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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 '