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- -,--- - . SUMMARY PROTOCOL 1601.007 Experiment Number _T-/:725.D0 ~ Initiation Late '/! 9/gZ . . : :. Completion Date 0Z >TOXICITY OF REFERENCE CIGARETTE SMOKE AFTER INHALATION IN MICE FOLLOWING 14 DAY SMOKE ADAPTATION PERIOD h 1.0 PURPOSE a The purpose of this study is to determine whether exposure to non-toxic doses of smoke reduces the mortality of known toxic doses of smoke. ."Adapted" animals will be challenged in a single acute exposure using doses from Protocol 160Z.001. These data will be used to establish doses and ~-regimens' for subsequent studies. 2.0 RATIONALE a Rodents are sensitive to certain components in smoke and .~ resistant to others and this sensitivity seems to be ,altered by previous exposures to smoke. The dose of smoke tolerated by naive rodents can, tl•llbrefore, vary from the dose of smoke tolerated by "adapted" or previously exposed animals. These responses may also vary depending upon the .cigarette type and exposure conditions. by exposing animals to known non-toxi-c (iero mortality) doses of smoke over 14 days, the animals can then be exposed to known toxic (60-100$ mortality) doses of smoke. The results from .`naive and adapted animals can then be directly compared. F~ . , . .... . . . . . . . .. . " , . -. . - , ._ . _ . . . . . . ' . . - ' _ ' aY 3.0 TEST CIGARETTE 2YPil~Z C'~ 3. 1 Name (or ~Code) : I~er . Date Sample Received : f ? Y. Z 3.4 Number of Cigarettes Received : 2000 ~.5 Prcposed n=.cer to ba usPd i:, t::'s Stud y N~~S .,k 3. 2 Registry Number :. .Z. 3.3 , V'..~. ~.n.: -Y~ ' .. .. .. .. . L . n. ^. - - , r1c4:~ ~ 1 . _ y T{M~ ~ MlCROBIOLOGtCAL C~ATES ASSO [

~ .0 SPONSOR,,. SUMMARY PROTOCOL 1601.007 a n 4. 1 Nam e L.4rr' l/a Yd Com1 ~~ ---.7 : . .-. 4.2 Address: q20 ~h liS~ ~, p0 f3ax 2/68~~ 6r~~hsboro . ~ ,V ~~ haa= r. ,,. 4.3 ~Authorized Representative: fJ. l`'li.nh cw, 5.0 TESTING FACILITY 5.1 Name : Department of Inhalation Toxicology; - Microbiological Associates 5.2 Address: .5221 River Road; Bethesda, Maryland 20816 = eahv/ ~ 9.eL,,.-1 i. 5. 3 Study Director : • ,T, , . . . . . . . . . . . . _ .. 6.0 TEST SYSTEMS 6.1 Animals : , , narian. K•y . 6.2 Smoke Generation: Fresh, whole cigarette smoke will be generated by means of a Walton Horizontal Smoking Machine .(Walton, . Process and Instruments Inc.). The operating princi- ples and use of this machine with mice have been de- scribed (Guerin et al, 1979; Henry et al, 19E0) Th~ Walto*~ gerPra*_~s smoke under the stan3ard _ . .. .. . .. f 1 ff 1 f 35 m pu vo ume, one , conditions of a 2-second pu { are under the direction of the Laboratory Veteri- r ~ ~~ . Female C57B1%Cum x C3H/Anf Cum Fl (BC3F1/Cum) mice have been selected because they: a) are available .commercially, b) can be obtained free of three impor- tant adventitious agents in the 1'ung, Sendai virus, pneumonia virus of mice (PVM), and Mycoplasma pu1-.z .monis, in addition to being free of 9 other common adventitious agents, c) have been characterized in , their response to smoke from nonfiltered reference cigarettes, and d) are compatible with the proposed >,<< smoke inhalation equipment. :.Animals will be obtained from Cumberland View Farms. Clinton, Tennessee, at t,4-5 weeks of age and held for 21 days quarantine. a: ;-Mice will be vaccinated against Sendai virus. The .•housing, cleaning, feeding and watering procedures - puff every minute. The smoke exposure times and. level of dilution air added to the smoke can be L , ;,i..~.. , -- ( .~, ~ i. ~ ~ s.-:a ~. \1: lY MICROBIOLOGiCA ASSOCIATES ~"~1MdM ~~1rltak ~~~

t 7.0 EXPERIMENTAL DESIGN Mice are restrained about the neck in stainless steel ;" stock-like holders for nose-only exposure to•the smoke aerosol. Mice are held in a neck-slot with a restraining spring. A chin rest ensures that the nose of the animal is aligned with the conical shaped opening of the exposure module. The nose of the ani- ~ mal protrudes through a dental rubber dam diaphragm ~ which forms a seal to prevent body exposure. Three mice are restrained per tray and 1 or up to a total of 12 mice•can be exposed to smoke at once.~ Six groups of 6 mice will exposed to reference cigarette , smoke using a dose and exposure regimen 5letermined_to be .approximately one-half the maximum tolerated dose (MTD) in ; the Acute ~Toxicity Study (1Ei01. 001 ) and which resulted in ;no mortality during the 14 Day-Repeated rose Study (1601.OOI-Mi). Animals will be exposed to this dose and regimen for over 14 days.` Cn the 15th day, these "adapted" animals will be challenged with the dose and exposure regi- mens known to be toxic to naive animals.-`A sham exposed control group will be treated daily on another smoking. machine. An inhalation route of administation for whole cigarette smoke is the only acceptable one.to the Sponsor and reflects the normal.mode of exposure. ;; 14. ; ;.~•._~ .. r. ,a.. ., . SUMMARY PROTOCOL 1601.007 precisely varied.` Mice are exposed "nose-only";,,(see Animal Holders below, 6.3) and breathe directly into the chamber where the smoke is generated. 6.3 Animal Holders s 7. 1 Smoke Exposure Regimen : 7. 1. 1 Smoke Concentration : Smoke concentrations of "10$. -20% and `30$ (v/v) will be used. These are attained by using 1; 2, or 3 cigarettes simultaneously on the Walton, respectively. A smoke concentra- tion of '30$ corresponds to "12 ug total par- ticulate matter (TPM)/mi smoke aerosol from typical reported "reference" cigarettes which are to have "16 mg TPM/cigarette. °` 7. l. 2 Smoke Exposure Ti:ne : ~~rY ,, Smoke wi 11 be generated during a 2 second N !.a a GJ lia ----~ puff and held in the chamber for an addition- ~ 3 ~ l MICROBIOLOGtCAL ASSOClATES 3..

L x . - . .~ Y . . -.1'77.{ , will be used.-'~,0ne consists of 30 seconds of smoke alternating with 30 seconds of fr?sh air per minute ,for.8-9 minutes; the cigarette butt is removed and extinguished. Air is provided to animals for 8-9 minutes before the next exposure is started.'The second type of inter- mittent exposure consists of two consecutive smoke exposures (as in continuous) followed by air for 8-9 minutes'before,the next exposure is started. 7.1. 2. 3 Number of exposures :':' c SUMMARY SUMMARY PROTOCOL 1601.007 i='-men Chart, exposures will be either con- :' « ) k ~~. t al 28 seconds; the smoke chamber will be pur ged with 30 seconds of fresh air. ,This will ~- be repeated each minute for .8-9 minutes . Butts will be manually removed,:extinguished with water, new cigarettes inserted and the ~j~,~r H; machine reset. As outlined in the Cose Regi ~ p, tinuous or intermittent. 7.1.2.1 Continuous exposures consist of 30 seconds of smoke alternating with ;hY¢ 30 second of fresh air per minute <for 8-9 minutes; the cigarette butt~ is immediately removed and extin- ;, guished , the next cigarette inser- ted and the exposures repeated over 140 minutes . <. - r ~ x}, A~{a'•' 7.1.2.2 'Iwo types of intermittent exposures ;iY k .Y-[, Maximum number of exposures . per day will.be based on 140 .;minutes/day of machine time. The number of total exposures will be based on the number of••ciga- rettes used, e.g .•, 'a maximum of "'14 t,a • for a continuous regimen, and '"9 0 , for an intermittent regimen. 7.2 Controls Exposure controls will be used which consist of age-' matched animals loaded into smoking machines, treated exactly as the test animals, the muchines set and run, but without cigarettes. .a : .7 . -. . . . . _ . . . . . . . _ . . . . . . . . f . rir;~.._,- . .- . x _;~.,.-. . . . "!`!~ A • F L.:J .... .. Jj, «.,(i} ;c. ~r* t. a f yD MICROBIOLOGICAL ASSOCIATES ~ /~, . wa ;n.Wn =s

aw: +F. b> SUMMARY PROTOCOL 1601.007 8.0 METHODS Animals are obtained from a source monitored and known to be free of adventitious agents, vaccinated against Sendai .virus, and quarantined for 21 days. Stringent disease control procedures are followed during quarantine to assure the use of healthy animals. -A11 animals have free access to certified laboratory diet and water ad libitum, ex- cept during exposure. Groups 1-5 will be exposed to smoke, and Group 6 will be a sham-exposed control. Groups .1-5 will be exposed to a known non-toxic dose of.smoke over 14 days (5 days/week) determined in Protocol 1601.001-M1:• -smoke concentrations: smoke/air cycle/minute: .;p/3 b s CaNdS number of puffs/ exposure: 07 continuous or intermittent : ;h mi l.trf- total length of time exposed to smoke :,~h 2i~n,~n ~oA total number of exposures : total number of puffs: ~ ~ length of rest periods between exposures : /6 M;h. ~" . :Group 6 will be sham exposed (no cigarettes) on another smoking machine. Cn Day 15 of the Study, Groups 1-5 will be challenged by,exposure to reference cigarette smoke as given in the ':{r pose Regimen Chart (page 7). Animals are randomly assigned to 6 groups of 6 animals each. The time of day at which - each exposure is initiated and completed, the number of the exposure, and the total number"of puffs are recorded. Im- mediately after the last exposure, survivors ~'-are bled from the retroorbital sinus and the level of car-~' boxyhemoglobin (COF:b) determined. Animals will be weighed 1 day prior to exposure, and on Cays 7, 14 and 15 immedi- :.ately after exposure. Animals are observed for their r~ general physical condition during and after the exposure. -.P.nimals are returned to their cages and a final observaticn recorded 24 hours after the final exposure. N., r .,: . . J s .. ,t. MICROBIOLOGICAL ASSOCIATES

„ . , ; Dose Regimen for Acute Toxicity for Reference Cigarettes- Length of Total Total time exposed No. of No. of ~ s ",,:, Group Treatment .`'-to smoke ` exposures puffsa ''30$ smoke "'36 min 9 "72 A"'12 ug TPM/ml ) , 30/30, 8 puffs/ t = cigarette inter ~ ;. mittent,`~min rest between exposures "2 0$ smoke '3 6 min (-8 ug TPM/ml ) , ; ` 30/30, '8 puffs/ .} ~ cigarette,' inter mittent,'8 min rest between ~~ ' .: . .. `/ ' . exposures .,..3 1 .. : ' . . "10$ smoke -3 6 min ; ("'4 ug TPM/ml ) , .~ 30/30,• 8 puffs/ ~ cigarette, inter- .mittent, 8 min : rest between ,exposures , "'72 "72 . `20$ smoke "48 min ~12 - "96 ("'8 ug TPM/ml). 30/30, 8 puffs cigarette, 8 min rest after every two 8 min consecu- ;. tive exposures ' "10$ smoke ~ ,'"48 min 12 `96 ('"4 ug TPM/ml ) , 30/30, 8 puffs/ '' . .p, . cigarette, 8 min rest after every two 8 min consecu- tive exposures . ' Sham exposed, NA NA NA 144 min, no cigarette aPhj.ffs are defined to be the number of times air is forced through the cigarette or cigarettes (2 or 3) placed in each cigarette holder. Cne -35 ml puffs of smoke will be generated from 1, 2 or 3 cigarettes resulting in "10$, 20$, or.30$ (v/v) smoke concentration. . .. I ~r= F:;.... _. , ~.. ! a E,= to L MICR0810LOGtCAL ASSOCIATES

v iai tviv r i" vv ~ bu ,r MriNxz t ~. v . . 9.0 EVALUATION OF TEST RESULTS » ,» . .. a? 9.1 The mean and standard deviation for the number of ° ;puffs received by each on Day 15 group is computed. :'The difference in mean number of puffs.between smoke . exposed groups will be compared using a t-test (Armitage, 1971). . .3 . . . - . . ,. . C . •- . 9.2 The mean number of puffs for each group determined in Protocol 1601.001 for naive animals will be pared (t-test) to the mean number of puffs for respective group determined here for "adapted" mals. Q com- its ani- 9.3 A group description of the survivors is given in . ..terms of their physical appearance, both immediately after exposure on Day 15 and 24 hours after the final - exposure. 0 , ~ 9.4F The COHb levels are determined to document level of smoke exposure. * 9.4.1 Data will be given in terms of a'mean and standard deviation.for.the'•groug on Day 7 and 9.4.2 A t-test will be used to compare pairwise the difference in COHb levels between smoke and sham-exposed groups. 9.5 The average weight for each group before exposure, immediately after and 24 Yiours after exposure ~ will be given and the differerices compared using a t-test . (Armitage, 1971) 10.0 . CRITERIA FOR DETERMINATION OF VALID TEST 10.1 Negative^ ( Sham-Exposed ) Control 10.1.1 None of the sham-exposed animals should die or show signs of toxicity.;: ~ 10. 1. 2'Sham-expQsed animals should not have COhb 1eve1s7 above 4%. . i ~ 10.2 Positive Control . ~ . , O W .;. MICAOBIOLOGiCA. ASSOCIATES ~: , . ~~'~r;~, w ~:,, ~:-

The level of COHb for survivors in the smoke exposed groups must be statistically (p <0.05) increased above the negative (sham-exposed) control. 11.0 RECORDS AND SAMPLE ARCHIVES , ;.. Records :Upon completion of the final report, retire all raw data and reports to the archives located at 5221 River Road, Bethesda, MD ,;t20E316. Lbcument all disposals of cigarettes by an addendum to the final report. 12.0 REFERENCES Armitage, P. Statistical Methods in Medical Research, .•New York: John Wiley and Sons, pp. 99-104, 1971. 11.1.2 All invalid data will be maintained as part of the raw data but will not be included in the final report. 11. 1. 3- The archives will be maintained by the Regu- latory Affairs unit. 11.1.4 Storage and retrieval of reports will be done in accordance with Microbiological Associates,- SOP 1506.422. _ .: Reference Cigarette Reference cigarettes which have been condi- tioned but riot used, will be destroyed by breaking cigarettes in at least two pieces, wetting with water and disposing in a sealed plastic bag. Guerin, M.R., Stokely, J.R., Higgin, C.E., Moneyhun, J.E., and F':olmberg, R.W. Inhalation Bioassay Chemistry - Walton Horizontal Smoking Machine for Inhalation Exposure of Rodents to Cigarette Smoke. J. Natl. Canc. Inst., 63, 441- Henry, C.J., Whitmire, C.E., Lopez, A., Dansie, D.R., Avery, M.D., Caton, J.E., Stokely, J.R., Holmberg, R.W., Guerin, M. R. , and Ko::ri, R. E. The Cosimetry and Cistribu- tion of Whole Cigarette Smoke Particulates in Inbred O

r Spons .~Apprwal - ( . Dat Study D' rector RAQA Da t e , Strains of Mice : Comparison of a Large Staoke-Exposure Machine (SEM) with a Snall-Capacity Smoke Exposure Machine (Walton).' In : Pulmonary Toxicology of Respirable Parti- cles, C.L.' Sanders, F.T• Cross, G.E. Dagle, and J.A. Mahaffey (Eds.), Technical Information Center, U.S. Department of Energy, NTIS. 177-192, 19800 S 7_ h, Date .Y MICROBIOLOGICAL ASSOCIATES ~~'srv-.7!