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Final Report 1-1729.005 Treatment: Animals were ear tagged and randomly assigned to 4 groups of 12 animals each using the stratified sequence randomization technique. Groups of mice (12 per group) were exposed to smoke from two test cigarettes and the Reference cigarette using the following defined exposure regimen on the Walton Smoking Machine (Henry et al, 1980, Guerin et al, 1979): 10% (v/v) smoke concentration (-4 ug TPM/ml) 30 seconds smoke/30 seconds air/minute 7 puffs/exposure 9 exposures/day 63 puffs/day 7-8 minute rest periods between exposures 31.5 minutes total time/day exposed to smoke -130 minute/day exposure period This daily (5 days/week) exposure regimen over 6 weeks resulted in: 270 total exposures 1890 total puffs 30 days exposed Exposure controls were used which consisted of age-matched mice loaded onto the smoking machine, treated exactly as the test animals, the machines set and run but without cigarettes. This sham-control group was also treated for approximately -130 minutes. The following designates the disposition of the animals: Group Description 1 Reference Cigarette 2 Test Cigarette 1 (D6) 3 Test Cigarette 2 (D10) 4 Sham-Exposed Control Animals were weighed 24 hours prior to the first exposure and weekly thereafter during treatment. Blood carboxyhemoglobin (COHb) levels were monitored in 2 mice/group at weekly intervals. 11

Final Report I-1729.005 Animals were observed during the test period for any unusual persistent conditions. Sacrifices: Animals were weighed to the nearest tenth of a gram prior to necropsy. Animals were anesthetized with Metofane. Blood samples from 3 animals/group at 3 weeks and 6 weeks were collected for clinical chemistry and hematology. Animals were killed by exsanguination. Necropsy: The necropsy is defined as external examination, including body orifices, and gross examination and fixation in 10% formalin of all of the following tissues and organs: Spinal cord (thoracic) Brain Eye (R) Pituitary Trachea Larynx Esophagus Adrenals (R & L) Thyroids (R & L) (with parathyroids) Lungs & Bronchi (R & L) Kidneys (R & L) Liver Spleen Thymus Any gross lesions Salivary gland (R) Mandibular lymph node Mesenteric lymph node Sternum (bone marrow) Zymbal's gland (R) Heart Thigh muscle (R) Sciatic nerve (R) Stomach Small intestine Pancreas Large intestine Turbinates Sk in Urinary bladder Females (R) = Right (L) = Left Ovaries (R & L) Uterus Mammary gland Gross observations made during necropsy were recorded on an individual animal necropsy form containing the animal identification number, group number, compound administered, date of death, necropsy date, signature block, study number, and other pertinent information. In addition, all tissues taken at necropsy were recorded on the necropsy form. Jars containing animal tissues were labelled with species, animal number, study number, date of necropsy and technician initials. 12

Final Report I-1729.005 The trachea was transected midway between lung and tongue, and the heart, thymus and extraneous tissues were carefully removed. The lung was weighed to the nearest milligram, and perfused with fixative via the trachea to approximate the normal expanded size. Histology: 0 Lungs - The preferred planes of section for rodents' lungs are illustrated in Dungworth et al, 1976. These were sections from the hilus along the axis of major airways for the cranial, middle and caudal lobes of the right lung and longitudinally through the main stem bronchus of the left lobe. Sections in this plane for each lobe revealed bronchial and acinar orientations of lesions much more readily. All of these blocks can be sectioned whole for microscopic examination. 0 Larynx - The larynx was transected from the trachea. Three transverse sections were taken from the larynx with the thyroid and parathyroids attached, one from the distal portion (vocal folds and caudal cricoid region), one from the middle portion (arytenoid projections and ventral pouch) and one from the proximal region (epiglottis). This will enable all major regions to be assessed. 0 Trachea and bronchial bifurcation - A block containing a longitudinal section of distal trachea (distal to larynx) was prepared to include the bronchial bifurcation. The bifurcation is a site sensitive to injury in inhalation studies because it is the major location where air flow changes direction and particles are likely to impinge. Only the respiratory tract organs were processed for histopathology at this time. Lung tissues were embedded in glycol methacrylate and sectioned at 2.5 microns. The remaining fixed tissues are being held until data are analyzed. Final disposition will be determined after consultation with the Sponsor. Blood Collections: Blood collections were made from retro-orbital sinus puncture or from anesthetized animals by cardiac puncture after access was gained by incision. Approximately 0.3 ml blood was used for hematology with potassium ethylenediaminetetraacetate (K-EDTA). K- EDTA was used as the anticoagulant since it does not adversely affect the shape of the red blood cells. Blood smears were prepared from a drop of fresh blood placed directly on a glass slide, spread, air dried and fixed in methanol. Blood for clinical chemistry was allowed to clot and serum separated from the cells. 13

Final Report I-1729.005 Clinical Chemistry: The following clinical Glucose (serum) Blood Urea nitrogen (BUN) Creatinine Cholesterol Triglycerides Calcium Protein, total (serum) A/G Ratio Hematology: The following hematological Segmented neutrophils count Monocyte counts Platelet cell counts Red blood cell counts (RBC) Basophil counts Hematocrit value Mitotic Counts: Alkaline Phosphatase Lactic dehydrogenase (LDH) SGOT SGPT Uric-acid (serum) Phosphorous Albumin tests were performed: Lymphocyte counts Eosinophil counts Reticulocyte counts White blood cell counts (WBC) Hemoglobin concentration For determination of mitotic indices in lung tissues, immediately after the last smoke or sham exposure, 3 animals/group were injected with 0.1 mg colchicine per 100 g body weight (Lamb and Reid, 1968). Four hours later, animals were anesthetized with Metofane and killed by exsanguination. Clinical chemistry and hematology were not determined on these animals. Necropsies were performed as described above and respiratory tissues examined microscopically. The mitotic activity of the bronchial epithelial cells was assessed. The number of nuclei of bronchial epithelium in mitosis was expressed per 12 diameters of a microscopic field using the 40x objective. Approximately 1000 cells were observed in each specimen. AHH Levels: For determination of AHH levels, 6 hours after the last smoke or sham exposure, 3 animals from each group were sacrificed by CO asphyxiation and lungs, liver, and kidneys dissected away from ?he carcass and weighed to the nearest 0.1 mg. Microsomes were prepared according to Burke and Mayer (1974) and stored at -70°C until assayed. AHH activity was determined by fluorometric chemistry tests were performed: 14

Final Report 1-1729.005 measurement of the 3-hydroxy-BaP (3-OH-BaP) formed according to Kouri et al. (1976). AHH units are expressed as pmole 3-OH-BaP formedTi-n7mg microsomal protein. Evaluation of Test Results The survival in each group is presented, as are any unusual observations during or after exposure over the 6 weeks. Blood COHb levels from 2 mice per group at weekly intervals are also presented. Individual animal weights 24 hours prior to exposure and at weekly intervals after exposure during treatment are presented for each group. Individual animal pathologies on the respiratory tract from 9 animals per group are presented, 3 animals/group after 3 weeks and 6 animals/group after 6 weeks). Total erythrocyte counts, total leukocyte counts, hematocrit, hemoglobin, differential leukocyte counts, blood urea nitrogen, blood glucose and other clinical chemistries from 3 animals/group sacrificed at 3 weeks and 6 weeks are presented. After 6 weeks of treatment, the levels of AHH in lung, liver, and kidney tissues from 3 animals/group and the Mitotic Index (MI) in lung tissue from 3 animals/group were determined. Statistical analyses of all the data presented were performed using BMDP-79 Biomedical Computer Programs, P-series (Dixon and Brown, 1979). Analysis of variance (ANOVA) was performed comparing the equality of means between groups and an F-statistic calculated. In addition, Levene's test for the homogeneity of variances was calculated, as were Welch and Brown-Forsythe statistics which assume unequal variances. . The data were entered as a matrix and the group means for each variable (column) compared, e.g. body weights of all animals on test for 7 days. Because the program only accepts data in matrix form, values that are not recorded must be entered into the computer as "missing" as opposed to zero and are omitted from analysis. Significant differences between individual means were determined by calculating a least significant range using a Studentized Range Test modified for unequal sample sizes (Sokal & Rohlf, 1969). Criteria For Determination of Valid Tests: None of the sham-exposed animals should die or show signs of toxicity. COHb levels in the smoke-exposed groups should be statistically (p < 0.05) increased relative to the sham-exposed controls. 15

Final Report I-1729.005 VII. RESULTS AND DISCUSSION A. General Toxicology B6C3F1 mice were quarantined for at least 21 days prior to use, during which time their health status was evaluated by observation. The animals were 12 weeks old and were judged to be healthy prior to the initiation of this study. Groups of animals were exposed "nose-only" to whole cigarette smoke generated on a Walton Horizontal Smoking Machine. Test cigarettes D6 and D10, and the Reference cigarette, were evaluated together using the 7 puff Reference Smoke Dose 1 (7 puffs, RSD1). This change from 8 to 7 puffs was adopted after consultation with the Sponsor because the results of the acute toxicity study (See Final Report, 1-1729.003) indicated that 7 puffs contained an amount of TPM that is equivalent to what has been found with other test cigarettes. Weekly determinations of TPM from the Reference and test cigarettes were taken and are presented in Section IV. Cigarette Characterization. The results indicate that the TPM varied only about 10-20% over a 6 week period and were within historical limits. The smoke-exposed groups were compared to a sham-exposed (no cigarette) control group. The Reference, D6, and D10 cigarettes had been previously evaluated using an 8 puff RSD1 regimen in an acute toxicity study. The results of that acute toxicity study suggested that a reasonably high dose of smoke could be given with good survival predicted in longer periods of exposure (see Final Report (1-1729.003) provided a change from 8 to 7 puffs was affected. Smoke and sham-exposures were initiated on March 24, 1983, and completed on May 4, 1983. Animals were treated 5 days/week for a total of 30 exposure days. One animal from Group 3 (#1173, test cigarette D10) died during exposure on Day 12 after 63 exposures, and one animal from Group 4 (#1187, sham-exposed) died during exposure on Day 19. This sham-exposed animal was found dead at the end of exposure and had not displayed any signs of struggling or toxicity. An autopsy did not reveal any overt cause of death. It is presumed that this animal died of accidental suffocation that was unrelated to the exposure. Aside from these two cases, the 3 animals/group scheduled for sacrifice at 3 weeks on test were sacrificed on schedule, with the remaining 6 animals/group sacrificed as scheduled after 6 weeks (Table GT1). Those animals sacrificed after 3 weeks received the scheduled 135 exposures and 945 puffs over 15 treatment days. While animals sacrificed after 6 weeks received the scheduled 270 exposures and 1890 puffs. Survival to the 7 puff, RSD1 regimen for the Reference and D6 cigarette smoke was 100% and 92% for smoke from cigarette D10. Survival to sham exposure was also 92% because of one accidental death. 16

Final Report I-1729.005 Animals were observed daily during and immediately after exposure for any unusual responses to treatment. No unusual responses were observed. Mice exposed to smoke were lethargic, ataxic, and felt hypothermic. There was tearing of the eyes, trembling, and shallow breathing observed after exposure. In addition, mice that had been exposed to smoke from test cigarette D10 were unable to walk and kept a prone position. Sham-exposed mice appeared alert and active after exposure, with the exception that occasional tearing of the eyes and rhinorrhagia was observed. The COHb levels from 2 mice per group determined at weekly intervals are presented in Table GT2. The COHb values for the smoke-exposed groups ranged from between 21.8 to 59.8% COHb. The smoke-exposed mice had significantly higher COHb levels than did sham-exposed controls (Studentized Range Test, p < 0.05; Table GT2). The individual animal weights 24 hours prior to the first treatment and at weekly intervals thereafter are presented for each group in Table GT3. Individual body weights for animals from each group are also presented in histogram form in Tables GT4 - GT10. Statistical descriptions for each group, as well as an Analysis of Variance, may also be found in Tables GT4 - GT10. The group means for each weekly interval are presented in Figure 1. All animal weights were relatively stable throughout the study and Analysis of Variance indicated that the body weights of the sham- and smoke-exposed groups were statistically the same (Analysis of Variance, p > 0.05). The lung weights of mice sacrificed after 6 weeks of treatment are presented in Table GT11. Lungs were dissected away from the thoracic viscera as described in the Methods, and weighed before perfusion with formalin fixative. The mean and standard deviations are presented for 3 animals/group. Analysis of Variance did not indicate any significant differences (p > 0.05). The AHH activities in the lung, liver, and kidneys of mice exposed to smoke for 6 weeks are presented in Table GT12. No increases were observed in the pulmonary or renal AHH activities of smoke-exposed mice. The AHH activity in the liver of smoke-exposed mice was increased above that in sham-exposed controls, but the increases were not statistically significant (ANOVA, p > 0.05). These findings do not compare well to results obtained in previous studies, which have always indicated induced AHH activity in the lungs of animals exposed to cigarette smoke. The level of enzyme activity is also not as high as had been obtained previously. This may be the result of the method used to isolate microsomal enzymes. Instead of ultracentrifugation, calcium chloride precipitation was used to pellet the microsomal fraction of the cell homogenates. This procedure was done because it is more rapid and does not require an ultracentrifuge. Preliminary results using a positive control of microsomes from 17

Final Report I-1729.005 animals treated with 3-methylcholanthrene had indicated that this method would be satisfactory for subcellular isolation of microsomes from smoke-exposed animals. However, the enzymes prepared in this way may be more susceptible to denaturation during freezing and thawing. As a result, calcium chloride precipitation will no longer be used to prepare enzyme fractions. Bronchial and tracheal epithelium were evaluated for changes in cellular proliferation after final smoke or sham treatment at 6 weeks. Twelve randomly selected fields from lung and 5 randomly selected fields from tracheal sections of 3 animals/group were examined at high magnification (400x) for the presence of mitotic figures. These fields represented approximately 1000 bronchial or tracheal epithelial cells, respectively. Very few mitotic figures were found in either the smoke-exposed or sham-exposed groups (less than an average of 4 and 1 mitotic figures/1000 bronchial and tracheal epithelial cells, respectively). These data suggest that increased numbers of mitotic figures could not be associated with this dose exposure regimen during a 4 hour colchicine treatment period. 18

Final Report I-1729.005 B. Clinical Chemistry and Hematology Clinical chemistry and hematology values for individual animals are presented in Tables CCH1-CCH4 for animals sacrificed after 3 weeks on test, and in Tables CCH28-CCH31 for animals sacrificed after 6 weeks on test. After 3 weeks on test, a few significant changes in the clinical chemistries were observed. Triglycerides were elevated in mice exposed to smoke from the Reference and D6 cigarettes as compared to sham-exposed controls (Studentized Range Test, p < 0.05; Table CCH7), but not in mice exposed to smoke from cigarette D10. However, inasmuch as the differences observed are only slightly beyond normal values, and there is no concomitant change in glucose or cholesterol levels, the results are not believed to be biologically significant. Serum creatinine and BUN were also elevated in all smoke exposed groups in comparison to the sham-exposed mice (Studentized Range Test, p < 0.05; Tables CCH8-CCH9). Elevation in both of these parameters is probably indicative of a possible decrease in glomerular filtration in the kidney. All the remaining clinical chemistries were within normal limits. Smoke-exposed mice also had significantly increased numbers of leukocytes (Studentized Range Test, p < 0.05; Table CCH2O). This appears to be a general response to smoke because the differential count of leukocytes did not indicate any abnormal cell populations. All the remaining hematology values were within normal limits. After 6 weeks on test, mice exposed to smoke from test cigarettes D6 and D10 had elevated triglyceride levels when compared to sham-exposed controls (Studentized Range Test, p < 0.05; Table CCH34). This was not the case in mice exposed to smoke from the Reference cigarette. Although similar results were observed after 3 weeks of exposure to smoke, the results do not appear to be indicative of any specific pathologic condition. As had been observed after 3 weeks of exposure, BUN was elevated in all smoke exposed groups (Studentized Range Test, p < 0.05; Table CCH36). An elevation in BUN without a concomitant change in creatinine may be the result of any number of 00 physiological effects and is not necessarily indicative of a m pathologic condition (Coles, 1980). Thus, the biological ~ significance, if any, of the increase in BUN is not known. ~ A significant decrease in the A/G Ratio was observed in mice W that had been exposed to smoke from test cigarette D6 when compared to animals that were exposed to the Reference cigarette and to sham-exposed controls (Studentized Range Test, p < 0.05; Table CCH40). No differences were found among the other groups. 19

Final Report I-1729.005 Since the differences observed were within normal limits, the results are not believed to be biologically significant. No differences in the hematology values of mice on test for 6 weeks were observed. 20