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I-172 5 . 007 VII. RESULTS AND DISCUSSION BC3F1/Cum mice were quarantined for 21 days prior to utilization, during which time their health status was evaluated by observation. The animals were judged to be healthy prior to initiation of this acute toxicity study. The animals were exposed "nose-only" over 14 days (5 days/- week) to whole cigarette smoke generated from the reference ciga- rette on a Walton Smoking Machine at approximately one-half the maximum tolerated dose for one day (r4TD-1 day). Groups of animals were then exposed for one day to five doses of smoke, varying in smoke concentration, number of exposures and puffs, length of time between exposures, and were compared to a sham-exposed (no cigarette) control group. Tables 1A-6A document the initial and final exposure time each day for rats on test in all six treatment groups. Exposures started on May 19, 1982, and continued daily, 5 days/week, (except for holidays and weekends) until June 3, 1982. Animals were not treated on May 31, 1982. For this study, the 14 day adaptation period is as follows: Day 14 is designated•--as the last day of adaptation, Day 15 as the challenge exposure day, and Day 16 as the final observation day. The number of exposures and number of puffs of reference cigarette smoke received by each animal in each group are pre- sented in Tables 1B-5B. No animals died in the sham-exposed group or exhibited any overt signs of toxicity. Mice exposed in the challenge treatment to 30% smoke, 9 exposures, or 20% smoke, 12 exposures did not survive the entire exposure (Groups 1 and 4, Tables 1B and 4B). Mice exposed to 10% smoke concentration for 9 or 12 exposures survived the entire challenge treatment (Groups 3 and 5, Tables 3B and 5B), as well as mice exposed to 20% smoke concentration for 9 exposures (Group 2, Table 2B). These data suggest that the MTD-1 day for adapted animals is the 9 exposures of 20% smoke used in Group 2. The individual body weights of the animals are given in Tables 1C-6C. Sham-exposed mice showed little change in body weight during the adaptation and challenge period (1% or less, Day 0 to Day 15, Table 6C), followed by an approximate 8% increase during the 24 hours after the final sham treatment. Mice in the smoke-exposed groups also showed little increase (3% or less) in weight from 24 hours prior to exposure (Day 0) compared to 24 hours prior to the challenge exposure (Day 14, Tables 1C-5C). The weights either at death immediately after challenge exposure on Day 15 (Groups 1 and 4) or 24 hours after final exposure on Day 16 (Groups 2, 3 and 5) also showed 4% or less increase in body weight in the respective groups compared to body we ights at Day 0. 11

I-172 5. 007 The sham-exposed group had COHb levels of less than 4% (Table 6D). The average COHb levels for the 5 smoke exposed groups during the adaptation period ranged from 22.3-25.5% COHb (Day 7, Groups 1-5, Tables 1D-5D). The COHb levels in 3 survivors immediately after challenge exposure on Day 15 were 56.3+14.4$ COHb for Group 2, 26.6+1.1$ COHb for Group 3 and 38.6+9.3$ COHb for Group 5(statistically different from sham-exposed control, p <0.05, t-test, Tables 2D, 3D and 5D, respectively). The COHb levels for Day 15 for Groups 2 and 5 were also statistically increased over their respective levels on Day 7 (Table 7D, p <0.05, t-test). A general description of the response of the animals to smoke is given for each group in Tables 1E-6E. All smoke-exposed animals demonstrated struggling and irregular breathing to some extent during the adaptation exposures. Mice which survived the challenge smoke exposures (Groups 2, 3 and 5, Tables 2E, 3E and 5E) were lethargic, uncoordinated, trembling, unable to walk and were lying prone, cold to the touch and had swollen noses. Twenty-four hours post challenge exposure, s urviving mice appeared normal. The number of puffs of reference smoke received„by the adapted mice to this Study were compared to the number of puffs received by naive mice in a previous study (I-1725.001). The means for each group are presented in Table 8. The adapted mice accepted significantly more puffs than the naive mice in 3 groups (Groups 1, 2 and 5). Group 3 received the same amount in both experiments. At the doses used in Group 2 and 5, 60-100% mor- tality was observed in naive mice, while zero mortality was ob- served in adapted mice. These results suggest that adaptation to a low smoke dose over a 14 day period has the potential to in- crease survival significantly. 12

I-172 5 . 007 VIII. CONCLUSIONS The criteria for a valid test were satisfied in that no sham exposed animals died or showed signs of toxicity and the COHb levels for the survivors in smoke exposed groups were signifi- cantly greater the sham-exposed group. The results of this 14-Day Smoke Adaptation-Challenge Study of Reference Cigrette show that mice are able to accept higher levels of smoke after adaptation than without adaptation. This increased tolerance resulted in 100% survival in two adapted groups (Group 2 and 5), where 100% and 60% mortalty, respectively, were previously observed (I-1725.001). While the dose of smoke which the animals received was increased in adapted Groups 1 and 4 (only Group 1 was statistically significant), the adapted animals did not survive the entire dose regimen used in these groups. Moderate levels of COHb were observed in the survivors in the smoke exposed groups (26-56% COHb). The mice which died did have convulsions and were quite agitated, similar to naive mice exposed to the same dose levels, suggesting that nicotine toxi- city was not altered (Cassarett and Doull, 1975). Animals gained little weight during the adaptation period. Survivors of the challenge treatment also gained little weight. Mice appeared normal within 2 4 hours post-challenge exposure, similar to naive animals which survived in the previous study (I-1725.001). The results suggest that the maximum tolerated dose for a single acute exposure will be higher for adapted mice. Thus at least two dose regimens of the reference cigarette (Groups 3 and 5) should be used to provide information for characterization or other test cigarettes. The dose regimen used in Group 3(10$ smoke, 9 exposures) yielded 100% survival after a single acute exposure of naive or adapted mice while the dose regimens used in Groups 2 and 5(20$ smoke, 9 exposures and 10% smoke, 12 exposures, respectively) yielded toxicity (as determined by mortality) after a single acute exposure of naive mice but not of adapted mice. The use of at least two of these dose regimens is suggested for all subsequent studies comparing the toxicity of test cigarettes with the reference cigarette. 13

I-172 5. 007 IX. REFERENCES Armitage, P., Statistical Methods in Medical Research, New York: John Wiley and Sons, pp. 99-104, 1971. Cassarett, L.J. and Doull, J. Toxicology, New York: MacMillan Publishing Company, Inc., pp. 237-437, 1975. 14

QUALITY ASSURANCE STATEMENT Study Title: Toxicity of Reference Cigarette Smoke Af ter Inhalation in ' Mice Following a 14 day Smoke-Adaptation Period Study Number: 1-1725.007 Study Director: Carol J. Henry, Ph.D. Initiation Date: 05 May 1982 Review Completed Date: 06 July 1982 This study has been divided into a series of phases. Using a random sampling approach, Quality Assurance monitors each of these phases over a series of studies. Procedures, doc- umentation, equipment, etc. are examined in order to assure that the study is performed in accordance with the Good Lab- oratory Practices regulations and to assure that the study is conducted according to the protocol. The following are the inspection dates, phases inspected, and report dates of QA inspections of this study. DATE OF REPORT SUBMITTED TO INSPECTION PHASE INSPECTED STUDY DIRECTOR MANAGEMENT 18 May 1982 Protocol 18 May 1982 18 May 1982 02 July 1982 Final Report 02 July 1982 06 July 1982 This report describes the methods and procedures used in the study and the reported results accurately reflect the raw data of the study. 06 July 1982 Quality Assurance Date RA/QA Department 15

Table 1A DAILY RECORD OF INITIAL AND FINAL EXPOSURE TIMES FOR MICE Group Number: 1 Adaptation Regimen: 10% smoke ("4 ug TPM/ml), 30 sec smoke 30 sec air over 8 min, 8 puffs/exposure, 8 min exposure alternating with 16 min rest, 6 exposures/day, 48 puffs/day. a Date Initial Time on Test Final Time on Test Total (Min/Day) 5/19/82 1:37 3:47 130 5/20/82 8:51 10:59 128 5/21/82 8:56 11:06 130 5/24/82 8:56 11:04 128 5/25/82 8:48 10:58 130 5/26/82 8:57 11:06 129 5/2 7/82 8: 58 11: 05 127 5/28/82 9:02 11:12 130 6/1/82 9:32 11:40 128 6/2 /82 8• 55 11: 08 133 Challenge Treatment Regimen: 30% smoke (-12 ug TPM/ml), 30 sec smoke/ 30 sec air over 8 min, 8 puffs/exposure, 8 min exposure alternating with 8 min rest, 9 exposures/day, 72 puffs/day. 6/3/82 9:27 9:54 27 a Animals not exposed on May 31, 1982.

Table 2A DAILY RECORD OF INITIAL AND FINAL EXPOSURE TIMES FOR MICE Group Number: 2 Adaptation Regimen: 10% smoke ("'4 ug TPM/ml), 30 sec smoke 30 sec air over 8 min, 8 puffs/exposure, 8 min exposure alternating with 16 min rest, 6 exposures/day, 48 puffs/day. a Date Initial Time on Test Final Time on Test Total (Min/Day) 5/19/82 1:38 3:48 130 5/20/82 8:51 11:00 129 5/21/82 8:56 11:06 130 5/24/82 8:56 11:05 `129 5/2 5/82 8: 53 11: 02 129 5/26/82 8:57 11:07 130 5/27/82 8:58 11:06 128 5/28/82 9:02 11:10 128 6/1/82 9:32 11:40 128 6/2 /82 8: 55 11:18 143 Challenge Treatment Regimen: 20% smoke ('"8 ug TPM/ml), 30 sec smoke/ 30 sec air over 8 min, 8 puffs/exposure, 8 min exposure alternating with 8 min rest, 9 exposures/day, 72/day. 6/3/82 9:15 11:32 137 a Animals not exposed on May 31, 1982.

Table 3A DAILY RECORD OF INITIAL AND FINAL EXPOSURE TIMES FOR MICE Group Number: 3 Adaptation Regimen: 10% smoke ("'4 ug TPM/ml), 30 sec smoke 30 sec air over 8 min, 8 puffs/exposure, 8 min exposure alternating with 16 min rest, 6 exposures/day, 48 puffs/day. a Date Initial Time on Test ~ Final Time on Test Total (Min/Day) 5/19/82 1:42 3:51 129 5/2 0/82 8: 55 11: 04 129 5/21/82 8:56 11:06 130 5/24/82 8:57 11:06 129 5/25/82 8:57 11:07 130 5/2 6/82 8: 58 11: 07 129 5/27/82 8:59 11:06 127 5/28/82 9:03 11:11 128 6/1/82 9:33 11:41 128 6/2 /82 8: 55 11:10 135 Challenge Treatment Regimen: 10$ smoke ("'4 ug TPM/ml), 30 sec smoke/ 30 sec air over 8 min, 8 puffs/exposure, 8 min exposure alternating with 8 min rest, 9 exposures/day, 72 puffs/day. 6/3/82 9:01 11:17 136 a Animals not exposed on May 31, 1982.

Table 4A DAILY RECORD OF INITIAL AND FINAL EXPOSURE TIMES Adaptation Regimen: 10% smoke ("4 ug TPM/ml), 30 sec smoke 30 sec air over 8 min, 8 puffs/exposure, 8 min exposure alternating with 16 min rest, 6 exposures/day, 48 puffs/day. a Date Initial Time on Test Final Time on Test Total (Min/Day) 5/19/82 1: 45 3: 54 129 5/20/82 8:56 11:05 129 5/21/82 8:56 11:05 129 5/24/82 8:57 11:06 129 ~ 5/2 5/82 9: 03 11:12 129 5/26/82 8:59 11:07 128 5/2 7/82 9: 00 11: 08 128 5/28/82 9:03 11:11 128 6/1/82 9:33 11:41 128 6/2 /82 8: 56 11:10 134 Challenge Treatment Regimen: 20% smoke ("'8 ug TPM/ml), 30 sec smoke/ 30 sec air over 8 min, 8 puffs/expos ure 8 min rest after every two 8 min consecutive exposures, 12 exposures/day, 96 puffs/day. 6/3/82 9:19 10:53 94 a Animals not exposed on May 31, 1982.

Table 5A DAILY RECORD OF INITIAL AND FINAL EXPOSURE TIMES FOR MICE Group Number: 5 Adaptation Regimen: 10% smoke ("'4 ug TPM/ml), 30 sec smoke 30 sec air over 8 min, 8 puffs/exposure, 8 min exposure alternating with 16 min rest, 6 exposures/day, 48 puffs/day. a Date Initial Time on Test Final Time on Test Total (Min/Day) 5/19/82 1:48 3:57 129 5/2 0/82 8:56 11:12 136 5/21/82 8:55 11:04 129 5/2 4/82 8: 58 11: 07 129 5/26/82 9:07 11:15 128 5/26/82 8: 59 11: 08 129 5/2 7/82 9:00 11:08 128 5/28/82 9:03 11:12 129 6/1/82 9:33 11:41 128 6/2/82 8:56 11:1C 134 Challenge Treatment Regimen: 10% smoke (-4 ug TPM/ml), 30 sec smoke/ 30 sec air over 8 min, 8 puffs/exposure, 8 min rest after every two 8 min consecutive exposures, 12 exposures/day, 96 puffs/day. 6/3/82 9:07 11:25 138 Animals not exposed on May 31, 1982.