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CTR HN 042229

'+e~se 97=. BIO-RESEARCH CO\TSli LTA\TS, Isc. :ON!V l'.TION EESEARCn 0!vEIOP H!Nf E. NO/iSJ2;a!R. .a. O_ Oi•ec'c. P. SERNf_-_O• V- 0-C;,ec1c. n R...x-.i May 14, 1974 Robert C. Hockett, Ph. D. Research Director The Council for Tobaccc Research - II. S. A. , Inc. 110 East 59th Street New York, New York 10022 Dear Bob: R.c.i~. 9 COw.+?iC~+t _~'?NJE Caaiit00E, v~SSaC-_'= i5 J:~t. (6~Tj33c.B:_5 Enclosed is the paper which a-e have prepared for o,_r co-tribc=on _to the Battelle, Seattle Sy=posium, `2E.cperi.^iental Respirato:v Carcinogenesis and 3ioassay-", Jt:=e 23-27. Please adc:se n•ie of a r changes or correcticns you may ~ s= :o make. Yours sincerelv, ~ r red3y Fior-'curger, ~L, D. President and Director rnak en:l. CTR CMTRRCTS 005721 11225462 C 6 T R H N 0 4 *22 2-23 (D

This in vestigation was suppor:ed by a contract from t5e Counci'_ for Tobacco Research - U. S. A. The s e.r2ressec in this paper are those o: the authors a^.d do not necessarily reflect the opinions of c^.e Couacil for Tcbacco Research. P. 3err_feld: Bio-Research Consulta:.ts, :nc. ,° Conunercial Avenue, Car.bridge, t:assac^use'ts (,'_14:. F. Horburger: 3io-Resea:ch ConrZI:ants, i: c. ,~ Com ierc`.z' °.•:c=ue, Ca:nbridge, Massachuse::s 02141; Resea.rc^. Professor of O^=o1ogy, Di•.:sior: cf 3as:_ Sc?emces, School o: Gra_'_ate Dentist:y, 3os'o.^. Cr.ivers`.ty: Research Professor of Pathology, Bostcn i:niversity School of `:edicir.e, Bostoc, \lassachuse:ts 031:S. A. B. Russfield: Pathology Departr.:ent, S:. Vincent 'r'ospita.l, Worcester, `tassachusetts 0:610. CTR CQHTRRCTS 0t~~ ~ ~1 11225463 M." b I a! e I~ I I y I •r•' I/ O/~ 2'M.^ ./w

-_ s.•sW -,-- _~ ~_-~ -; _ _,- io-Research Conaultanta ' - Cigarette Smoke Lnhalatioa Studies . - =_In Inbred_ Syiia~n •Hamsters -' _ ~ _y ~ Hag~sters are desirable t' est sub'ecafoi .tobacco emoke . _inhalatioa studies since they have a niuch greater iesistance to toxic effects of nicotine than•rats or mice (Bernfeld and Ho:nburger, 1972). . Inbred hamsters 1 were used in the present study in order to achieve reproducibility of results and to detect possible strain- related differences in response to tobacco smoke. Two strains, the BiO 87. 20 a•-id BIO 1;. 16, ct•ere selec:ed for chronic toQcity experiments. Al' animals weighed 108 =-; g at the beginni=g of tae chror_:c studies. Only males were ueed, l:oused in groups of six i:: 12 x 14 x 6. 5-ir.ch poly-prop:lene cages. Sz_n-I-Cel, Deodor grace, was used as becdina ma:eriz. T~r aYit-i~ roor7 te ioe: a u-e R-ae keo_ between 72° a.Z on F. F1•:oresce=- oullbs wer e the orl;: lizcc source an-? were acc~-._rca'.:~ cor..r oL'ed :o ooerate be-•.een 7:c0 a.^. and 7:00 p.-. T=.e ~.•.a1s recei-.-ed Wa:"n- Vlo--se Breecer Blox and fresh ta~ water ad .Ntodif:ed Walton reverse srno•<f_a :racc:-es were use:, as des- cribed elsew-_ere (Ho:f:nazui and V.';r.der, i'170'. In these :.^.achines, air is pushed through lighted cigarettes by zpoly-ing posit:ve pressure at the burning end. All ar.i:naIs wore well fitting, prr-na=ertly ac:ached felt rings around their necks (3/16" th:c'.<, 2" outside cila.-neter, and i/8-3/Y" inside dia•^ieter, depending or. the size o` ::e ha-r:ster). The felt r .. .r. ~ CTR CONTRRCTS 005723 o 11225464 v...' TR HI ~I 1...• ' inv f v 32

_ _Bernfeld, _= Cigarette Y .~~ ,, ._. ~ ..... ...~~ _ . . Homburger, Russfield__ _Bio-Reeeireh-Consultante .- Smoke I= :aiatioa Studies. were reinforced on both 'ides by. thin, ring-'shaped aluminium plates of slightly emaller dimensions (1-7/8•' outside diameter and 15/16" inside diameter)• The heads of the a.nisnals -,,eie-inserted into the smoking machine by means of these collars, so that only the heads were in contact with the smoke. The bodies remained outside the machine, firmly restrained by the collars, thus elimina- ting the need to confine the animals in tubes with attendatnt excessive sweating and stress. Six a::mals could be et?osed simtiltaneoasly to smoke inhalation. As the felt collars p-e•:ented the a_mala from grooming their eyes, these .+•ere •xashed pe:ucillin =ol,_::on on a cotton s•rb ab once a day, 5 cays /week. S:z:oke wae genera:e_ from four Ip.l P'encuck. Reference ciga- :eaes2 burning srnultaneously and •xas adt i-:s.erec :o :::e a_mals in a o0-second putf cycle. =ach cycle consis:ed of a 2-_eco:_ period of s:noke -e_eratio: fol:o%ved S%-a 1=-secoc:~ period of a~ci::o_al s:r.ole ezposure a,c a 43-seccnd period of expos-.:re to :h es:^. air. D.:rinz the 2-second period of smoke ?eneration, 35 rr.: of air %vas pushed through each of the four ciuar ec:es, Yie:d:ng a tc:al volume of 140 rnl sr oke which was introduced into the -'_i rr1 exoosure cham- ber. A magnetic stirrer pro•.-ided instan: The smoke dilution factor was 725:140 or 5. 2:1. The s:no;ce concentration izhalec by the hamsters was therefore appro-nmatei.- 1c. 2'7 of that lea:ir.g the mou:i - 2- CTR CdNTRaCTS 00157224 11225465 WW T R I I/ I 0 4 <t.~t 16•a.M :r.' r.'

of smoke in the lung of a hwYian smokez is'7-10°,6 (aveiage puff volume,~35 ml; human tidal volume, 350-500 ml). It took the smoke less than 1 second to traverse the o i.nches; between the mouth end of the cigarette and the hamsters' heads, assuring that the harn.ter inhaled smoke no less fresh thz,. that reaching the lu=gs of the hur.:an smoker. Each hamster was e_•nosed twice a day, 5 days/v+eek, to 8 consecutive pufi cycles from the Kentucic.• Reference cigarettes. The first through e:gL:h puffs fro:r: the cigaret:es were use=, re- sulting :n a butt lengt:. slightly longer than 30 :rz-.. T'--e -)eriod cf e:cposure ranged from 35_ 100 weeks. Cage-held c_ctrol animals frorr: each i.^.'--=edU-ne. o: ce::Zpa-ab:= age a^.d initial weight, were maintaine,_4 simulta=eousl: in ::e same a-ma1 :oom a-d were handled :n the same way as all other I:ar.isters %:ith regard to weekly we:g:^.ing, per:naneatl}- weari= g fe::-alumini•:.^~ collars, etc., but these con:rol har.-.sters did no: come in:o contact with the smoldng machines. A second control grou? consisted of comparable hamsters exposed to "sham srnoki ng" in the machines, which were operated under standard cnnditions, except that no ciga^ rettes were inserted ir.to ther.i. For each of the two lines, BIO 15. 16 and BIO 87. 20, 102 hamsters •xere ex,oosed to :^:oke, 60 were exposec - 3- C TR CCHTRRCT S C1Q5?25 11225466 CTR HN 0"°'~ 222~"~ ~

Bernfeld, Horaburger, Russfield ' -~Bio-Reseaich Consultants Cigarette Smoke Ir':alaticn Studieg. , to shar, smoking conditions, and 60 were Lsed as cage-held controls. . -i~ : - _ . .... -: .'. Since it was impossible to obtain all 222 hamsters required for each inbred line at the same time, aniznals of the proper initial age and weight were introduced into the experiment in lots of 6, staggered over a period of 37 wceks. As far as possible, 1 lot of 6 aaimals of each of the controls was started at the same time as 2 lots of sr-:oke-exposed harnsters (12 a=Smels). Aaimels were exposed to smoke or con:rol conditions for fro^ 7= to 100 weeks. They were sacr`ficed ear i:er when they a??ear ed moribund, as judged by cocs'_sten: weigh: !oss or the appearance of ede:na. Some hamsters of all groups were zso sacriticec: a= 45 and 60 .:•eeks. Cc: :plete autopsies •.vere ?er:or:-ec. The cntire res?:r- a:or. tract and any other organs or tissues ....._^ appearec _rossl.: aonor-a! were studied ^is:ologicalr;. i..zs irSia_ec aad f.xec in Te1!yes:iczi•:'s fluid in an aopara:us es: rciev~; de%:se= b}• Dr. Sadarnc ishika.~a (1972). Resulta 1; l.forta'_ity: As si:o•.vr: in :er fi¢ -es ! a^.d 2, r.-.or:ality was very low in all groups ur.til about the o0t'r. week of the ewerimer.t, a: x+-vch time the animals had a c!^: or.niogic=_' age of appro.'-ia:atel}' 73 weeks. Thereafter, mortality increased r:.~id1}• i- all groups, CTR CaNTRACTS 4C15%'26 11225467 CTR IlN 4'~.~42~` 35

- T--- . ~ --- . .. .- • ~ Bernteld,-Homburger; Russfield ~--Bio-Re earch Con.nltants ~-- - - - - - • ,.-~ ~ -- - - - - - _'__~- '--. --- -___ -- c-r•r•. •:Cigarette Smoke Inhalatioa Stndies... ' -•-- c _ - i.w..;o ~i. --~- - . _ __ -.~reaching 100he at about 100 weeks. Neither smoke ezposure nor _-_ _-._ bham smoki-ng had a significant effect on rnortality. - There,waa no ....~.:.:ri-C difference in survival between the two strains. ' Text-figures 1 & 2 - 2) Changes in Body Weight(te.art-figu.res 3 and 4): In both li.nes of hamsters, individual body weights were a~ected by smoke in- Text-figures 3 ~ - & halation and, in addition, by the mere e=perimental manipulations, ouch as sham smoking eonditions and/or stress produced by the latter. Isitial weight in both groups ave: aged 108 g. The 9I0 87. 20 cage-held controls attained a final weight of 144. 3 g, whe: eas t:: B10 15. 16 ani:na.ls attained only 125. 0 g. ExTosur e to cigarette s:noke recuced bocy weight of both inb: ed strains by about 10 g during the first 2 weeks of t:7~atment. Subse- cuently, weights increased again, o,.t so:ne-x•^•a: more e1o-:-r in the BIO 15. 16 t:-aa in the BIO 87. 20 z=a•=a'_s. Because of the :ow final weight of the cage-held BIO 15. 16 : ar.:ster s, the weigh: di:=er ence between the controls and smoRe-e:cposed h,-nster s was much less in . the BIO 15. 16 line than ia the BIO 87. 2C, •L"^.e. S^a.:n sr^o:c:ng produced body weights i:ter:nediae between those of the cage-held controls asd the sr:oke-exposed animals in li: e BIO 15. 16; it had no effect on body weight in line BIO•87. 20. It thus appears that the long-term response to s:noke inhalation, i. e. , the failure of the smoke-esposed a nirnals to gain weight, was - 5- CTR CONTRRCTS 005722% 11225468 C~'"~' H~'` `.~4-: }~`~~~

Z~i, - : = = ~ Cigaie e Smoke Inhalatioa Studies... :: _ _ _ _~._ _ _~~due, at least in large part, to direct eficcts o? smolce inhalation = -. z'.~, =' `Berafeld. Honzburaer. Russfield - iwReeearch Conaultants Table ? = ieurc r -7-==iather than to nonspecific stress. - _ . -- _ -,~. - - - - - .~, - ' By far the most important findings in this _eaperiment occurred in the larynxes (Table I), approcir.zately two-thirds of which were . studied histologically (illustrated in Figure 1). (The remair.der were transplanted into ha:nster cheek pouches for an experiment which - will be reported separately. ) Among the sr.,oke-exposed ha^ns:ers, only 2 out of 48 larynxes in the BIO 15. 16 line (4%) and 3 ou: of 45 frorrm the 3IO 87.20 line (7%) were regarded as bistological;y normal, as opposed to 6?_90170 of the la=yr-ces fror.: tl:e corresponding control animals. ?a:hological c:.anges irn the sr.io:e-e_:posed hamster s were classified as follows: Hyperplasia •xas de:i.^-ed as h7perplast'.c thick- eniaK of tSe scuarr.eus e-_itheliu:r 7r.?ld =•_clear dysplas:ar The 'oasal layer re^:a:::ed i=:ac: and fa?rly strai3^t. Tl::s chin3e was see: i.^. 407) of the of sr.:o<e-e_-,-posed B?O 15. 10 hamsters and in "s3?, of e^e BiO c7. 20 1:ne, b•1: :r. aone of mntrol animals. ?se,_ZoeY_:heiicnatous hyper?:a<_:a Tas ce:ine= as proliferation of the sq'_amous epithelium in wh:ch thr.ra v:ts a da«=growth of cells into the dermis, often in a re:icu:ar Nuclear d}splasia ar.d mitotic activity tended to be :::ore ir.te-se than irn s:r.tole hy'?erplasia. This type of change was more frequent in 3I0 1i. 1 o ani-^als -6- CTR CCh{TRaCTS 005728 11225469 C ~m ~' I-I~~~ 0~-~ '`,''ti ~~~`

------ --.-----~-;'~si==~ Hornburser, Russfield.y "Bio-Reaearch Consultants ==Cigarette Smoke Inhalation Stndiep... N:eontrols. In many cases of this type of change, the basal layer -'-of epidermis wae clearly intact.. In some, it became v~ery irregular and apparently isolated cell clumps were seen in the de: mis, a few suggesting lymphatic invasion. In advariced stages, pseudo- epitheLiomatous hyp^rolasia is exceed?ngly dif.ficult to differentiate from early invasive carcinoma. This occurred im 19. 6:b of the BIO 15. 16 animals and in 4"0 of the BIO 87.20 harr.sters. It should be emphasized that no distant metastases were found in any a-':naL :.one of these tissue c-azges occurred in the con::ols. Small squa_-nous papillor :as simular to t: ose occurri=g in some of the trachea anc bro_c.`.i %•e: e founZ in the lar; =:'eal epit--elium of 25ro of srnoke_e-r~osaa 'Ma:-:ste:s a=3 9= 15', of t^e 5=O 15. 16 an:rnals. Occasionally, :here was pseudoepithe_o:::atcus c' cells at the base o_' nanil'.orza. A few larynxes o* sr.:oRe..ex2osed hamsters also s:-c••%-ed chronic in:lamma:ion and!or squamous metap:asia of m.:ccus Siands. C^rccic inflamznation also occurred in a few cont: ol animale a=c '.vas associated wzth slight epithelial thickening, but cever with frank h.-perplasia, pseudoepitheliorr.atous change, or pap?lloma fo-mat:on. TF-ere Was no significant difference between sharr srr.oked and cage-held controle :a this respect. _ r- 2 L cTR caNTRACTS aQ5%'29 °` 11225470 C~~ R ~'°~~` `'", ~ ~ 0'~~ ~ ~ ~~ ~