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\ I • / rr BIO-RESEARCH CONSULTANTS, im == CON3YLTATION == /(j!AlCN 0lVil0PAlNT t. MOr1Ui61l, Robert C. Hockett, Ph. D. Resea:ch Director The Council for Tobacco Research - U. S. A. , Inc. 110 East 59th Street New York, New York 10022 Dear Bob: • CoA,Y!/CIAL AVtNY! CArlN00l, PuitaCMV3T73 071A1 (sIT) esa.ef3t March 28, 1974 Enclosed is a tnanuscript, toye•Ler .c-ith tables and photographs to follocr up on your suggestion rsade on March 6th. I hope this will meet the "scientific requireme=:s" ahich The Council for Tobacco Resear ch consider s appr opriate to permit publication, for example, in t'--e Journal of the ra:ional Cancer Institute, and also to serve as a basis for a contribution te the Battelle June Syraposium on Irhalatio:. At rr.}• paper on Az-31 8's FASE3 meeting I sha11 not inclLCe a:.:h::.g %rh:;_ You ::ave =ot previously approved for publi- C!^.0=. FI::j*.L: hianuscript .-I 5"ours yincerely, Freddy Hcmburder, M. D. s Pr esident and,Director ~ r'1 ~ Z CTR COh{Tf2RCTS 005769 ON 11225510 CTR HN 04242-8

a 91 ; ~~C APP, ' 1974 aP .e IIt__ .rG Ii :s _ SY'1LAIN DLFFYRENCES IN THE RESPOlLSE OF INBAM -_~ ._ . . SYRIAN HAMSTgRS TO CIGA3tBTTL sMOi+E IIiHALATION 1•_ P. Berafeld3, F. I3oanhnsses4 aad A. B. 3tussIIelds 1 Recet.ed. 2 This in.estlastioa was supported by a contsact from the Couacil for Tobacco Researcb - U.S.A. The riews e:pressed ts, t3is paper ase those os the aothors aad do not teIIact the opinious of the Coancil for Tobacco Research. 3 Bio•8eseasch Consultaats, lac.. 9 Commercial Avenue. Cambrid;e. 3da.saachusetss 02141. Bto-Reseasch Consultants. Iac. o Research Professor of Oncolo;y. Di.islon of Bssic Scieaces. School of Graduate Deatlstsy, Boston L'alversity: Research Professor of Pathology. Boston University School of Mediciae, Bostaa, Massacbwetts 02118. Lndependent comsnltast at 34A Park Street, Broohliae, Massachusetts 02146. 4 5 r ~ ~ CTR CCINTRRCTS 005770 ~° 11225511 CTR NN 04~42-*9

Aernfeld et al. Pate 1 SUMMARY - Onf. bundred two (102) maL hamsters of eaci of 2 inbred hamstcr li.nes were subjected to chroolc cidaretta smoke exposure twice a day. S days/week, in a modiiled Waltoa rs.erse smokity asacLlae. Sixty sham-smoked and 60 caje-b.ld cosesoL were ased for each strain. Saso,ce exposure for tip to 100 weeka Lad ^o etleet oe mor- talitT in eit3er strs.ia, but tsaded to redsce body.sijht. Carbo:ylumo- slobia levels increased markedly iasam edlstely atter each smoke atepo- su:e, but returned to baseline levels in L.s tha.z 24 hours. No change was lound ia s.rum trijlyceride ievels or virus profiles of smn3u- exposed ant-at•. Chronic smoke espoeure incseased relative weidbt of the lungs and heart in both strains. but to different degrees. Over 90'k o( the smohe-ezposed animals of both strains sho.ed hTperplastic changes in the larynx. Ho.vever, severe hyperplasia was nearly S times more Lrequent in I strain tban in the other. Tbe inbred llne, more sus- ceptible to Laryngeal hyperpiasia, also developed 2 nasopharyngeal ramors. 1 of them mali;nsnt. Smoke eairposuse induced rare beni=n squamous papillomas in the alr passages of both strains. The strain less susceptible to Laryngeal byperplasis exhibited more pulmoaary adenorru- tosis, incidence of which was not siS.niScantly affected by smoke ezposnre. Occurrence of clumped pulmonary macrophaqs was proof that smoke bad sctually reached the lungs. One strain was more susceptible to this pbe- nomenoa than the other. la neither stTaln did amake exposure alt.ct the incidence of tna:ors arisina outside the respiratory tract or the deteesr- atiw changes characteristic oi aging hsmsters. r -r. VZ CTR COHTRRCTS 005771 26 11225512 CTR. IIN 042-430

. Bernfeld et al. Pajs2 A model system for studYiad the sllects oL ehronic la3aLtloa of cijaretts Smoke bac been de.eloped by pontsawill atd co-worksrs using random-bred Syrian joldsa hamsters (1). The purpose at tLds paper is to rsport tse effects of Sea.tie difte:.nees bet...en 2 lines ot inbred Syrian hamsters in a somewbat related modal system. Tbe most appropriate methods tor studying biolosie eliects ol tobacco smoke in expertmsatal aalaaals are aecessarll7 tLose based om chronic iahalatioa simulatiaa the conditions ssistiaj in the hsmaa smoisr. In aeeordanee with oae of the main principles ot toaieolosic experisnsntatioa. it is desirable to use doss levels in aaimals many times hi;ber than t`ose salely tolerated by basaaa subjeats. In the case of tobacco smoke, it is ditiicult to achieve thls soal, siace the smoks coot.ains not only s::bstsaees of possible chronic tmdcity but also coat- poaads of kaown acuts tasiclty such as nieotiat, carboa monoxide. ete. , v.hich sbarply reduee the tolerassd doss Lrvels (2). Hamstsrs are desirable test subjects for tobacco smoke iahsLtioc studies sisue tbsY has a much greater resistance to tmic effects of nicotine than do rats or mics (3). Inbred hamsters were ased in the present study in order to iaaprove reproduciblllty ot results and to detect po.sible stsain• reLted ditfersnces in responss to tobacro smoke. I r .j. ~ CTR CC1NTRaCTS 005?72. ~ 11225513 CTR HN 0422431

Bernteld et al. 3 PaBe .MATERIALS AMD METHODS MimaL All bamsters used were SIO` Lsaast+ers purcLsoed lrom TJ:LACO, 8ar 1•iarbor, )laine. Fi.e strains. ths inbred BIO 2.4. BIO 87.20, BIO 82.62. and 310 IS. 16 lines, aad the A8 (randam- b;ed) line, were used in pret+=+&&+y ar.ite toxicity studies (to be reported elsenhese). On the basia of tbsss studies, two strains. the BIO 87.20 sad BIO 15. 16. were selected for chronic toxicit7 experiments. All aaimals wei=hed 108 +4 g at the be8Lsa1a8 of the cLranic studies. Oaly males .vere used, since msle haaesters, unllke other uule rodeats used ia laboratory work. are less beL118eren: thaa females so that ssveral s^-^=ls can be housed in 1 cage. .vhsreas female hamsters require indlvid..a1 carind. In tae present study. hamsters were housed Ln 8roups of o in 12 x 14 x 6. S-lncb pol7Propylens boses covered with well fittin8, wirs-screen lida. Sa.n-I-Cel. Deodor jrade, was used as bedding material. It was changed once a veek, at which tirae the cages were washed in 1e7o F .vater and deter jent in a commercial ca=e-washis; taachlas. The ani:aal room was continuously v.otilated (32.2 esshan=es/bous). Temperature was kept between 720 and ioo !. Fluorescent btalbs were the only lisht source and were autoautically controlL.d to operate between 7:00 a.m. and 7:00 p.m. CTR CONTRRCTS 00~~t3 11225514 CTR NN 042-43-2

. 1h /, Bernfeld et ai. pa No other aaimals of any sort were kept in the room bousiag the esperim+ental hamstsrs and no smoke was e.er ginerated within tbis room. Tb. saimals recei..d Wayne Mouse Breedsr Blaa and fresh tap water ad llbltum. Twice a day. S daTs/week, bam.tsrs were transferred to a nearby laboratory for exposure to smoke. Generatioa of Cigarette Smake Modiiied Walton rs'.erse smoking macbines were nsed, as dsscribed elsewhere (4). In these machinea, air ts pushed through li&Med ci;arettes by aPplyiad positive pressure at tbe burning end. This is in cont:ast to the conantlonal saoking machine or the buT=^ sasolcer. who sucks air through the eigarette by applying negative pressure at the mouth end. wll aaisaL wore ..ell fittiaj, *permanently attached felt rin;s around t2+eir necks (3/16' tAick, 2" outsids dLaaeter. and S/8- 3/4 • inside diameter, depeadins on the eise of the baa+ste4 Tbe felt rin=s were reinforced on botb sidss by thin. ring-shaped alumia:am places of slightly aaoalber dimensions (1-7/d' outside diameter and 15/16" inside diasneter). The bsads of the aaiaals were inserted into tbe smoking taschine by means of these collars, so that only the beads were ta contact with the smoic.. Tbe bodles rematrud outside the tsucbine. IIrmly restrained by tbe collars, thus elimlaaHn= tha need to confine the aaimala ia tubes with auendant auceasl.e sweating and stress. Six animals could be exposed simultaoeoosly to smoke lahalatioa. CTR co~{Tt~RCTS o05<<4 11225515 I CTR HN 042243-3

I Ih /. Berafsld et al. Page 5 As the teit eollars prev.nted the animals from Rroomlai tbeir eyea, tbese were srasbed with penlclllia solution on a cotton swsb once a dsy. S days/sek. Smoke was =eneratsd from 4 1R1 Kentucky Refereace cigarettes 6 burning simultaneously and was administered to the 6 IRI Kentucky Reference ciIIarettes are produced by. and were obtained froa. the University of Kentucky. Tobacco and Healtb Rsseascb Institute, hentuccy Research Foundation. Composit:oas ol the P.elereace ei1a.rstt. (IRI) at 123C moisture .rere as follws (5): flue-cured la=iza. 40. 1Y.; flue-cured stem, 14.2L; Burley ' laaiina. 24. y7:; Tur;cisD (wLole leaf), 11. 6S; Maryla.nd lamlaa, 1. 1;.: glycerine. 2. i.•.; invert s_crose. S. 356. a^i--!s in ov-secoad )caf cycles. Each cycle co=sisted oI a 2- aecond period of str.oie generation followed by a 1i-second period of additional sa:oke exposure and a 43-second period of ez?os,:re to fresh air to mtniaise the toxic effects of carbon monosade. CTR CCNTRRCTS 005,57175 72' ~ N 11225516 C TR MN 042434

. h /I nerateld et al. Page 6 During the 2-seeond period of smoka generation. 35 ml of air was pushed tbrou2h each of the 4 cijasettes. yielding a total volume of 140 ml emoke which was iatroduced into the 72S m1 ezposure chamber. A ma=netic stirrer provided instant mixing. Tbe smoke dilution factor was 725:140. or 5. 2. The smoks concentration Lnhalsd by the hamsters was therefore approsimatsly 19.2>i od abat leaving the znouth end of the cigarettes. In comparisos., the avsrase concentration of amoks In tbe lung ol a human smoker Is 7-10% (average puff volume. 35 eal; human tidal volume. 350-500 ml). It took tA- amoke less thaa 1 second to traverse the 6 inches between the mouth end of the ci2arettes and the hamsters' beads. assuring taat the hsoster indaled smo:ce no less lresh t.`ia that reaching tbe ha-nae smoker. Chsonic Inhalatioa Study A preliainary study was performed to detsr::Une a dose schednle at which mortality would be negligible during the initial months of the ex"riment. It was fouasd that an e:po.ue o1 ap? rori- mately SY6 of the LDSO. previously determined under ideatical es?otuse conditions. constitoted the upper sale limit. This varied •omewbat with the strain of hamster. For obvious reasoas, exposure time suitable for the most sensitive strain was used for all aalmals in the long-term study. CTR COHTt~RCTS 005 ~ ~ 6 11225517 CTR MN

Ik /k Berateld et aL Paje 7 Accerdiagly, each hamster was utpoeed twiee a day. 3 day./week, to a coetsecutis p+Lff cycles trom the Kentstcity Refersnce ci`arettes. The first through eighth pvils tsom tbe cigarettes were used, resultiaj ia a bult length slijhely lotWr tbaa 30 tam. The period of exposure ransed trom dS-100 weeks. inbred line used. One control sroup consisted of use-b.ld assmals of comparable age aad initial weight which were matn•-ined simul- taneously in the sams aaiasal room and were bandisd ia the saaae way as all other bas-sters with regard to weekly weishiaa. permanently wearing felt-alsainuxn cultars. etc.. but whicb did sot came into con- tact with the ataoldnS machines. The secood coatrol group consisted of ccsparaDle bamsters exposed to ''shas saololnQ" on the ta.achiaes, which were operated under standard cooditioni, except that no cigarettes were inserted into tbsm. For each of the 2 lines, 310 13. 16 aad BIO 87.20, I OZ haassters were exposed to smoke, 60 were exposed to shars smoidni, and 60 were used as case-beld controls. Two simultaneous control groops wese maiataisad !os each Since it was imposslble to obtain all 222 hamsters required for each inbred llne at the same tkne. aalrnals od tse proper iraitial aje and weight were introduced into the experiment in lots of 6, stajeered over a period of 37 weeks. As far as possible. 1 lot of 6 of each of tbe controls was stasted at the same time as 2 lot.s (12 =n{^w&U) of smoie- exposed hamsters. I ~ m S r C TR CCHTRAC T SZI 005777 -1 ~ s 11225518 CTR HN 0422243-6

. r /. Bernield et al. Page 8 , Obaervations during lahalatloa Stndy Iadi.idual body weights were recorded once a week throuQh- oat the esperiment. Carbozybemojlobla lewL were measured by a mlcromethod (6) in hamsters ait.r tarioas periods o( smo3ce es.posure". Blood was obtained by heart puactnre tmder light Nembutal aaesthesia. In order to avoid possible deleterious eUects of heart puncture oa hamsters in the chronic isLalatloa study, carbo=ybemojlobin le.els were determined ia 6 male hamsters (100-120 g body weight) of each inbred strain which were exposed to smoke for 1-3 weeks, but which were not included in the chronic inhalation study. In addition, carboxy- hemoylobin levels were determined in a staall aumber of rand,.mly selected amsters from the chronic study atter 13-45 weeks od e:posure. iermiaation of Scudy A,zimals -ere sacriticed wheo they appeared asorib:usd, as judged by consisteat weight loss or the appearance ol edema. Some hamsters of all groups wers also sacrificed at 43, 60, 75 and 10 weeks. Complete autopsies were pertoraaed. The entire respiratory tract and any other orsaas or tissues which appeared abaormal grossly were studied hlstoloSlcally. Lunts were inflated aad tised ia Tellyesaicaity's fluid ia an apparatus especially d.vised by Dr. Sadamu Isbikawa (7). CTR CCHTRRCTS 005778 X Ln 11225519 C "t~~' ~'IN 0~'~ ~~'' ~ ~