4172 t 707 1721 s 241 2.42 24 66.6 592 t135 620 t103 0.95 4841 t 325 2433 3 259 1.99 41 134.2 532 t 81 539 * 59 0.99 5151 = 459 2618 t 343 1•97 : aAnlmals were exposed to 102 smoke and 8 puffs/cigarette for all three cigarette types. For 2A1 and 3A1 cigarettes, exposure time of 30 seconds per minute for 8 consecutive minutes were used. For 2R1 cigarette exposure times of 20 seconds per minute for 8 consecutive minutes were used. Five such exposures were given In the morning and repeated again In the afternoon. UDS and DNA replication assays were performed by Dr. R. Rasmussen. bDeposltlon determined from dosimetry results and dally smoke records. eUDS was determined after treatment of lung slices with methylmethane sulfonate (n the presence and absence of hydroxyurea. dDNA replication was determined from lung slices Incubated 3H-thymldine (3H-TdR).

TABLE 39 Lab_elling Index (LI) in vivo 0etermined by Autoradiography - of Lung Tis'3ue'from BC3F1/Cum Mice Following Exposure" to Smoke from 3 Reference Cigerettesa Weeks of Exposure Cigarette- type L1' (~) 9 2A1 0.80 3A1 0.97 2R1 0.29 Sham- 0.19 Shelf- 0.44 i3 2A1 0.82 3A1 0.89 2Rt 0.28 Sham- - Shelf- 0.39 ~Mice were Injected with %t00 1tCi 3H-TdR immediately after treatment and sacrificed 30-60 mjnutes later. LI is percent... _ _ of 3H-TdR labeted cell,s divided ,by ch4 Ro.tal number of cells ~. _ counted x 100X• Approx(mately, 2.500 cells from 18-20 randomly ^ selected f(eids were counted. ` -67- CTR CONTRRCTS 026333 11248083 C TR HN 0 44 4, 11

TABLE 40 A QUALITATIVE ASSESSMENT OF TIIE DEGREE OF LI OBSERVED IN VARIOUS TISSUES OF MICE FOLLOWING A 13-WEEK EXPOSURE TO 2A1, 3A1 AND 2R1 CIGARETTE SMOKE AND IN SNAM- AND StiELF-CONTROLS.a TISSUE Intestlne Exposure Conditions Lungr' Trachea Bladder Crypc V1111 Liver Kidneyb Spleen 2A1 ++ + + +++ + + + +++ ; 3A1 ++ + + +++ + + + +++ : ~ n w 2R1 ++ + N.D. t++ + + + +++ ~ "'{ Shelf ++ + + +++ + + + +++ ~ n Machine + + + +++ ++ + + +++ a. The assessment was made on the basis of examination of a minimum of 5 fileds per section at a awgnlfleatton of 400x per field. ~ b. Most of the label was 1n the covtex c. += Lightly labelled = < 5 labelled cells/field ++ - Moderately labelled = 5-10 labelled cells/field +++ - Heavily labelled => 10 labelled cells/field

TABLE 41 • Kinetics of the Plaque Forming Cell (PFC) Response Strain of mice Days post SRBC Injection Number of PFC/106, spleen cellsa 8C3F1/Cum 3 42 BC3F1/Cum 5 1787 BC3F1/Cum 7 13 BALB/c Cum 3 21 BALB/c Cum 5 1553 BALB/c Cum 7 12. aAverage number of PFC's from five individual animals per group. -69- CTR CONTRRCTS 028335 11248085 C TR I"IN 0444 13

TABLE 42 Effect of Cyclophosphamide on the PFC Response of BC3FI/Cum Micea Age of mice Dose of Cyclophosphamide (mg/kg body weight) Number gf PFC/10 spleen cells 22 weeks• 0 1554 22 weeks 75 mg/kg 360 22 weeks 250 mg/kg 30 59 weeks 0 1379 59 weeks 75 mg/kg 307 59 weeks 250 mg/kg 75 aAnimals were given cyclophosphamide ( lp ) 1 day prior to the administration of SRBC. PFC assay performed 5 days post SRBC infection. c -70- CTR CONTRRCTS 028336 11248086 4.J' I R / I 1 1 ir1' 6)• 4-1, 14

TABLE 43 Effects of Acute Cigarette Smoke Exposure on the PFC Response.of BC3F1/Cum micea Strain of mice Treatment Group PRC/106 Expt I Spleen cells Expt Ii BALB/c Cum Shelf 1553 1211 Sham 1504 1275 2R1 Smoke 1848 1222 BC3F1/Cum Shelf 2156 1727 Sham 1933 2064 2R1 Smoke 2363 1810 aAnimals were exposed to 2R1 cigarettes for 5 days prior to SRBC infection and 5 days post SRBC injection. The exposure regimen was 4 cigarettes/day; 8 puffs/cigarette; 20 sec/puff. cTR caNTRRCTS 028337 11248087 CTR IIN 04•441ED ~

TABLE 44 Effects of Chronic Cigarette Smoke Exposure on the PFC Response of BC3F1/Cum Micea PFC/106 Spleen Cells Treatment Experiment I Experiment II Shelf 1348 1840 Sham 2231 1980 2R1 Smoke 2199 2340 aAnimals were exposed to 2R1 cigarettes on the SEM II machine for 52 weeks prior to immunization, and 5 days post immunization. -72- CTR coNi"RRCTS 028338 11248088 CI / 7 I pI I 1 'M' / f I/ I• M W

TABLE 45 Determination of DNA Elution Rate Constants for MMS-Treated Pulmonary Cellsa,b• L Treatment Animal # Estimated k Value X DNA Remaining on filter at 1.0 hour Control 1 0.150 86 2 0.223 80 3 0.274 76 MMSc 1 1.34 26 2 2.30 . 10 3 2.52 8 a Cells were isolated from pulmonary tissue frosn BC3F1/Cum female mice, 20 weeks of age. bLung cells were isolated according to procedures given in Progress Report Grant 11241, 1980. cMMS (120 mg/kg) was given 5 hours prior to isolation of lung cells. -73- cTR caNTRRCTS 028339 11248089 CT~'~ t-IN 044-4 17, " .

TABLE 46 THE NUMBER OF SCE/METAPHASE CHROMOSOME IN BONE MARROW CELLS FROM BC3F1/CUM MICE EXPOSED TO 3A1 CIGARETTE SMOKEa. (I reatment Number of Metaphase Chromosomes Counted Number of SCE/Metaphase Chromosome Number of SCE/Chromosome Sham-exposedb 50 4.7 0.11 ( 8% > IOSCE/metaphase) Smoke-exposedc 25 8.4 0.21 (40% > 10SCE/metaphase) a. Exposure conditions as described for CTR 1016.and discussed in the text. b. Results from two animals, ti 25 metaphase chromosomes analyzed from each. c. Results from two animals, ti12-13 metaphase chromosomes analyzed from each. f -74- CTR CQNTRRCTS 02e340 11248090 CTR t-IN 044418

TABLE 47 Relative Lu'rg=ileights of BC3F1/Cum Female Mice After Exposure to Smoke from 3 Reference Cigarettesa Relative Lung Weights Weeks of Smoke Exposure Hours after last exposure 3A1 Cigarette 2R1 2A1 9 3 1.41 1.07 1.41 .6 1.32 1.01 1.36 9 1.26 1.10 1.37 13 3 1.59 1.05 1.48 6 1.48 .97 1.41 9 1.47 1.08 1.56 aExposure conditions were as described under CTR-96. Relative lung weight - lun weiaht/bod wie ht of imoke-exoosed ung weight/body weight of sham-exposed (I -75- CTR caNTRRCTS 028341 11248091 C TR NVI 0 44 4 1 c.)

TABLE 48 ,Status of the C57BL/6J - DBA/2J Recombinant Inbred Lines Strain Designation AHH Number of Mice Induciblea Availableb 1 12 + 17 males, 11 females i 25 + - , 5 females i 27 + 2 males, 2 females i 29 + 8 males, 11 females i 21 - 3 males, 1 24 - 29 males, 21 females aAHH inducibility was determined at Jackson Labs by the DMBA skin ulceration test. b Strains which did not breed successfully: i1, i15, 023, and #24. C -76- 'CTR CC1NTRRCTS 026342 11248092 CTFZ I-IN 04'44220

TABLE 49 AEROSOL CONCENTRATION DETER,4INED AT VARIOUS SOLUTION CONCENTRATIONS Chemical Solution Concentration (percent, W,V) Calculated Aerosol Concentration '(U9/1)a Recorder Values (MV)b Phenolphthalein 0.2 66 ± 1 0.2 0.4 138 ! 1 0.4 0.6 245 ± 5 1.0 0.0 323 ± 4 1.5 1.0 265 *- 100 2.1 Catechol 0.2 20 * 4 0.2 0.4 75 = 5 0.4 0.6 165 = 5 0.7 0.8 247 t 7 )..1 1.0 320 t 10 1.4 aAerosols were sampled at 100 mi/minute for 3 minutes, collected onto Cambridge filter pads, chemicals eluted from the pads and concentration determined by UV absorbance. blalues determined from direct response of optiael sensor during generation of aerosol. CTR CaNTRACT5 416343 11248093 CTR 111q 0444' 2-1

1 ~ z M W y 60 50 40 30 20 10 0 / FIGItRE I MEAN BODY NEIGH, JURING CHRONIC 2R1 CIGARETTE SMOKE (0) AND SHAM (*) EXPOSURE AND MATCHED SHELF CONTROL (0) BC3F1/CUM FEMALE MICE. T i 20 40 , WEEKS ON TEST I -L 60 80 i

FIGURE 2 PERCENT SURVIVAL IN 8C3F1/CUM MALES AND FEMALES AFTER CHRONIC "CONTINUOUS" EXPOSURE TO 2R1 CIGARETTE SMOKE 100 ~ 80 ~ ~ A ~ ~ v 60 o " ~ '.'° cfl ~ ! 1 ~ ~ i 40 ~ ~ ~ 70• i ~ T ...~u f ~ 20 ~ ~ L ~ N ! ~ : (A . ~ ~ .~ -p Ul1 2R1 Smoke d 2R1 Smoke 9 • --r 10 20 30 40 50 60 70 80 Weeks After First Exposure C-117 9/13/80

FIGURE 3 CHROMATOGRAM OF NICOTINE IN SMOKE-EXTRACTED IN CAMBRIDGE FILTER PAD I I t t 32 28 24 , r...~ j . 20 16 1I2 I 4 0 (M1n.) . r _ CTR CQNTRACTS 028346 11248096 CTR NN 04,~'~4,24,

/ FIGURE 4 CHROMATdGRAM OF NICOTINE tN SMOKE-EXTRACTED FROM GRAB SAMPLE , C: L T-"~ ; 20 16 1~2 8 I 0 -81- (Min.) CTR CQNTRRCTS Q2634?' 11248097 CTR VIN 04442E5

9- 7J rr FIGURE 5 FLAME IONIIJITION DETECTION OF YAkIOUS NICOTINE CONCENTRATIONS • 0 4- 3- S- • • u l ` I .l .2 .3 ~ .4 .5 .6 .7 .8 .9 1.0 ' Af4T. NICOTINE INJECTED (m9) CTR CaNTRACTS 028348 11248098 CTR HN 04,442G

/ FIGURE 6 ._ pt7A1tTITATION OF NICOTINE ON CAMBRIDGE FIL,TER PADS USING 3-METHYLINOOLE AS•INTERNAL STANDARD 9- • 8- 7- 6- r- E ~ ~ a ~ 5- 0 V .- z 0 4-t 3-~ 2-j 0 1-i • L • ~0 20 30 40 50 60 70 Peak area NICOTINE RATIO - ^-- , ---- . r CTR coNTRacTS 028349 11248099 CTR Vff-l 0444,27

i 50 FIGURE 7 TPM IN LUNG, TURBINATES, LARYNX AND TRACHEA AS A FUNCTION OF TIME AFTER INHALATION OF 3H-CATECHOL LUNG TURBINATES • . v a ~- 40 30-i LARYNX 0 0 0 TTuQ /uI-\ 0 10 CTR CaHTRRCTS 026350 11248100 CTR- IwIN ~".44.42'~

FIGURE 8' OPM PER ML OF BL000 AS A FUNCTION OF TIME AFTER INHALATION OF 3H-CATECHOL • 11 0 2 8 10 12 14 :CTR CONTRRCTS 028351 11248101 CTR NN 04' `.', 4. 29

FIGURE 9 OPM PER WHOLE ORGAN OF LIVER, SPLEEN AND PANCREAS AS A FUNCTION OF TIME AFTER INHALATION OF 3H-CATECHOL LIVER SPLEEN PANCREAS ° C., 3 1 Q O\ 2 4 • 6 8 10 12 -TIME (Min) CTR CONTRACTS 028352 11248102 CTR W"'1 04-4`-'~• 30

FIGURE 10 DPM PER WHOLE ORGAN OF KIDNEY AND BLADDER AS A FUNCTION OF TIME AFTER INHALATION OF 3H-CATECHOL KIDNEY ' f BLADDER° - " I °.- 8~ a 0 2 4 6 8 10 12 CTR coNTRRcTS 028353 11248103 CTR NN 0444.31

FIGURE 1] 2- I DPM PER WHOLE ORGAN IN BRAIN AND•HEART AS A FUNCTION OF TIME AFTER INHALATION OF 3H-CATECHOL BRAIN o~o HEART , 6--0 2 4 6 R ~q,~ e! ~ -, i , 8 10 12 TIME (M1n) ` -88- CTR coNTRaCTS 028354 11248104 . CTR I-If°I 044"4,...° 2

FIGURE 62 CHhOROFORM-METHANOL.SOLUBLE AND INSOLUBLE RADIOACTIVITY URINE OF MICE EXPOSED TO 3H-CAT-2R1 CIGARETTE SMOKF ORGANIC.SOLYENT. SOLUBIE _ 3H LABEL ORGANIC SOLVENT „_, IN504U4E*,.__ ' 3H LABEL 5.7% - 2 94.3% Ij' 11 { 1 l ~ I ~ I 1 1 1 I ~ ~ I i ~ i ~ ~ , ~ , , ~ ~ ~ , CTR coNTRacTS 028355 11248105 {r.f' I R I l I ! 044,433

PROGRESS REPORT Contract CTR-0030 9/1/79 - 8/31/80 Fiaure 13 (a). HPLC chromatogram of reference CAT. A Waters Associates modular HPLC system was used for these.studies under the following conditions: A 3.9 rom x 3 cm micro porasil column operated at ambient temperature and a flow rate of 1.5 m./min..,an isocratic solvent system of ethylacetate: hexane (60:40, v/v), fractions of 1.5 ml were collected each minute and were monitored by both UV absorption at 254/nm and liquid scint- illation counting. Figure 13 (b). HPLC chromatogram of an ethyl acetate soluble urinary extract from SC3F1/Cum mice exposed to 3H-CAT-2R1 cigarette smoke. -90- CTR CCNTRRCTS 028356 11248106 C! l ! ! ~) ) ) (D44,4134

FIGURE 13 TIME(MIN) _o1_ a 1 2 1 I I t 1-__I 1-- _ 1 L-t 4 5 6 7 8 9 10 1 1 12 13 CTf2 CONTRRCTS 026357 11248107 CTR HN Cf 4- 44,-35

PROGRESS REPORT Contract CTR-0030 9/1/79 - 8/31/80 Fi ure 14 (a . HPLC chromatocraph of a sample of 3H-CAT-2R1 cigarette smoke generated during exposure of BC3F1/Cum mice on the 'dHSH. Conditions for the HPLC analyses are given in Figure 13. Peak is identified by UV absorbance (solid lines) and radioactivity (hatched bars). Figure 14 (b). HPLC chromatograph of 8-glucuronidase/aryl sulfatase treated urine from BC3F1/Cum mice after exposure to 3H-CAT-2R1 cigarette smoke. Urine (5 ml) from several mice was collected, adjusted to pH 5 with sodium acetate and glacial acetic acid.. A solution of 8-glucuronidase containing aryl sulfatase (10,000 units, P.L. Biochemicals; Inc.) was incubated with the urine at 370C for 36 hours. Urine samples were extracted with 3 volumes of ethyl acetate at 0°C. The extracts were dried under a stream of nitrogen and stored at -60oC until analyzed by HPLC as described in Figure 13. \ cTR CoNTRRCTS 028358 11248108 C 7 / -'! H! 1 0 4 ) -4 4 43 6

RESPONSE UV 254nrn Icv N j ® V9 ® ® 0 6; ® ." il ® ` a .V N WdCI~OI X l ® -93- -4,0 CTR CQNTRRCTS 028359 11248109 CTR HN 044'437,

PROGRESS REPORT Contract CTR-0030 9/1/79 - 8/31/80 Fi ug re 15. HPLC chromatograph of 9-glucuronidase/sulfatase enzyme treated urinary extract from shelf control BC3F1/Cum female mice (CTR-1Q1B). -94- CTR CaNTf2RCT5 026360 11248110 CI I l VI • I y/' -4441r+' •r^

0 n o. .• 00 r- M .• .. .~. .~ C r ~ 0 v wub9t A (1 .~. ., .r h ir CTR CaNTRRCTS 028361 11248111 CTR IMIN 0-1-4~-~39

PROGRESS REPORT Contract CTR-0030 9/1/79 - 8/31/80 Fiaure 16. HPLC chromatograph, of B-glucuronidase/sulfatase enzyme treatea urinary extract from sham exposed BC3F1/Cum female mice (CTR-141B). Q -96- CTR CQHTRRCTS 028362 11248112 CTR MN 044440

CTR COHT[2RCT5 028363 11248113 CTR HN 04,444, 1

PROGRESS REPORT Contract CTR-0030 9/1/79 - 8/31/80 Fi re HPLC chromatograph of B-glucuronidase/sulfatase enzyme treated urinary extract from 2R1 cigarette smoke exposed BC3F1/Cum femalefnice (CTR-101B). -98- CTR CCNTRRCTS 026364 11248114 . CTR HN 04.4`4. 42

~-.,. J7 V ~~ ~'-..0 NI-I N,L 3 SLL8bZLL 81~~~iN03 2~1~ S968Zo -66- RESPONSE UV25dnm

FIGURE 18 INDUCTION OF PULMONARY AHH IN BC3F1/CU?/ MICE BY CIGARETTE SMOKE NINE MONTH SMOKE EXPOSURE 2A1 0 0.25 O 5 A1 • e 0 : 0 0.20 O • . 0 O o 2 R1 ~ ~. ~ I E O O 0 ~ ~~~ ~ ..~ ~.. ~.. - .~. U ~ ~ 0 e C  ~ `~ a E 0.15 ----E-- E O 0 : . i. ~ 0 ° OJO a. • c 6 m S O P7 - 0.05 0 E c . •.. ~:. ., ~- 0 2A1 M.C 2A1 2R1 2R1 OA1 3A1 LtC 2A1 2R1 ~A1 M.C 3 HOUR 9 HOUR 6 HOUR C. t -100- CTR CQNTRRCTS 026366 11248116 CTTR t-IN 0444-44

PROGRESS REPORT Contract CTR-0030 9/1/79 - 8/31/80 Ficure 19. Rate of atresia in 5 inbred strains and one hybrid strain of mouse. L -101- .cTE2 coNTRacTS o2s3s "1" 11248117 CTR IIN 04,4445

FIGURE 19 Q• ~ > I 4 3 2 1 0 '4 3 1 0 4 2 1 4 3 2 0 4 3 2 .1 0 4 C57 BL/6N . Strain 3 T 2. 1 0 10 20 30 40 A(3 E ( weeks l CTR CONTRRC;S 028368 11248118 I . T a Strain C3H/Anf Cum Strain BC3F1 /Cum € ~ ~ § C. S~A~~ Straln ~ Stnln Strain D I r I Tq 1 T Y i L ~ ~ Stnln Strain L I I L ~ T p ~' I. 57BL/6 Cum BA /2J DBA/2N CTR HN 0444. 46

PROGRESS REPORT Contract CTR-0030 9/1/79 - 8/31/80 Fiaure 20. Rate of atresia in BC3F1/Cum mice exposed to 2R1 cigarette smoke (as described under CTR-101A) or sham exposed. . -103- CTR COHTRRCTS 028369 11248119 CTR VIN 04444f

8Y f" Vb,0 NI-I 4..j 1t../ OZ68tiZll U-£8Zo 510U&N03 &0 -tiot- NO OF OOCYTES / /OVARY I X10 31 O W 0 A O tn O w N O I .)

PROGRESS REPORT Contract CTR-0030 9/1/79 - 8/31/80 Fi ure 21. Alkaline elution of DNA from mouse pulmonary cells following long term smoke or sham exposure. Animalc were exposed to smoke daily (5 days/week) from 2A1 cigarettes for 40 weeks. t1MS (120 mg/kg) was given IP ti 3 hours prior to sacrifice to aged-matched positive control animals. See text for further details. -105- CTR caNTRaCTS a2Q3?` 1 11248121 /, /• W' C/ R I II 4I 04I.

I 100 90 80 70 60 50 40 30 20 I- 1 FIGIIRE ?1 \ \ ~ SBAIi EZ°OSED SMOKE EXPOSr.~~. l4LS T3SdTID ` 12 24 36 48 1 , 1 ~ .CTR CaNTRRCTS 028372 11248122 CTR I-IN 044-4,50

PROGRESS REPORT Contract CTR-0030 9/1/79 - 8/31/80 Figure 22. Alkaline elution of DNA from mouse pulmonary tissue following thronic smoke or sham exposure plus either BaP-7.8-diol (180 vg) or Bap (1.2 mg). Animals were exposed to smoke daily (5 days/week) for 40 weeks and sacrificed 7 hours after the initiation of the last exposure. BaP- 7,8-diol or BaP treatment was given ti3 hours prior to sacrifice to age- matched positive control animals. -107- CTR caNTRacTS 028373 11248123 CTR HN 04-44'51

FIGURE 22 • \0 12 24 36 48 60 CTR CCNTE2RCT5 028374 11248124 CTR HN 044''°'~' 52 .

FTGURE 23 , EFFECT OF 2R1 CIGARETTE SMOKE L p W p • N CI 0 p S d O K d W W W Y Y 0 N ~ •4 00 i<- C-. N Q C U Q 0 ON ODC LEVELS INLUNGS 0 .. 24 DAYS 44 DAYS 150 DAYS - L;,,l,1J.~ t 1 r r t r r.'. t 1 t i t t r.~ 1 t t t r~.: ~ 1 t r t 1 t.~ 2 4 6 810 24 2 4 6 810 24 ~ 4 6¢10' 24' 2 4 ~ 810 ;4 2 4 6 8 10 24 Hours. After Last Exposure to Clgaro-tte Smoke CTR CONTRRCTS 028375 11248125 CTR HN 0'4,44~,.'°.t...,"

PROGRESS REPORT Contract CTR-0030 9/1/79 - 8/31/80 Fi ure 24. ODC induction by 2A1, 3A1 and 2R1 cigarette smoke 9 weeks smoke exposure).' -110- CTR CQNTRACT S 028376 11248126 CTR f-It',l 04`4-4,S4

FIGURE 24 ` cv z ~ ~ z ~ ~ 0 J 4 W N O C. sc W / c W Vf O a K W LLA M O ~ f d N N ~ Z o-r t..l 0 Z V C O 1~.. O N.. O ~- O ~ 1-- h- 2 ~ ... ~ > F-- u a u ~ Y- u a U ~ ~ ~ ~.1 W d V W I ~ N N 0 i ..... .... .... . ... 1 s 0 0 HARVEST PERIOD (HOURS POST LAST EXPOSURE) . CTR CONTRRCTS 028377 11248127 CTR MN 0444!5t.:5

PROGRESS REPORT Contract CTR-0030 9/1/79 - 8/31/80 Fi ure 25. ODC induction by 2A1, 3A1 and 2R1 cigarette smoke 13 weeks smoke exposure). r C. -112- CTR caNTRRCTS a2B3?'a 11248128 CTR HN 044456

FIGURE 25 z .. n 0 ~ 0 ,- s ~- ~ 5 4 0 3A1 ~p 2A1 zn1 . .e.~- ~ I ._........ .o- ~l __+.f~~' ........... .....................Sf......... 0 ` ' t t ~T J~ 3 6 9 24 ~ HARVEST PERIOD (HOURS POST LAST EXPOSURE) L i113- CTR CCNTRRCTS 028375 11248129 1 CTR HN 044457<

PROGRESS REPORT Contract CTR-0030 9/1/79 - 8/31/80 Fi ure 26. ODC induction by 2A1, 3A1 and 2R1 cigarette smoke 1 weeks smoke exposure). -114-• CTR C0NTRRCTS 028380 11248130 CTR PIP"1 0+44bB

FIGURE 26 s r t7 C7 ~ Z v O W N ~ J O O n- h 4 O w x W 4 x 1 W • Y S N W t vi Z ~ .T- r. rr .-. G O W 8 O U. O a- O Z Y .. .r ~ .~ > > ., f- ~..e d .. c~.~ a c.~ u 1 r ........ . ... ~ M u- ~..~ W CU W C- N G. • N \ ~ `Q 2R1 ... . .. ...... .. .... ... 2A1 I 1 Z a e a C HARVEST PERIOD (HOURS POST LAST EXPOSURE) CTR CaNTRRCTS 028381 11248131 CTR MN 04• 44• 59

PROGRESS REPORT Contract CTR-0030 9/1/79 - 8/31/80 Fiaure 27. Seven stage Cascade Impactor. The following specifications are given for 1 liter/minute: Stage Number Jet Diameter (cm) Effective Cutoff Diameter (wM) • Nose 0.2362 4.5 1 0.1778 3.0 2 0.1422 2.1 3 0.1168 1.5 4 0.1889 1.0 5 0.0686 0.7 6 0.0406 0.3 L -116- CTR CONTRACTS Q2g382 11248132 Ci / V VIN 0/' ! 4 )' 60

FIGURE 27 Aerosol Entry g H ® m ^ Vaccuurs Applied L -117- CTR CaNTRRCTS 028383 11248133 CTR PIN 044461

PROGRESS REPORT Contract CTR-0030 9/1/80 - 8/31/80 ` > PUBLICATIONS 1. Oinowitz, M., Nims, R.•Bhooshan, B., Kouri, R.E., and Henry C.J. Induction of Ornithine Oecarboxylase (OOC) by 12-0-Tetradecanoyl- phorobol-13-acetate (TPA) in Pulmonary Tissue: A Model System for Tumor Promotion in Mouse Lungs. Proc. Am. Assoc. Cancer Res. 21: 31, 1980. 2. Henry, C.J., Avery, M.D., Dansie,.D.R., Lopez. A., Breth, L.A., Billups, L.H., and Kouri, R.E. The Effect of Exposure to Whole Cigarette smoke on 3-Methylcholanthrene (MCA) Induced Lung Tumors in BC3F1/Cum Mice. Proc. Am. Assoc. Cancer Res. 21: 126, 1980. 3. Henry, C.J., Billups, L.H., Oinowitz, M., Rasmussen,. R.E., Avery, M.D. Dansie, D.R., Lopez, A., Breth, L.A., Mullinax, H.D. and Kouri, R.E. The Effect of.Exposure to Whola Cigarette.Smoke on Pulmonary Mixed Function Oxidase, Ornithine Oecarboxylase, DNA Repair Capacity and on 3-Methylcholanthrene (MCA) Induced Lung Tumors in BC3F1/Cum Mice. Symposium on Cocarcinogenesis and Biological Effects of Tumor Promoters, Castle of Elmau, Klais/Bavaria, Federal Republic of Germany, p 56, 1980. 4. Henry, C.J., Lopez. A., Dansie, O.R., Avery, M.D., Whitmire, C.E.. Caton, J.E., Stokely, J.R., Guerin, M.R., Curren, R.D., Kouri, R,E. Distribution and Clearance of Three Cigarette Smoke Constituents, Dotriacontane (OTC), Nicotine (NIC), and Benzo(a)pyrene (BP), after Exposure of Mice to Whole Cigarette Smoke. Proc. Soc. of Tox., 1981. Distribution of Chemical Carcinogens. In: Genetic Differences in Chemical Carcinogenesis. R.E. Kouri, ed., CRC Press, Boca Raton, Fla., pp. 1-20, 1980. , 6. Kouri, R.E., Schechtman, L.M., and Nebert, O.W. 'Genetic Control of Carcinogen Metabolism. In: Genetic Differences in Chemical Carcino- genesis. R.E. Kouri, ed., CRC Press, Boca Raton, Fla., pp. 21-66, 1980. 7. Schechtman, L.M., Henry, C.J. and Kouri, R.E. Exposure, Uptake and 5. Kouri, R.E., Rude, T„ Whitmire, C.E., Henry, C.J., Sass, 8., Billups, L.H. Correlation of.Inducibility of Aryl Hydrocarbon Hydroxylase with sus- ceptibility to 3-Methylcholanthrene Induced Lung Cancers. Cancer Letters 9: 277-284, 1980: 8. Henry, C.J., Whitmire, C.E., Lopez., A., Cansie, D.R., Avery, M.D., Curren, R.D., Caton, J.E., Stokely, J.R., Holmberg, R.W., Guerin, M.R., Kouri, R.E. The Dosimetry and Distribution of Whole Cigarette Smoke Particulates in Inbred Strains of Mice. Comparison of a Large Exposure Machine•(SEN) with a Small Capacity Smoke Exposure Machine (Walton). Hanford Symposium, Richland, WA (in press) 1980. 9. Kouri, R.E., McKinney, C.E., and Henry, T.J. Genetic Control of Breast Cancer Susceptibility in Animala.. In: Genetics and Breast Cancer, H.T. Lynch, ed., Van Nostrand Reinhold Co., New York, NY (in press) .1980. CTR CONTRRCTS 028384 11248134 C TR VIN 044'462

rnvunw o Rcrunt Contract CTR-0030 9/1/79 - 8/31/80 10. Rasmussen, R.E., Boyd, C.J:, Dansie, D.R., Knuri, R.E., Henry, C.J. DNA Replication and Unscheduled DNA Synthesis ih Lungs of Mice Exposed to Cigarette Smoke. Can. Research (submitted) 1980. 11, Kouri, R.E., Wood, A.W., Levin, 'd.,. Rude, T.H., Yagi, H., Mah, H.D. and Jerina, D.M., Conney, A.H. Carcinogenicity of Benzo(a)pyrene and Thirteen of its Derivatives in C3H/f Cum Mice. J. Natl. Cancer Inst. 64: 617-623, 1980. L CTR CONTRACTS 026365 11248135 CTR IIN 0 4-4•463

I r { CTR CoHTRRCTS 028386 11248136 CTR IaIN 04446 4