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I-1725 .007 y L 7:1-.~ ~ .. . .. ~ ~ .~ .. ~~ .,. . . . .. . . . . . . . . .~ ..~ .._..~ :.~ X L. 0 ? i 0 e Toxicity of Reference Cigarette Smoke After Inhalation in Mice Following a 14 Day Smoke-Adaptation Period .-Fina1 Report For Lorillard Company 420 English Street Greensboro, North Carolina 27420 By Microbiological Associates "' 5221 River Road Bethesda, Maryland 20816 r

, .;; , I-172 5 . 007. q g Table of Contents Pa ge ,. i«&i~; . Data Page 3 . '`,'Introduction 4 ,:Purpose 5 F' t . r_Reference Cigarette Characterization ~~ V. x..-Test Description ., VI. ',Materials and Methods a : . :.. . : ~ ~ . ~iS~-c~~f~ VII. ";Results and Discussion 11 ~ 13 IX . References 14 ~. . X. Quality Assurance Statement 15 VIII. ```_ Conclusions

ai N 1 i zr 10. : ~;. ~ -172 5 . 00 7 , I. DATA PAGE Cigarette Identity: Reference y•D,ates"Samples Received: March 26, 1982 Date Sample Returned: Retained in storage Initiation Date: May 5, 1982 Completion Cate: See Review Completed Date, page , MA Experiment Number: I-172 5.007 15 :114A Notebook Number : 172 5.007 ~ y~Ni. ~,"".Archives Location: . 5221 River Road, Bethesda, Maryland 20816 r~~ Sponsor: , Lorillard Company 420 English Street _. P.O. Box 21688 Greensboro, North Carolina 27420 r Authorized Representative: Harry Minnemeyer, Ph.D. Connie Stone, Ph.D. Testing Facility: Microbiological Associates ~~N > 5221 River Road : `' Bethesda, Maryland 20816 Lead Technician: David R. Dansie ~.y~.L2ti Study Director: Carol J. Henry, Ph x. r ~-~O~1~ X-.. -J6'vv-ii:

•°'Rodents are sensitive to certain components in smoke and by previous exposures to smoke. The dose of srioke tolerated resistant to others and this sensitivity seems to be altered j3by naive rodents can, therefore, vary from the dose of smoke be exposed to known toxic (60-100% mortality) doses of smoke. - The results from naive and adapted animals can then be directly mortality) doses of smoke for over 14 days, the animals can then conditions. By exposing animals to known non-toxic (zero :responses may also vary with the cigarette type and exposure ,'tolerated by "adapted" or previously exposed animals. These compared.

;The purpose of this study was to determine whether exposure These data will be used to establish doses and regimen for sub- to single acute exposures using doses from Protocol 1601.001. of known toxic doses of smoke.;•:"Adapted" animals were challenged to non-toxic doses of smoke over 14 days reduced the mortality of vsequent studies.

. CIGARETTE CHARACTERIZATION "Approximately 2000 reference cigarettes were received by a reqistrv number of 2. Preliminary characterization of the Microbiological Associates on March 26, 1982, and were assigned confirmed by the sponsor. The sponsor specified storage at room particulate matter (TPM) per cigarette. These data were verb ally rette filter, yielding an average of 22.1 + 1.6 mg wet total ~'required to smoke the cigarette to within "'0.5 cm of the ciga- mm water. When 4 cigarettes were sequentially burned on the Walton Horizontal Smoking Machine, 8 puffs/cigarette were reference cigarette indicated a resistance-to-draw (RTD) of '"100 temperature and no expiration date was provided. h h i l of t i For t e purposes s study, t e test art e was stored c h ~° in the original cellophane sealed boxes in a secured area at room L temperature. ~ ~

~'"` In'order to determine the maximum tolerated dose (MTD) that will be used in long term studies, it is necessary to perform rpreliminary experiments to assess the lethality of the reference ,cigarette smoke through acute toxicity studies. This was ex- pected to yield a dose of smoke which would be toxic to the ani- mals during a one day exposure. It was then necessary to es- tablish whether the toxicity and resulting mortality observed in Knaive animals after a single acute exposure could be altered if animals had been "adapted" to smoke. This was accomplished by exposing the animals over 14 days (5 days/week) to a dose deter- mined in Protocol 1601.001 and 1601.001-M1 to be approximately one-half the MTD over 1 day (MTD-1 day). The animals were then °challenged with single acute doses known to be toxic. Five doses I of smoke were used for the challenge, varying in smoke concen- tration, number of repeated exposures, and number and length of rest periods between exposures. Mice were observed for mortality and toxicity responses, weight changes, level of carboxy- hemoglobin (COHb), and general physical conditions during the 14 day adaptation period and during and after the challenge ex- vposure. Smoke-exposed groups were all compared to a sham-exposed 'tcontrol group, which was treated identically to the smoke-exposed ;groups but no cigarettes were used in the smoking machine.

BC3F1/Cum mice,'females 8-12 weeks old Sendai vaccinated Cumberland View Farms, Clinton, Tennessee Smoke Generation Equipment: Walton Horizontal Smoking Machine (Walton) Stock-like neck holders Process and Instruments Corp. Brooklyn, New York Materials: Heparinized blood collecting tubes (250 ul) IL-CO-Oximeter Metophane anesthetic Methods Animals were obtained from a source monitored and known to be free of adventitious agents, vaccinated against Sendai virus healthy animals. Mice were observed for signs of illness, un- usual food and water consumption and other signs of poor health. The animals were judged to be healthy prior to utilization in this study. and quarantined for 21 days. Stringent disease control pro- cedures were followed during quarantine to assure the use of .libitum, except during exposures on the smoking machines. environmental contaminants. Water and food were provided ad certified laboratory rodent chow which had been analyzed for cage in polycarbonate autoclavable cages with filter top cage .lids. Corn-cob bedding was used and animals had free access to humidity, and a 12 hour light7dark cycle. Mice were housed 6 per Animals were housed in an AAALAC-accredited facility with a controlled environment of 74 + 5 oF, 50 + 2 0% relative Animals were ear tagged and randomly assigned to 6 groups of 6 animals each using the stratified sequence randomizaton technique. Smoke exposures were performed over 14 days (5 days/ week) for Groups 1-5 using the following dose regimen: 10% smoke concentration, 30 seconds smoke alternating with 30 seconds air over 8 minutes, 8 puffs/exposure, 8 minutes exposure alternating with 16 minute rest, 6 exposures per day. This dose had been determined in Protocol 1601.001 and 1601.001-t41 to be approxi- mately one-half the MTD-1 day. Sham-exposed (no cigarette) con- trols were exposed simultaneously on another smoking machine. On the 15th day, the mice were challenged using 5 doses of reference

cigarette smoke (see Dose Regimen Chart, page 10) for approxi- Animals were observed for any unusual general physical condition exposure on Days 7, 14 and 15, and 2 4 hours post final exposure. weighed 24 hours prior to the first exposure, immediately after . the level of carboxyhemoglobin (COHb) determined: Animals were 'and 3 sham-exposed mice were bled from the retroorbital sinus and :on Days 7 and 15, 3 surviving smoke-exposed mice-fran each group during adaptation and challenge. Immediately after the exposure at which each exposure was initiated and completed, the number of exposures, and the total number of puffs were recorded both mately 140 minutes or until all mice/group died. The time of day '~'during and after the exposures. Exposure controls consisted of age matched, weight matched animals, the machines set and run, but without cigarettes. mice loaded onto smoking machines, treated exactly as the test posure. A description of animals which died is given in terms of estimating the causes of death. COHb levels are presented for 3 survivors from each group immediately after exposure on Days 7 and 15. .'immediately after exposure on Day 15 and 24 hours post final ex- survivors is given in terms of their physical appearance, both P ;,using a t-test (Armitage 1971). A group description of the ences between naive (I-1725.001) and adapted groups were com ared by each animal in each group during adaptation and challenge are presented and.discussed. The mean and standard deviation for the number of puffs received by each group is computed and differ- The The number of exposures and total number of puffs received survivors 2 4 hours post final exposure. .The weight at death is given for animals which died during an exposure. posure, immediately after exposure on Days 7, 14 and 15 and for Controls The individual animal weights, the group means and standard deviations are presented for 24 hours prior to the first ex- Criteria for Determination of a Valid Test None of the sham-exposed animals should die or show signs of toxicity. COHb levels in sham-exposed animals should be less than 4%. The COHb levels in the smoke-exposed groups should be statistically (p <0.05) increased relative to the sham-exposed controls. -

Dose Regimen for Acute Toxicity for Reference Cigarettes Length of Total Total time exposed No. of No. of Group Treatment to smoke exposures puffsa 1 '3 0$ smoke ( "' 12 ug TPM/ml ) , 30/30, 8 puffs/ exposure, inter- mittent, 8 min rest between exposures 2 `20$ smoke ('"8 ug TPM/ml), 30/30, 8 puffs/ exposure, inter- mittent, 8 min rest between exposures 3 "10 $ smoke ("4 ug TPM/ml ) , 30/30, 8 puffs/ exposure, inter- mittent, 8 min rest between exposures 4 '20$ smoke (-8 ug TFM/ml), 30/30, 8 puffs exposure, 8 min rest after every two 8 min consecu- tive exposures 5 -10$ smoke ('"4 ug TFM/ml ) , 30/30, 8 puffs/ exposure, 8 min rest after every two 8 min consecu- tive exposures 6 Sham exposed, 136 min, I:o cic?ar?tte '"36 min 9 "72 '36 min 9 `72 "3 6 min 9 72 "48 m.in 12 "96 '"48 min 12 '"96 NA NA NA Puffs are defined to be the number of times air is forced tr.rough the cigarette or cigarettes (2 or 3) pd aced in each cigarette holder. One "35 ml puff of smoke wil1 be generated from 1, 2 or 3 cigarettes resulting in '"10$, 20%, or 30% (v/v) smoke concentration. i 10 '

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.

Table 6A DAILY RECORD OF INITIAL AND FINAL EXPOSURE TIMES FOR MICE Group Number: 6 Adaptation Regimen: Sham-exposed control, no cigarettes a Initial Time Final Time Total Date on Test on Test (Min/Day) 5/19/82 1:55 4:10 135 5/20/82 9:00 11:16 136 5/21/82 8:48 11:05 137 5/24/82 8:45 11:01 136 5/25/82 8:43 10:58 135 5/26/82 8:45 11:01 136 5/27/82 8:44 11:00 136 5/28/82 8:46 11:11 145 6/1/82 9:20 11:36 136 6/2 /82 8: 46 11: 00 134 Challenge Treatment Regimen: Sham-exposed control, 6/3/82 no cigarettes 8:44 11:06 142 a Animals not exposed on May 31, 1982.

I-1725 . 007 Table 1B TOTAL NUMBER OF EXPOSURES AND PUFFS RECEIVED BY 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. Animal Total Number Number of Exposures Number of Exposures/day Total Number of Puffs/Received Number of Puffs/Day 0217 60.0 6.0 480.0 48.0 0218 60.0 6.0 480.0 48.0 0219 60.0 6.0 480.0 48.0 0220 60.0 6.0 480.0 48.0 0221 60.0 6.0 480.0 48.0 ~ 0222 60.0 6.0 480.0 48.0 Number of Survivors: 6 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. 0217 1.0 - 8.0 - 0218 1.0 - 8.0 - 0219 1.0 - 8.0 - 0220 2.0 - 16.0 - 0221 2.0 - 16.0 - 0222 1.0 - 8.0 - Group Mean: 1.3 - 10.7 S.D. 0.5 - . 4 1 - ~ Number of Survivors: 0 O QD

I-172 5 . 007 Table 2B TOTAL NUMBER OF EXPOSURES AND PUFFS RECEIVED BY 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. Animal Total Number Number of Exposures Number of Exposures/day Total Number of Puffs/Received Number of Puffs/Day 0223 60.0 6.0 480.0 48.0 0224 60.0 6. 0 480.0 48.0 0225 60.0 6.0 480.0 48.0 0226 60.0 6.0 480.0 48.0 0227 60.0 6.0 480.0 48.0 0228 60.0 6.0 480.0 ` 48.0 Number of Survivors: 6 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, 72 puffs/day. 0223 9.0 - 72.0 - 0224 9.0 - 72.0 - 0225 9.0 - 72.0 - 0226 9.0 - 72.0 - 0227 9.0 - 72.0 - 0228 9.0 - 72.0 - Group Mean: 9.0 - 72.0 - S.D. 0.0 - 0.0 - Number of Survivors: 6

I-172 5 . 007 Table 3B TOTAL NUMBER OF EXPOSURES AND PUFFS RECEIVED BY 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. Animal Total Number Number of Exposures Number of Exposures/day Total Number of Puffs/Received Number of Puffs/Day 0229 60.0 6.0 480.0 48.0 0230 60.0 6.0 480.0 48.0 0231 60.0 6.0 480.0 48.0 0232 60.0 6.0 480.0 48.0 0233 60.0 6.0 480.0 w 48.0 0234 60.0 6.0 480.0 48.0 Number of Survivors: 6 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. 0229 9.0 - 72.0 - 0230 9.0 - 72.0 - 0231 9.0 - 72.0 - 0232 9.0 - 72.0 - 0233 9.0 - 72.0 - 0234 9.0 - 72.0 - ----- ----- ----- ----- Group Mean: 9.0 - 72.0 - S.D. 0.0 - 0.0 - Number of Survivors: 6

I-1725.007 Table 4B TOTAL NUMBER OF EXPOSURE AND PUFFS RECEIVED BY MICE Group Number: 4 Adaptation Regimen: 10% smoke ('"4 ug TPM/ml), 30 sec smoke/ 30 sec air, 8 puffs/exposure, 8 min expo- sure alternating with 16 min rest, 6 expo- sures/day, 48 puffs/day. Animal Total Number Number of Exposures Number of Exposures/day Total Number of Number of Puffs/Received Puffs/Day 0235 60.0 6.0 480.0 48.0 0236 60.0 6.0 480.0 48.0 0237 60.0 6.0 480.0 48.0 0238 60.0 6.0 480.0 48.0 0239 60.0 6.0 480.0 48.0 0240 60.0 6.0 480.0 " 48.0 Number of Survivors: 6 Challenge Treatment Regimen: 20% smoke ("8 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 0235 8.0 - 64.0 - 0236 1.5 - 12.0 - 0237 1.5 - 12.0 - 0238 1.1 - 9.0 - 0239 1.5 - 12.0 - 0240 1.4 - 11.0 - Group Mean: 2.5 - 20.0 S. D. 2.7 - 21.6 Numbe r of Survivors: 0

I-172 5 . 007 Table 5B TOTAL NUMBER OF EXPOSURES AND PUFFS RECEIVED BY MICE Group Number: 5 Adaptation Regimen: 10% smoke (8'" 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. Animal Number Total Number of Exposures Number of Exposures/day Total Number of Puffs/Received Number of Puffs/Day 0241 60.0 6.0 480.0 48.0 0242 60.0 6.0 480.0 48.0 0243 60.0 6.0 480.0 48.0 0244 60.0 6.0 480.0 48.0 0245 60.0 6.0 480.0 48.0 0246 60.0 6.0 480.0 "" 48.0 Number of Survivors: 6 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. 0241 12.0 - 96.0 - 0242 12.0 - 96.0 - 0243 12.0 - 96.0 - 0244 12.0 - 96.0 - 0245 12.0 - 96.0 - 0246 12.0 - 96.0 - ----- ----- ----- Group Mean: 12.0 - 96.0 S.D. 0.0 - 0.0 Number of Survivors: 6

Table 1C BODY WEIGHTS OF MICE Group Number: 1 Animal Body Weight (g) a b b c d Number Day 0 Day 7 Day 14 Day 15 Day 16 0217 17.0 15.3 16.8 16.8 0218 20.2 19.1 20.2 20.4 0219 18.6 18.1 18.9 18.9 0220 16.7 16.0 16.7 16.7 ~ 0221 17.9 17.3 18.2 18.0 0222 20.2 18.3 20.7 20.5 ----- ----- ----- ----- ----- Group Mean: 18.4 17.4 18.6 18.6 S.D. 1.5 1.5 1.7 1.7 a Twenty-four hours prior to first exposure b Immediately after daily exposure c Immediately after challenge exposure d Twenty-four hours post final exposure

Table 2C BODY WEIGHTS OF MICE Group: 2 Animal Body Weight (g) a b b c d Number Day 0 Day 7 Day 14 Day 15 Day 16 0223 0224 0225 0226 0227 0228 Group Mean: S.D. 19.3 19.2 18.9 19.4 18.6 20.7 ---- 19.4 0.7 18.4 18.3 17.1 18.0 17.7 20.1 ---- 18.3 1.0 19.7 19.7 19.0 20.0 19.0 21.1 ---- 19.8 0.8 19.1 19.5 18.3 19.3 18.7 20.2 ---- 19.2 0.7 19.3 20.2 18.9 19.2 18.5 21.1 ---- 19.5 1.0 a Twenty-four hours prior to first exposure b Immediately after daily exposure c Immediately after challenge exposure d Twenty-four hours post final exposure

Table 3C BODY WEIGHTS OF MICE Group: 3 Animal Body Weight (g) a b b c d Number Day 0 Day 7 Day 14 Day 15 Day 16 0229 19.4 0230 19.5 0231 21.0 0232 19.2 0233 16.4 0234 18.5 ---- Group Mean: 19.0 S.D. 1.5 18.3 18.7 20.7 17.5 16.2 17.3 ---- 18.1 1.5 19.9 19.2 21.7 19.4 18.0 18.4 ---- 19.4 1.3 19.1 19.0 21.2 19.0 17.0 18.0 ---- 18.9 1.4 19.4 19.4 21.6 18.8 "17.4 19.0 ---- 19.3 1.4 a Twenty-four hours prior to first expos b Immediately after daily exposure c ure Immediately after challenge exposure d Twenty-four hours post final exposure

Table 4C BODY WEIGHTS OF MICE Group: 4 Animal Body Weight (g) a b b c d Number Day 0 Day 7 Day 14 Day 15 Day 16 0235 20.3 19.6 20.3 20.7 0236 18.3 17.6 18.3 18.3 0237 19.9 19.6 20.7 20.7 0238 16.7 16.7 17.7 17.9 0239 19.9 19.0 20.8 20.8 0240 18.1 17.3 19.4 19.1 Group Mean: 18.9 18.3 19.5 19.6 S.D. 1.4 1.3 1.3 1.3 Twenty-four hours prior to first exposure b Immediately after daily exposure c Immediately after challenge exposure d Twenty-four hours post final exposure

Table 5C BODY WEIGHTS OF MICE Group: 5 Animal Body Weight (g) a b b c d Number Day 0 Day 7 Day 14 Day 15 Day 16 0241 0242 0243 0244 0245 0246 Group Mean: S.D. 18.5 19.5 18.5 18.6 20.6 18.9 ---- 19.1 0.8 18.2 18.5 18.6 17.7 19.2 18.7 ---- 18.5 0.5 19.5 18.7 19.2 19.1 20.3 19.1 ---- 19.3 0.6 19.5 18.5 18.7 18.7 ~ 20.1 19.3 ---- _ 19.1 0.6 19.4 18.8 19.1 18.2 20.2 19.8 19.3 0.7 a Twenty-four hours prior to first expos b Immediately after daily exposure c Immediately after challenge exposure d Twenty-four hours post final exposure ure

I-172 5 . 007 Table 6C BODY WEIGHTS OF MICE Group Number: 6 Animal Body Weight (g) a b b c d Number Day 0 Day 7 Day 14 Day 15 Day 16 0247 20.3 18.7 20.0 18.8 20.6 0248 19.0 17.2 19.0 18.4 20.2 0249 19.5 18.1 19.9 19.4 21.3 0250 18.2 18.0 18.7 18.6 20.0 0251 19.5 18.6 19.7 19 5 20.9 . ~ 0252 15.8 15.2 15.8 15.7 17.3 ---- ---- ---- ---- ---- Group Mean: 18.7 17.6 18.9 18.4 20.1 S.D. 1.6 1.3 1.6 1.4 1.4 Twenty-four hours prior to first exposure b Immediately after daily exposure c Immediately after challenge exposure d Twenty-four hours post final exposure

Table 1D CARBOXYHEMOGLOBIN LEVELS IN MICE AFTER EXPOSURE TO REFERENCE CIGARETTE SMOKE DURING ADAPTATION AND CHALLENGE TREATMELdT Group: 1 Adaptation Regimen: 10% smoke (-4 ug TPM/ml), 30 sec smoke/ (Day 7) 30 sec air over 8 min, 8 puffs/exposure, 8 min exposure alternating with 16 min rest, 6 exposures/day, 48 p uffs/day. Challenge Treatment Regimen: 30% smoke ("'12 ug TPM/ml), 30 sec smoke/30 (Day 15) sec air over 8 min, 8 puffs/exposure, 8 min exposure alternating with 8 min rest, 9 exposures/day, 72 puff day. Animal Percent Carboxyhemoglobin (COHb) Number Day 7 Day 15 0217 23.3 a 0222 24.4 a 0218 28.8 a Group Mean 25.5 S.D. 2.9 a Animal died during challenge treatment.

Table 2D CARBOXYHEMOGLOBIN LEVELS IN MICE AFTER EXPOSURE TO REFERENCE CIGARETTE SMOKE DURING ADAPTATION AND CHALLENGE TREATMENT Group: 2 Adaptation Regimen: 10% smoke ("'4 ug TPM/ml), 30 sec smoke/ 30 sec air over 8 min, 8 puffs/exposure, (Day 7) 8 min exposure alternating with 16 min rest, 6 exposure/day, 48 puffs/day. Challenge Treatment Regimen: 20% smoke ("'8 ug TPM/ml), 30 sec smoke/ 30 sec air over 8 min, 8 puffs/exposure, (Day 15) 8 min rest after every 8 min exposure, 9 exposures/day, 72 puffs/day. ~ Animal Percent Carboxyhemoglobin (COHb) Number Day 7 Day 15 0227, 0225 27.9 43.0 0224 23.8 54.4 0228 21.2 71.6 Group Mean: 24.3 56.3 S.D. 3.4 14.4

Table 3D CARBOXYHEMOGLOBIN LEVELS IN MICE AFTER EXPOSURE TO REFERENCE CIGARETTE SMOKE DURING ADAPTATION AND CHALLENGE TREATMENT Group: 3 Adaptation Regimen: 10% smoke ('"4 ug TPM/ml), 30 sec smoke/ (Day 7) 30 sec air over 8 min, 8 puffs/exposure, 8 min exposure alternating with 16 min rest, 6 exposures/day, 48 puffs/day. Challenge Treatment Regimen: 10% smoke ("'4 ug TPM/ml), 30 sec smoke/ (Day 15) 30 sec smoke/30 sec air over 8 min, 8 puffs/ exposure, 8 min rest after every 8 min exposure, 9 exposures/day, 72 puffs/day. Animal Percent Carboxyhemoglobin (COHb) Number Day 7 Day 15 0229 27.7 26.9 0232,0230 24.8 25.3 0234, 0233 22.6 27.5 Group Mean: 25.0 26.6 S.D. 2.6 1.1

Table 4D CARBOXYHEMOGLOBIN LEVELS IN MICE AFTER EXPOSURE TO REFERENCE CIGARETTE SMOKE DURING ADAPTATION AND CHALLENGE TREATMENT Group: 4 Adaptation Regimen: 10% smoke (-4 ug TPM/ml), 30 sec smoke/ 30 sec air, 8 puffs/exposure, 8 min ex- (Day 7) posure alternating with 16 min rest, 6 exposures/day, 48 puffs/day.ay. Challenge Treatment Regimen: 20% smoke ('"8 ug TPM/ml), 30 sec smoke/ 30 sec smoke/30 sec air over 8 min, (Day 15) 8 puffs/exposure, 8 min rest every two consecutive expos ures, 12 exposures/ day, 96 puffs/day. Animal Percent Carboxyhemoglobz`n (COHb) Number Day 7 Day 15 0239 23.2 a 0235 19.9 a 0240 23.8 a Group Mean: 22.3 - S.D. 2.1 - a Animal did not survive challenge treatment.

Y I-172 5 . 007 Table 5D CARBOXYHEMCGLOBIN LEVELS IN MICE AFTER EXPOSURE TO REFERENCE CIGARETTE SMOKE DURING ADAPTATION AND CHALLENGE TREATMENT Group Number: 5 Adaptation Regimen: 10% smoke ("4 ug TPM/ml) 30 sec smoke (Day 7) 30 sec air over 8 min, 8 puffs/exposure, 8 min exposure alternating with 16 min rest, 48 puffs/day. Challenge Treatment Regimen: 10% smoke ("4 ug TPM/ml), 30 sec smoke/ (Day 15) 30 sec air over 8 min, 8 puffs/exposure, 8 min rest after every two min consecutive exposures, 12 exposures/day, 96 puffs/day. ~ Animal Percent Carboxyhemoglobin (COHb) Number Day 7 Day 15 0242 21.5 35.3 0244 23.9 31.5 0245, 0246 21.8 49.1 Group Mean: 22.4 38.6 S.D. 1.3 9.3

Table 6D CARBOXYHEMOGLOBIN LEVELS IN MICE AFTER EXPOSURE TO REFERENCE CIGARETTE SMOKE DURING ADAPTATION AND CHALLENGE TREATMENT Group Number: 6 Treatment Regimen: Sham exposed control, no cigarettes Animal Percent Carboxyhemoglcbin (COHb) Number Day 7 Day 15 0247 1.9 2.1 0248 1.5 1.3 0249 2.2 2.7 Group Mean: 1.9 2.0 S.D. 0.4 0.7

I-1725.007 Table 7D-Summary CARBOXYHEMOGLOBIN (COHb) LEVELS IN MICE AFTER EXPOSURE TO REFERENCE CIGARETTE SMOKE DURING ADAPATION AND CHALLENGE Group Mean Percent COHb + S.D. a Grou Challenge p Treatment Day 7 Day 15 1 30%, 30/30, 25.5+2.9 8 min/ expos ure 8 min rest, 9 exposures 2 20%, 30/30, 24.3±3.4 56.3+14.4 b,c 8 min/ expos ure, 8 min rest, 9 exposures 3 10%, 30/30, 25.0+2.6 26.6+1.1 c,d 8 min/exposure, 8 min rest, ` 9 exposures 4 20%, 30/30, 22.3+2.1 8 min/exposure, - 2 exposures fol- lowed by 8 min rest, 12 exposures b 5 10%, 30/30, 22.4+1.3 38.6+9.3 ,c 8 min/ expos ure, - - 6 2 exposures, followed by 8 min rest, 12 exposures Sham exposed, .9+0.4 .0+0.7 144 min, - - no cigarettes Standard deviation bSignificantly different (p <0.05, t-test) from Day 7. cSignificantly different (p <_0.05, t-test) from sham-exposed control (Group 6). dNot significantly different (p >0.05, t-test) from Day 7.

Table 1E GENERAL OBSERVATIONS Group 1 Adaptation Period: Day 1-14 Mice demonstrated some struggling and irregular breathing during exposure. Immediately after exposure, mice were generally lethargic, uncoordinated, trembling and had swollen noses. On occasion, the mice had runny noses and were lying prone after exposure. Challenge Period: Day 15 Mice demonstrated severe struggling, gasping, irregular and labored breathing during exposure. Mice went into convulsions before death. All mice died in this group. ~'

Table 2E GENERAL OBSERVATIONS Group 2 Adaptation Period: Day 1-14 Mice demonstrated some struggling and irregular breathing during exposure. Immediately after exposure, mice were generally lethargic, uncoordinated, trembling and had swollen noses. On occasion, the mice had runny noses and were lying prone after exposure and cold to the touch. Challenge Period: Day 15 Mice demonstrated struggling, gasping and irregular and labored breathing. All mice survived in this group.,_Immediately after exposure, the mice were severely lethargic, uncoordinated, trembling, unable to walk and were lying prone, cold to the touch, and had swollen noses. Twenty-four hours post exposure, mice appeared normal and fairly active.

Table 3E GENERAL OBSERVATIONS Group 3 Adaptation Period: Day 1-14 Mice demonstrated some struggling and irregular breathing during exposure. Immediately after exposure, mice were generally lethargic, uncoordinated, trembling and had swollen noses. On occasion, the mice had runny noses and were lying prone after exposure and cold to the touch. Challenge Period: Day 15 Mice demonstrated some mild struggling, and shallow breathing during the challenge exposure. All mice survived in this group. Immediately after exposure, the mice wer`e severely lethargic, uncoordinated, trembling, cold to the touch and had swollen noses. Twenty-four hours post exposure, mice appeared normal and act ive .

Table 4E GENERAL OBSERVATIONS Group 4 Adaptation Period: Day 1-14 Mice demonstrated some struggling and irregular breathing during exposure. Immediately after exposure, mice were generally lethargic, uncoordinated, trembling and had swollen noses. On occasion, the mice had runny noses and were lying prone after exposure and cold to the touch. Challenge Period: Day 15 Mice demonstrated struggling, gasping, irregular and shallow breathing during exposure. Extreme struggling and c6hvulsions were observed before death. All mice died in this group.

'I-172 5 . 007 Table 5E GENERAL OBSERVATIONS Group 5 Adaptation Period: Day 1-14 Mice demonstrated some struggling and irregular breathing during exposure. Immediately after exposure, mice were generally lethargic, uncoordinated, trembling and had swollen noses. On occasion, the mice had runny noses and were lying prone after exposure and cold to the touch. Challenge Period: Day 15 Mice demonstrated mild struggling and labored and shallow breathing during expos ure. All mice survived in this_group. Immediately after exposure, mice were severely lethargic, trembling, uncoordinated, unable to walk and were lying prone, cold to the touch, and had swollen noses. Twenty-four hours post exposure, mice appeared no rmal.

~ ~, I-172 5 . 007 Table 6E GENERAL OBSERVATIONS Group 6 Adaptation Period: Day 1-14 Mice demonstrated no unusual behavior during the sham- exposures. Immediately after exposure, mice had swollen noses. Challenge Period: Day 15 No unusual observations were made during sham-exposure. Twenty-four hours post exposure, mice were normal.

Table 8 COMPARISON OF ACUTE TOXICITY FOR NAIVE AND ADAPTED MICE Mean Number of Puffs Received + S.D. Group Treatment 1 30%, 30/30 8 min/expos ure, 8 min rest, 9 exposures 2 20%, 30/30 8 min/exposure; 8 min rest, 9 exposures 3 10%, 30/30 8 min/exposure, 8 min rest, 9 exposures 4 20%, 30/30 8 min/exposure, 2 exposures followed by 8 min rest, 12 exposures 5 10%, 30/30 8 min/exposure, 2 exposures followed by 8 min rest, 12 exposures Standard deviation. Naive (1-1725.001) Adapted (1-1725.007) 7.8 +0.4 10.7+4.1 b 8.0+0.0 72. O+O.Ob - 72.0+0.0 72.0+O Oc . -~ 11.0+2.5 20.0+21.6d 79.8+17.7 96.0+O.Ob bSignificantly different (p <05,_t-test) from naive animals (I-1725.001). cAnimals received scheduled total number of puffs in each experiment. By definition, there is no difference between naive and adapted animals. dNot significantly different (p >0.05, t-test) from naive animals (1-1725.001).