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CTR t Cnrra, t#s Bio-Research Consultants, Inc. 9 Comr.:e rcial Avenue Cambridge, Massachusetts 02141 _ inal Report on Contract C- 191 A Pzoiect Entitled "The Dete.^: :nat:on o-' t^.e L seiuLzess of the S: rian GoiZe:: Haras:er as Model :nimal for L-ihalatior. St•.:dies" Sd b:r.itted tc The Cc,....::-.,- =o-a=cc Resea-ch - _.S.A. Dated S=~-ter.:ber 11-7 3 Prepared by Peter Bernfeld, Ph. D. ~^TP ~~it-•-!~'~c~;'~"r' ! CTR. MN 042474

Table of Contents I Page Lntroduction Materials and `Setnods i ~ Animals 2 Acute nicotine toxicity 2 Generation o: cigarette smoke .. Acute tcxicit: of whole sr::oke - Chron..c ir :a'.a:ior. st•,:d•. - l 3e sL ts --kcute ..e c c::r.e *.cx: c i t•: 2. Acute sr_:oxe ir.hala:ion s.`.:d.• Chrer.:c ...:.aiatio^ stuc.%• 3. `:ortal. , _ o. C :ances ... 5od•.• weie:^.ts _ c. Carbc-: :::or.c:cide content :.. biood trislti•cer.ce levels _::':c5!e exposure e. .. _s or.._.ies c: test an.~:ais c: smor.e exoos•.:re crn absci,t_. crgan weie:^.:s at aL•tocs': g. of smoke exposure o:: re:ati•:e organ weights 2 -.s: catl:oloeica: f:ndin es _ar =~ :.e;:hoolakia ?seLdoeoitheiic:::atcus i.•perpias:a Papilloma for: :atior. Lungs Clumping of macropha¢es Ectopic bone formation Adenomatos is Trachea and bronchi c ~- i-i e a d s Heart Liver lt --A r*'~ ~ Spleen ~ Kidneys Adrenals 2 r, N Histopathological findir.¢s in untreated young control animals 0 -J ~.n ~+"FD ilr)i, j`F`11= n(l'•'r f^~nC ('sic ') CTR MN 04224"S

/1 DiscLs sion Pace 2C A. 5. Effects of smoke :atalation on various sites or organs of the aamster Difference in response of two inbred strains of zc C. hamsters to smoke inhalation Resocr.se of han:s:ers to smoke inhalatiorn as a :un ct:or. of :: e c•~ration of exposure 2? D. L^-fiuer.ce o: srro::e -.=.alation by ha:nsters or. .::e:r ....,.:a:. '_- E. Sier.:':car.c= cf :e :roper cor.trc: zroups .....-ec hamster . . s^^.oke ... S•........:. -a =- : -..roug: X_.= _e=e-c ... _ eures t::roug: c 7' (1, ~ ~. ~ L1 T 11 H ) ) 0 42 i 76

The most appropriate methods of studying biologic effects of tobacco smoke in experimental animals are necessarily those which are based on chronic inhalation and, thus, sisr:ulate the conditions existing in the human smoker. In order to make such chronic inhalation studies as significant as possible, it is necessary to adhere to one of the main principles of chronic toxi- cologic experimentatior., that is to use dose levels in animals wh°:ch are many times higher than those safely tolerated by hu^:an subjects. I : or oractica: p•_:rposes, there are strineer.t lir::iz- tions, ^owever, tc the use of high concentrations of smoice in an:- r•.:als. As it is well r::.ow•n, tobacco smoke is a very complex r: :a- ture which, :r additicn to t: e principies to be tested for chronic to:.icir;, also conta:::s some s::bstar.ces of extremely hi?:^n acute toxici-,: s,,:c'rh as n:cot: ne, carbon monoxide and hydroc•:anic acid, which ccnsice-abl: red, ce , e dose levels tolerated bv tt:e ar.::::ais. T1is ce.^.sidera:'.c n is o: tr.e 1::Z:ost imDortance in the seiection c' ar. aoC-o7rtate anir::ai s?ec:es w: Sch, because of a oart:c;:lari.: ^?;: tolerar.ce for r.:cotine, •sculd be t::e most s•.:ita5le :::odel for =:::c::e t:,alat:cr. s:::cies. . vvas tne :)_r~:se ci .ce present pro;ec: .,, ce::-.c.. strate ...e ieas:b:ii ..: or. the effects of chronic :obac__ s:-:cke -..ra:a::cn _.. _ :e eolder. ra:r:ster ... 2ener_.. a::_ ... ,nbre= -ines o: .:at scec:es ..• oarticular. :t t:.e t::: e.• :_.. t:.;s :o-.. .:as :a s.t;c ar._ star-__, no reports c: ir.:esr.¢a:icns of t^e sa:::e ..:-ic in other '.aocra::r:es were a•:ailabe irn the sc:e: t;::c :*e a._re . ?t was ._._.. , ....u•ever, that the ha::zster .s a rr• _.. cc t:e. ~r.....:.. ...ode: :or sr::.,:ce inhalatiorn studies tian c:rer ._ cEr.., ... ?ar..c~'.a ...:ce anc rats, because of its co.^.s:cerac•. creat_- r_s:stZr.-_e .: t::r ._xic effects c: r.icotine. The avaiiability of numerous highly inbred lines of han:sters was an important feature in the planning and exec;:- tiorn of the present study. Inbred animals are, indeed, of the most beneficial influence, since reproducibility of the results in animal experimentatio.^n is possible only with the use of in- bred lines, where changes in genetic influences with time, as well as changes fror : one animal to another, are reduced to a .^.:iniznum. Reproducibility is, thus, at an optimum wher. inbred lines oi anir.:als are used, althou¢h it may occasionally be achie ved to so:ne deEree even with noninbred animals. r+ m, ,r % r'! • t i 1R ,.-% u q* f .1 -~~~ . ~ r r-'~' . Cf R i f f "i 0422477

_%LkTErZL4:_S AND %:ET :ODS Animals -A11 hamsters used were Bio?-Hamsters; they were obtained com.::•iercially from TEL.ACO in Bar Harbor, Maine. Two inbred lines of hamsters, i. e. Bio1F 15. 16 and Biol 87.20, were selected for the chronic studies frorr. among a total of five lines (four inbred and one noninbred lines), according to their tolerance to nicotine and whole smoke, determined in pre- liminary acute toxicity tests. All hamsters weighed 108 = 4 grams when ch ron'c smcfe exposure was started. Cn1}• :::ale animals were used in the present st•.:cy , because :: aie : a:::sters, ;:alike males of other rodents used in laboratorv work, are less belligerent than female hamsters. = or this reason, multiple male hamsters can be housed in one ca¢e without eccu:•r:- ar.:or.g the anir :ais, whereas fer::a:e l:amsters ca•_s_ - iuries or even deat'rh to their cage mates ar_ would r.ave tc be :o;:sec ..n sin2le cages. All a n::r.a:s were : oused six in a cage. The ':azes :o^siste-_: ..: ooivcroo.viene boxes of the dimensions i2 x 14 a c.: : eieh:' ...:.`.es. _^ey were co:erec with well-fittias .~:re-s:-een Sar.-i-Ce:. Deodo- :•ad::. servec as .^e oecd:nz r.:ater:a:. Caees •a•ere cr.ce a week with deter¢ent and hot vrater c: .03c = ... a :::ac::.ne. At t::e sar::e bedc;n :::as reoiacec. a;:: :ais were keot ..n a co::t:r.uo•,:s :eati'.a:e_ a:r exc: asees per hour) ar.d ter.:oerat•,:re cor.:-c::e_ r:.orrm t'-_"--c`' F.with a•_...:r:aticaliv controlled .:ucresce::t . ~-:uC a..... .:. 7:CC p.... j•.vithout any outside iicht. =...s a:.~.a_ . _,,... .....:ainec or.:} ha:r.s:ers, and the room was li....:ec t:, _... :rais ..sec -.. ...= present -oiect. The haz:^sters were t.~ice a=a;:, .. = cavs eacr: week, to a nearb}• lallcratcr•: fcr ex- posur._ .,, sr: oke. :t no r.:ne, was there any smoke generatec ... t:ze ar.i:::a? roo:rm ir. u•::ic: the animals were housed. All hamsters received Wayne Mouse Breeder Blox and fresh tap water ad '.ibitu: :. Acute nicotine toxicity - Ten male hamsters iro^: each of five lines, weighing from 100 to 120 g(aporoximatel;: t^ree months of age), received intraperitoneal injections of nicotine tar- trate at five different dose levels. The dose necessarv to kill 50=': of the animals (LD50) within 30 minutes (no animals died from r.ico- tine toxicity after that length of time) was determined by interpola- tion of re¢ressicn lines cor•.:outed for the relationshio between lcza- rithmm of the dose (expressed in mg per kg of body weight) and percent e. r?°P rn fuT t•.''-n(''T CTR Nt`~ 0~-~~-`,~ S

lethal respor.se ::: each strain. For comparison, analogous LD:C determinations were made in male rats (Charles River CD rats, six weeks old, with body weights ranging from 100 to 120 g), as well as in male mice (Charles River CD- 1 mice, three months olc;. Generation of cigarette smoke - In both acute and chronic inhalation studies, Walton- Morrissey reverse smoking machines (U. S. Patents \os. 3, 476, 119 and 3, 528, 435) were usec to produce smoke from cigarettes. The principle of reverse sme:;- ing consists in air being pushed through the cigarettes by appl;:ing positive press,-re at the burning end, rather thar. beine sucked through the cizarettes by creating negative pressure at the r: out:•. end, as is the case in :: e conventional smoking machine, or in t:a: of the hurr.an s^:oker. ':'his is achieved by lowering a plastic pis- :on over the _:hte= c:gare:tes, whereby seai is made w:tr: :he p:a:- for:rf that hc1cs t:e ?n this w•a•, air ,:nder :)ositi•:e press;:re is a:::.w-ec to Dass from the head of the piston thro,,:gh :-._ lighted cigarettes; a ccrrespondir.e arnount of smoke egresses a: the r.:out : end cf .::e c:garettes an d enters into the exposLre c:a~- ber d:recti}• Lr.-;er::eat: :ne c:garettes. _: e exper.:::er.:a: an:r::a:s perraanentl}• wore •.t•e:. -...in: ielt . . _ _ a-o ..._ .ae:. r.e c.a (3 i . :che s thic~c, two ir.c.._ _ outsice diame:e- and :iS :c 3/4 inches . nside diameter, de?e::d- .a c::tne s:=e .. _= were rei -_~crce_ o .. oot.n s:ces b; .... ,.. _ s:apec al,:^:'.._... ^'_ates of siig:^.tlv _r.:a:.:- cir::ensicr.s (. -,_ -....ne- J•-ts:de diame:e' and 1-5/16 inches ;ar e:er). ' ear.s c: .: ese collars, t=e heads of t:e an:r.:ais co;:lc oe easi'.•: ar.= t`.ial.: nserted in:c t: e exposure c::a^:cer an=. F_: ...~ same t:~.e, ::e re:-ainder of t::eir codies was le't cu:sid: ,.._ c=a:r.ber •.n a...... • restrair.ed oosi:ien. The two ta¢es c- t: is _ r~cec• re w•ere tnat (1) cni%• t::e 'r.eads of the ar.::r.a:: w•ere ..n contact with smoke ar.d (2) their bodies were not encasec in narrow tubes, thus protecting the animals against excessive sweating and minimizing the stress imposed on them. In this r^an - ner, six animals could be exposed to smoke simultaneously. Smoke was generated from IRI Kentucky referer.ce cigarettes and was administered to the animals in 60-second puff cycles. Each puff cycle consisted of a two-second period of smc::e generatior., followec by a 15- second period of additional smoke ex- posure and a=3-secor.d period of exposure to fresh air (to rr.inim:ze the toxic effects c-' carbon monoxide). Four cigarettes were srr.c:<2c r_''?°P"* r_1 n f-•.f T ~~SO f Tn a a CTR HN 04224 . 7'~

simultaneously; after one set of cigarettes had been consumed te the desired butt length, or had been smoked for a predetermined number of puffs, another set of four cigarettes was started, and their first puff was generated exactly 60 seconds after the last puff of the previous set of cigarettes. During the two-second period of smoke generation, a volume of 35 ml of air was pushed through each of the four si- multaneously smoked cigarettes, after they had been lighted with the first puf:. Hence, a total of 140 ml of smoke was introduced into the expos•,:re c'.^.amber, where a magnetic stirrer instantane- ously causec eeua'. cistribution of the smoke throughout this cha- - ber. The latter :ac a ca?acit•• of 725 ml and, her.ce, the smoke was diluted by a factor of 725:140 or 5.2. Accordingly, the smoke concentration :nhalec bv the ar.imals was, approximately 19.2To of ::zat e¢ressing at t:e ... ~t^ end of the cigarette. Ln compariso::, the average ccnce::tra::sn of cigarette smoke in the lung of a hu::.an sr: oker ranges from seven to ten percent, as arrived from the a•.•- erage •.$iue- :or t::e p,::-' volume (35 ml) and for t: e human t:ca: :OiL•.^.:e (~_. to -.~r...:). _: e c:s:..zr.ce be:«•eern the mout: end of the cigaret:es and t:= ..;acs ....e an ::-ais ..n t: e exposure c::a^-.cer was less t na:. six inches; :t took t-e sr-.:cke less than one second to traverse t::at c;stance. =.e ex?er.r.:ental a:::mais breathed smo::_ v ..icc u•as cer:a:: ..... :ess resh t::a.^n that co:rimon 1y .:.haltc .-.e ..,.^:a : sr::ct;er. : a:r.eter: Ac;::e tolicitv of whole smoke -=weive male .ro^: eac.. .,. ....r ..:es, weigi.ing .rc^: iC0 to 12C g (approx:: :a:_ . ...ree :::ont::s oic) ar.c, for comparisor., 3o rr.aie rats (Char:es ..•er CD rats, six weeks o: ase), also weighir.g from 100 to 12C €, were :... exposec to intermittent cycles (see above) of whole smoke from Kentucky reference cigarettes until death c-ccurred. In the acute studies, cigarettes were smo::ed to a bstt length of 30 mni, thus pro- ducing ten puffs or ten consecutive smoking cycles from each ciga- rette, i. e. from each set of four cigarettes smoked simultaneously. All ten puffs of smoke were offered, one by one, to the test animals. Survival time c•f each animal under these conditions was recorded. Chronic inaalation study - Before the actual chronic inhalation sn:dy was started, a preliminary chronic study was per- formed to establish conditions under which the mortality of the test animals during the initial weeks or months would be negligible. It -„ f''TP r' .1f••i i°P Qr`?'C. (1(1c:,() a CTR HN 042-480

was, thus, four.d t:at appr oxirnately 517o of the exposure time, at which 50°0 of the anir: als died in the acute experiment, constituted the upper safe limit of smoke inhalation which caused no mortality when the animals were exposed repeatedly, i. e. twice a day, five days a week. This limit, like the mearn survival time in the acute experiment, depended to some extent on the strain of hamsters. For obvious reasons, the exposure conditions suitable for the mos: sensitive stra:n were used for all strains in the long-term study. Accordin_:v, each hamster u•as exposed twice a da•:. five days a.:•eek tc eiz::t consecutive puffs from Kentucky '.R1 re'- erence ci¢arettes, eac: pLf: consistine of a two-second period of smoke generation, an acditional 15 seconds of actual exposure of the anir :als tc smoke and =3 seconds of exposure to iresn air. L'ader te ese cc :ca:cr.s, a'.: .rst through eighth puffs o: the ci¢a- rettes were usec; ...is -es•.:aed in a outt length sorne•u :at lo.^.cer t::an :0 n-r.:. : xoosure of t:e animals to sr::o:ce was continued fcr ocriods of tir.:e rare~-e _-.,... Y! to 100 u•eeks. = eac : inbred line of hamsters stLdiec in .r.is rr.an- n er, contro: groups were rr:aintained. On e con:r_. ; c cor.s::tec .,_ :a_e-..-._ ar..:::als o: conparable ar= and wei: :. .: at rece:%•ec no .-ea:::._r.. exceot th at these ani^:a:s .%ore t!:e sa-r.= iea-aiu ri^ c„'_'-ar_ as :r.e : ar.:sters exposed to cisarette sme:::. of ar..: :aLs o: cc=aratie a£c ar.d we:c :. .:.a: v:ere e>-_sec tc "sl:ar.: smok:r.3" cond:.'.ons icen..:-. •.c•:ti: ...c<_e c: ti.- _.n t::_ experimer.ta: grcup:, except :._ : zare::es ::ere nto t::e smokin ¢ r.:achines. _: e Ee e-a_ desien of the cr:ronic s:: o::e sr_ ...c.___ _..._ cc...-_. groups, is shown ... Taoie a:s= :catc_ t::e -._...=er c: an.mals ir. each grouo. Since :: was not possible to obtain the recuired nu:::- ber of hamsters from each inbred line (222 animals) at the same time so that all animals would be of the same age and weight, the experiment was started gradually, as hamsters became available, but always in lots of six a:.irr.als. In this manner, the beginning of smoke exposure was staggered over a period of about 37 weeks. Inasmuch as possible, for approximately every two lots of six hamsters starte,_4 in the experimental groups, one lot of six ani- mais in each of t^e two control groups with the same inbred line was started si:r.•.:ltaneouslv. ir.~some ir.star.ces, whole lots of six ra.^.:sters were lost ir•.:t:ally because of carbon monoxide poisoning ~ ~tp Ina nr- CTR NN C"422481

t .r• the two experin:enta: _:•oLos, ;.e. sever.lots or42 animals in Group A, and t•,vo lots or 12 anir::ais in Group D. These animals were subsec;:ently replaced b}• new lots of hamsters. The sched- ule of the staggered beiinning of the experiment is shown in Ta- ble II. Because the hamsters wore their felt-aluminum collars per^:aaently, t:.ey were unable to reach their faces with their fee: and. hence, could not groom their eyes. L^n order to crevent ens•_:n _ i r-iec::cr•s, the eyes of each animal were washed w•it: a cctton s•r:ao. d:•e_ c::ed with a penicillin solution, or.ce ever;: day, five ca•: s a w•ee::. .r.ci... ~a: bod•y wei¢hts of each ha:r:ster were deter- .^inec cr.ce awze- t::e exoeri:r.er.t, Car.,ox-..._••. ••'c.,... leveis were r.:eas:.r_- .•n a randcrniv se:ec:ec ar•:^:ais a:ter various periods ..- sr::c:~e ex:)er.r::ental and control gro•.:ns were as iLd¢e= o•.:: r.c: e::- c__ .re: ,--,,.-n cons:___... %vr:g:: icsses or gains (ece rai sc.•era: cc::sec_.••;e . Ec.._. :...~ als from all groups •.vere a:so =a .... __ Z..C~ ~ ......-.C: ~_, cC. 7: and ~~ %t•eei:s of e:•:oc- s•..re -.. :.Mc::e, _r r.an= ::c_- control ccnditicns. .--, sac:--•c?. etc a;.s p=:__ v.ere per:o----ed. r:.istopathologic o-' en.-re -es:~ra:or;: .rac: were done. For this pl:r7cse, fluid in an esoeciai'• ce~•isec and ?ersor.al cor:^=unicatior. ..- :)r. Sa~an ._ applicatior, o" a constant an.. %ti•e:' c=f:ne= ...n the lung (and trac^ea; ..c... t~_ ,-- stde a: ti:= sarr.e t:e:ped to prevent damages to tre c:ssLe oy enciosi.^.g the w•: ole organ in a chamber, thus creating an a?pro- priate and constant air counterpressure against the liquid from the outside of the lung. iiistopathologic studies were also done c n all abnormal tissues. Organ we;rl:ts were determined ir a certain number ~ • of cases and changes in seru^: triglycerides were measured irn m ~ some instances. Virus profiles were studied in a portion of the C1' a::i^:als fror:: experir.:ental and control groups. : inaily, the larv:x frorr: certain animals, that had N.+ C N -6- r"" TG'' !~~~.)~'~!'?r ('~~L~.~'~L r1 V TR I I 1 I 0422482

1h A 1 oeen exccsed ::. smoke or to control conditions for extended per:c--:s of time, ran_:;.3 from 60 to almost 100 weeks, was i:::plantea the cheek po;:c: of animals of the sa:r.e line. L sually, the laryr_\ was cut transversally into three approximately equal sections, ea::n of which was implanted into a different recipient animal. The c^eek pouch ir.:plan:s were inspected once a week,. In the ever.t that a re- cipient anirra: appeared sick, the implanted tissue was transierre;: to a new hos:. riistopathologic studies were made on t: e irr.plante= tissues at seiec:ed ti^:e intervals. RES:: LT~ ,cL:e icet.ne tox:c:t•: - Intraperitoneai:•: .._ez-__ n icotine tar.r_:e was ...:nc to be r:-:arkedl; and significantl-:es_ ._s:c ..n r.a::.s:er: ... :-a:= an_ ....ce, as s~c•.,.. ..n the data c: - --': .. of t^e data frcn: : icure :.D- value_• as .=_.ca:ec ... =abie Ii?. It is apparent that --_0 -. ...__ ne .:...-a -.n all har::ster lines taar. .n ....__ (p 5 C• - ) a=--: ..:c: er ... three o: the .^.a-:ster ..ne: :an irn ra-_ . .. _. ... .:. . (~ _ ". 0: j, i o2. 62 (,p = C. C=) ar.= .-. - = (p = V.). _ _-°s= e8:e a-sG stat:St.e811•. fere-.ces .: .... ...: ...._ .cx:,..:v a^•ior.g s=:era. C: tre -norec a::.. ....,--_ ::ar~.-.:. ...._s. _.. s. anta:als o: :::e .5. 1c in..rec ..ne a-= or_ -e _.::a_. ._ ...._...•_ ...c.. Z . - cr _'.': : ar::ster s a:ir a:s c: _ot.. .:e 8_..,. ar._ ?_ 1ir.es ex:.:bi: greater -csis:_..:_ ._ . -.:CGt'.'.e .-.a= .:.cse G: .::e ...- s:'a:.. (D = S . "'5) _. s.^:Cr:e ^ha:at:o:• s-1C\• - All a'...':.a-_ .r.... .:e e:iecte of smoke ir.::a:a::or. -:- .r....cs: ...stances ic:• s::Ostantial•' lc ger ---•-_- t:an ra:s 3: -e sa^'.e GGC v:e:Eats, as ts e• 1Ce n. ....... .: C aa:a .:. 2 igl:re 2. ce r.z::cas The r.:ear, survival tirnes and their standard are shown in :abie IV. Hamsters from all lines tested were cons:s- tently and r-:ar:<edly less s;:sceotible to the acute toxic effects c- :.:ga- rette smoke than rats (p 5 0. 0005). In addition, animals from t-: 15. 16 inbrec strain of han:sters showed considerably higher res:s- tance toward cigarette sr:oke than the three other hamster l:nes tested (p ra-:ged frorn 0. C1 to < 0. 0005), and both the 87. 2C and __. 62 inbred lines oi :^.a:::sters ex::ibited sig.^.ificantl}• higher tolerance :c cigaret:e sr.:eke taan the nor.in bred P.B strain (p 5 0. 0C05). CTR HN 042-483

When the results from both studies on the acute toxicitv of iatraperitoneally injected nicotine tartrate and acute s::zoke ir.:-:ala:ior. toxicity are taken into account, there remains no doubt that the 15. 16 line of inbred hamsters is the most appro- priate experimental at:irr:al model among the species and strains of rodents studied (see summary data in Table V). It is also aD- oarent that the 82. 62 and 87.20 inbred lines of hamsters share t: e second place with respect to tolerance toward nicotine admin- istratio^ cr w:^.ole smoke ir.halation. Since the animals from the 82. 62 strain are known to exhibit a high incidence of cardiomyo- pathic lea'cr.s and. _.,-see::ent1}•, to have a much shorter average survival e -:an 5 '. 2 C' :arnsters, the latter strain of anirr:a:s is uc° r:..,:•e s•.:i:az:> :cr ione-.erm st-,:dies than the former. _.._ _..ic ::ilalation sr,idv a. `.fortalit•. -;here was a verv low :ali.-: in a_: _r.til the animals were on exoerirner.tai z...._.=s ac:,•_: __ weeks (see Figures _ and 4); at that :::•:e -e:- aie was 73 w•ee;cs. _::ereafter, mortal:t;. c-easec .n --e e::oer.....ntai as well as in the control and .n e.._...cc- be ass:::::e_ by e::traooiaticn .., :.ati - reac-ec c_-c a:te:• .00 weeks. It is ev:dent ..,,... .: _ cata ... -_ -__ __..- .na: exoos;:re to s^:oke or to s::a:: -s....,.... ~ -._..._.._ _ :.c: . ne ......ta:tv c: the animals. C:anges in bed•. •.t•eiehts - _:ere v,er, cetw•een the two .nbred 1: nes of to c: a n;:es in 5odv weig::t '3DO:: exDesL're to _.:e _... c- cc :tr_:,. ..itions, as seen in Fie•.:res 5 anc ..._ _ .,_ _.-. r.es were started on the experir-:e nt at a..rnc wr.er. was approxin:ately 108 grams. The :age- r.e:c cZ'...r:.'. ar.::::a_s of tt.e 81.20 strain increased to an averase boc}• u•e:_::t of i-;-;. 3 grams, whereas the weight of the corresDcnc- inc controi a ni:::als of the 15. 16 line leveled off at a much lcu,er :::ark,. i.e. at 1 25. 0 grams (see Table VI). Exposure to cigarette smoke reduced the body u•eiz:: o: t:^.e ar.::::als of „ot : inored strains by abc•:t ten grams during t*-e first two weeks of treatment. Subsequently, their weights increased agair., but some~c•i:at more slowly in hamsters of the 15. 16 than in t::ose cf the 67. 2 0 l:ne. Because of the fact that the 15. 16 con:re: anir.:a:s cained cr.'. 1i:tle weie:^.t, but the 87, 20 control hamsters ir.creasec rr.ar kec:;: weight after the beginning of the expertr.:en :, -S- CTR HN 0422-484

the difference between sr::oke-exposed and cage-held control ::arr:- sters became much more important in the 87.20 than in the 15. 16 line. As shoa-n above, these marked differences in body weights did not affect, however, the mortality of the animals. Exoosure to sham-smoking conditions yielded bod.• weights that were intermediate between those of the smoke-exoosec hamsters and of the cage-held control animals in the 15. 16 line, but aoproac'.^.ec and even slightly exceeded the weights of the cage- held control anir•.:als irn the 87. 20 line (Table VI). :he d?-°ferences ir. body weights between the ar.i: :a':s of the experimental grot:> and those of each of the two control 2rc•.:ps were highly significant b;: stat:stical criteria (p S 0. 01) in the 87.20 inbred line at all tir::es (see :able VI, columns i and g); in the i-. :o line, the ditfer_..ces were sicni`.icant at certai., tir::es oniy..e. between -C and o~ wee:<s, and above 90 weeks of trea.r.en:. -r. s:~.e instances, t:ere was a significant difference betweern t^e two ccr.:r:~: groups (see =a*--.'. ee %•i, column n). .t t:::s acoears that the bod•: weight iosses of the e:.:- oer,:r:e::ta: an .•._a:<_ .,r :r.cre exactly the'.r failure .,: za:.. was d•.:e to t-.e e:'ects o. _..:o:e inhalatic n, rather .: an to n e s:`e== suffered by .: e an:ma:s _.;rir handling in the sm.o:ci ^:ac: ne. .:ss .,as not 1il::ited: notce:e~, .,. .:e an. :::als ^: t.Ae exDer-^:e::tal Crol:Ds. JoT.':ewRa: less r.:ari:e6 lossez were also O:~ser•:EC ln DO:: Ero.1Ds o: ::a^:s:ers ex:~csed tG s:.a:' :.- s^:o:..,E con d:.:en s as well a_ irn cage-neld contro: ar.-mals c: o: the two .. nes (o 7. 2:). _:is pheno:r:enon r::a.• be attr-butec. .:._ r :cre. at leas. ..n par ,.c stress endured 'o} t: e a::.r.:a:s ,•n en «...-- ec._stc^ ec ._ ..ear.. - tr.eir 'elt-alw:.i.^.ur collars, .r to stress __- soci~tec eti_,e-.^:e::tal conditior.s of forcec i= cb::;zat::-. t::e anirra.s ..n the s:-okinc chamber. c. Carbon monoxide content in blood - Car- boxyhemoglobi:: levels were determined in six male hamsters (1~_- 120 g body weight) of each inbred line at selected time intervals. This was performed by heart puncture of animals lightly anesthe- tized with nembutal (with 100~*o survival), before and ir:.mediatel;; after smoke exposure, four hours later (just before the second ex- posure of the day), 24 hours later (before exposure on the follow- ing day), as well as on a Monday morning before exposure (after srr:oke inhalation, twice a day, on four days during the preceding week). The animals used for this purpose were not the same as -9- r%l !~1 ~rl r" TIP r-nWTPC Q%1 Tc~ CTR HN 042 4BEEz

/ those used in the chror.ic inhalatiorn study, because the former were given a muc: ::igier smoke exposure than the latter (20 min- utes of exposure tu-ice a day, as opposed to eight minutes twice a day, each minute consisting of 17 seconds of 1:5.2 diluted smoke and 43 seconds oi fresn air inhalation in both instances). It was known that such severe conditions of smoke inhalation, as admin- istered in the blood carbon monoxide study, would not allow the animals to survive ::•tore than a few weeks. In addition, subjectio : of any animals of the long-range, chronic inhalation st•,:dy to the stress and possibla of repeated heart punctures was avoided. Sin ce E: c'.d not aonear indicated to carrv out pie heart puac:,:res a: s:ort :i^-ie intervals on the same animals, the data or. ca-box•:-e:::oglobin levels at the various staFes oi smoke :•hala:ion re=lec: •: e results obtained wit : six ir.divicual ha r.sters .n eac.c inbrec s::own in :ables VI: and ViII. _- :.ar.:ster_ c. bot :.•nes, :er e ras a mar::ed increase of carboxv: e:::ozie_:.. to between 20 and -_ ,.r:rr:ediately after exposure to s-r:oxe (see animals - and -: on ca.• C... =a~le VII, and ar.:rr.als _ and - crn da;: : in Table 1'ili;. -^ese nizh ie•re:s decreased within fo•sr ::ours to oet.c•een - and :::-, (=ee a ni:r.a:c 5 and b, on day 0... Table t'I: a nd on da~: :•.. Table se;.ond smoke exoosure on .ne same cav a.ain :-a:sec :ne ca.=c~ : e cglcbi .-aiues ir: .ne bicec •., be:•-A•e: 3G ar.c _ (see e..~..___ = ar.c c on ca. 7 in Table %'Ii, an c ar.::ra-s 2 anc - o r: _a•. ' .r. _ a :._ _ ... ter t«•o exposures _:ca-e:•: smo- -e w::i::m .'ou:• •., e •r.o;::•s and 15 to 1Chours of rest c,:r; , :e .. , caroc~::::e:r. -lob:r. ::ad r et•,:rned to near ore:-eat •_:e:_ ..n ai: ._s:an:_s ksee ar..:r.ais . and 2 o^ day r. =ao._ _ an,a cG 14ay - ... .co'-° V_:I). Carc-.. :r.or.ox:ce .vas al«•avs co.^.:7lete:•: ?5.....:c:?C c._.._ a:ter exposure, on one or on se._ra: co.. secur'.•.•e da•: s.o t•.%•o and or.e-half times greater doses _. cigare::e smoke than used :^ t: e long-term inhalation study (see all data a: 9:30 a. m. in Tables VII and VIII). Nevertheless, the mortality irn the 87. 20 line was considerably higher under these conditions than in the 15. 16 line of hamsters. It may be assumed that this differ- ence between the two inbred strains was not related to the toxic ef- fects of carbon monoxide, but rather to those of other smoke in- gredients, possibly including nicotine (see the higher susceptibilit;• of the 87.20 hamster to ir.traperitoneally adr.zir.istereC nicotine tartrate, _ ioure . and Table IV). E:COos::re of hamsters to cigarette smoke for m,.c.. r"11711-81 t,.,r f R f f N 0422486

shorter tirr.e Deriocs, i. e. for only eight minutes, twice a da;:, as used in the chronic in: alation study, may thus be assumed to cause no lasting accu_-nulation of carbon monoxide in the blood of the ex- perimental animals. This was actually confirmed experimentally in a few anirnals of both inbred lines, as seen from the data in Ta- ble IX. i d. Changes in serum triglyceride levels durinc s:no:ce exoos.::•e - The differences between the mean tri- glyceride leve'ss of ~.e four experimental groups of each of t:ze two inbrec 'i=es are not statistically significant, excep: that t::e;: were lower in .:e case of the young 877.20 ha:r:sters (Group :: ), as seen from tne cata of Table X. The increase of serur:: triF_'.;:c- erides during arir.c is a co.:.rmon occurrence in man and ani^:ais. T'-.e fact .:a: :....- an ._.crease ._n serur^ tr:£1yceride ecc;:rrec ..n the c'. _C ..r.e ar.c not ..n t~:e :3. 16 lin e of : a.^.:ste r s r:at• be a:- ....,,:tec to .: e..._.: s-~:ailer oody weight in.reases in .^e .~. ._ -:ne c•... .^ it: e..:::e ...te r ral s t•.:diec. =xoes--:re of the hamsters tc ci:arette smoke c:c not a::ec- serur.: .-igiycer.de 12•:eis in ei.ner of .:_ ..:o _...__ _..=e. .r.~est ::o e. _s -o:iles o: test ar ^:a:- _-c..: ea_.. crou~ were =ac=.--cec ar.c s rc:.._ :as s:__:ec a'ter ._ weeks on etiper.::•:enta: ccr.c: .,::s; e:ccosur_ t: s:~ok=_, expos.:re to s::ar::-smoi:ing ccnc:- .:o::: cr :aze-nc:c:. - ... acc.aon, s:•: an.rnals of eac:: .::bre_ _. e. «•: . .. =ec .._ • _ • oeer. ....der any exper.^:ental co :citio: c an_ -e,ocr.cec ... %ve:. .. and age to t: e ezper.r.:ental an :r::a.s a: %rere a:so i.^.ciudec n .ae virus s:...:es. ... Tables XI and XII. These data .'-ear. i::cicate that, ,•• a:i .r.stances, the virus pro:.le of the an.r::a'.s .:as not changed b;: exposure of ha^:sters to cigarette smoke or to co: - trol conditicns. f. Influence of smoke exposure on absolute orean weights at autoosti• - After 45 weeks of smoke exposure, or exposure to s::a:::-s:^oking conditions, or maintaining the anirnals under control conditions (cage-held controls), six hamsters -°rorrm each of the six groups, as well as six young untreated control an:- r.:ais frer:: each c.° the two inbred lines w•hic : never had worn fel:- alumi nu~: collars a nc whose a¢es and body weights were analogo_s to those of the anirr.a'.s at the beginning of the study (107 = 3 gra:::s r-"TP n'O~,O`I%I c`TR 11N 04*22487

body weight), were sacrificed and autoosied. The results on the weights of their organs are shown in Tables IX and X. The fact that the organs of the initial control ar.i- mals (identified as Groups CC and FF) were significantly lighter in many ir.stances than those of the animals from all other groups, can be interpreted simply on account of age differences. These findings were to be expected and are without importance. Apart from these organ weight differences, d•.:e to age differe::::a:s c: t.^e anir::ais, there were only a few instanc_s in whicFh sn:o:e exoos•.:re or sha:r.-smoking conditions may have induced sig ni::can t c'r.anges ... organ weights. These are (:) ir.- creased adrenal .c•ei¢hts in aniraals exposed to sharr.-smoking con - _itions o: ..ct :.nbrec lines, out not in the smoke-exoosec har.:- s:ers; (2) ......rease.. •x•ei_^.ts of the lun gs (ir.ciuding trachea) ... ._. 16 harr.sters exposec to s::a:r:-srr.okinc conditions; (3) decreasec 1:•:er and :kic=e•: w•eig-ts in sraoke-etoosed hamsters of the o". _- _:ne ori\• an: f.=j Cecreased soiee:: •.i•eigt:ts ..n sr.-.oke-ex7:sed ..'. 2: ha: :sters a nc. .., a....:c : lesser exter.t, ..n the contre': an irrals c' ....s iir.e, e>--sec t; _:a: s,--:e',.ir:g cor.d:tions. -: esc _a:a are di-_....., to ir.teraret. 5•.:bsec_e-.. aa corr_par:s_=s were :r.ade crn bas:s cf .^e reiatire organ s, aii•.:ence o: sn:ore exoos•.:re c:: re:a::%•_ zar. ~e:-- -..-- ..-zan w•e:ghts were referred to a star.carz ..cd; .v~ ; .. c: ~DO g-a: :s, by di •id:^c eac.h organ \ti•eig :t b-: .: - .: F sarr.e a:::r.:ai on aucops: (in ¢rar s), and ... ... .:C. The results are st...unari:.ec in :a_.=z data allow tLe fcl:ck•in¢ conc:~sior.s: (:) There were no apparent di-'fer- ences in relat:•:e organ weights between ar -mals of the two tnbrec lines; this is t:-ue for each of the four age groups studied, as a•el': as for all s:x major organs analyzed in this manner; i. e. liver, spleen, kidnevs, adrenals, heart and lungs, the latter includinc also the trachea. (2) Relative organ weights tended -7 to increase with advancing age in all instances, experimental and centr ei Ere::ps alike; they also became generally more variable m 2 .c•it:: increasing age, in olde r an'.:: a'-s. as evidenced by higher standard deviation s t.f1 N ~ ~ CTR I I I I 0I2•8WJ'

(3) The increase in relative organ weights with advancin g age was particularly marked in the case of the kidneys, and was equally observed in both inbred strains; this was due to deposition of amyloid; renal amyloidosis was, in fact, the leading cause of death in hamsters in this experiment. (4) In the adrenals, the increase in relative organ weight upon aging in both inbred hamster strains was the conseoLence of a variety of processes which could be rec- ognized microscopically. The prevalence of these age changes precluded use o_' &crenal weight as a measure of stress. (5) There occur lower relative spleen weights in s:: oke-etpcs2d and in "shar:.-smoked" ha.^.zsters of the 57.20 strairn t: an :n t: e ca¢e- :eld control animals, esoecially be- tween -C and ZG ..ze::s ..` the exoerir::ent; this differer.ce :-:aN• rec- resent a stress effec: (cissoiuticrr of 1,,-mphocytes), but disaopears later in the experi:::en. (over 90 weeks) when diseases affecting :::e soieen becan :.: oe :::a:::fest; no sLc:: effect is d:scernible in the _. lo lir.e of : a:: _-_:•s. (o) There were no w•eig:^.t changes :n liver, spieen,..-cne~: s.,s adrenais, .vhich couid be attributed to sr::oicir.g, w•:t: a.e x....r.or ezceptions whic : are probabi.; of no im- ocrtar.ce, these _..ce- . ..s were increases ... liver and 'r.idne.• o: both strains ir. s:::c::e-exoosec ar.ir als, but only during the ea rl:er stages ci the ezner...._r.. (40 to 60 weeks); ..n most ir.star.ces~ .::ese d::ferences ~~er_ s:.a::s.icallv s:_ni-icar.t only at the :r; cC^.ri~denc cdencc level (see Tab:es X'•: and X",'ii). (7) Relative heart .veigi:s of =arns:e-- exoosec: to S:::o:ce :or .-.,... -:0 to 60 weeks were markedlv and si,-- nifica nti}• greater (. ~ G. 01) in both strains than those of the a::r:ais of both of the ccrresponding control groups (Table XIX); in 87.20 hamsters, the hearts of smoke-exposed animals were still signifi- cantly heavier than those of the cage-held controls aSter from 60 a nd 75 weeks, but this difference disappeared as the experiment pro- gressed; in contrast to the animals of the 87. 20 line, there were many extremely large hearts in hamsters of the 15. 16 line after ~C• weeks of smoke exposure, but none in the control groups; this ob- servation may represent an important, smoke exoosure. strain-limited effect of ---I m ~ (8) Lung weights showed no syste^:- U'1 N atic differences between experimental and control animals of the ~ -13- ~.~ra n t-n Ci R f i i 3 0422489

-5. 16 line Lr:i: a:er ~0 weeks, when the lun¢s of smoke-expesec hamsters became si¢nificantiy heavier (p 5 0.01) than those o: tae control ani:r.als (Table XX); i.^n the 87. 20 hamsters, however, h,:ng weights of smoice-exposed anir: als were consistently higher than those of the control animals and, by 90 weeks, exceeded the weie::ts of the lun¢s of caoe-::eld controls by about 50°,'0; because oi the rather large variability of the individual values (high standard devia:ior.s) during the later stages of the exoeriment, tae statistical significan ce of these d:f-erences :s somewhat reduced. ... Listooatholo¢_ical: ndin¢s - Rela::%•e'.•: few anir•.:a's_ s'_ e e-e :cs: ._. :istclogical stucies; losses were c::e t: cannibalis::•: ar.cic: c_ca.: of carcasses o: an imals t-at had ciec soor.taneou, siy. ~e:er. eless, 7S. Y": of a-i hamsters, with .he exoeri.•....: ha_ oeen beeu::. could be used for ..:stooa:4ol-_g:c s:,:dies (see =azoie ::1:), a : :ax:r.-ur.: loss in Croup C (s.. _. _- evoosec 15. - :a:-s:ers; •.c•:ere s:iil two-:...ras c: all ani:::a:: V:_r: s:adied Of .r.e 7:'r•a::zs:e: s wi:ic: c:ec soon:a':eous., cn.~ were s• i:ac:e :os ..:s:ciogic s ~c; , cause c' =eat c_ :d be es.ac- ..s:ze. .-s:ar.ze_. as seen . =ab:e ?:X~. : ena: a: .~ic:cos._ -.tas t: _.•_cs: c:..:se .,- dea:.. .•n prac.Ecaii}• a:_ gr,,,::)s. here ex:s:- .•.. ac-=-=-•. re:a:ior.s::o between cause of dea::: an~ ..._ exce=:~r. „- t~_ .,..re:::.e c: :V:c na:_-:a:,.ncea: ._:::cr: ... ~.:rc:.. :. (s:r.c.Ke-eX- Zse:. .:. . _ :.ar.:_-_. . oose: :a:.cns ,.n t=.e ._ss.._- _- c.. :_- r.a:-:=.,-s are rece.cec ... =acies Xti:_ .... ..=se cat ~: _ a-ra^zec crZar. cr sae ('.z~ .rac :ea. r.eart, '.'.:er. sp_ee^, k:cr.e: = and ac. _ ,...- _-rain c'`r.a:nsters (15. 16 or 87.20 li.^.es), b}• t::e experimental conc:t:cns used (groups of smoke-exposed animals and of "sna:::-smokec" or cage-held control animals), and t::e•: are subdiv;ded into four arbitrarily selected time per:ods of ex- posure to the exoeri:-:ental conditions (40 to 60 weeks, 60 to 75 weeks, 75 to ~0 weeks and over 90 weeks). _.ar%-nx - Bv far the most impcrta::: ~ indings in ...is exper.ment occurred in the larynx. Sixty-six per ~ cent of the larvnxes .rom the 15.:6 line of hamsters and 6-1-; cf those _ror:-: :e S7.'_C line were examined histologically. Among t;-.e sr^oke-exposed l:a:-:zsters, only two out oi 48 tissues from t`:e tr'1 N %Z O ,~ .~ _"'Y~.wy L.~ Tf"G ~ f i`~ S..A `~' L' ~ 0

15. 16 line a nd t:ree out of 45 tissues .ror.: tae 87 .20 line (7~~) were reeardec as '.istologicalir normal, as opposed to .rom 68 tc =~~, of the lary :xes fr orr the corresponding control animals (see Tables XXIII and XXI%', colurr.n v). The pathological changes, limited ex- clusively to the lar}-rLx of smoke-exposed hamsters, were of the fol- lowing three kinds: Leukooiakia - This was defi::ec (loosel,: after po:aen•wiil) as t::ickening and hyperplasia of the so;:a- rr.ous epithe::•,:::°: c' t: e larvr_x in which all the growth occurred up- tt•ard tcw•arc .: e-_--...... _: e basal laver remained intact and :air-* straiCat. ?_ .. :a::ia ceL'.c be associated with nuclear dyspiasia tt•:ic': was us•.:a:'.•: r:•::'_c tc :::ec•rate in de¢ree. This was seen in =G~c of ,r.e iar• :xe s o: t::e 15. : c 1ine, and ..- B 317o of thcse of t:e c'. ? C __r.e (colu:r:.. . . -c.._-_ XX:._ ar.d XXIN'). Pseurioeoithelio-:atcus :asia -:'.::s tt•a_ :.e -.n_z as , ration of t: e squar::ous epit::e:•_..e c: t:e :ar} _.n ...c-. -: e =asa: :a;"er of cells becar::e .-recuiar, a: :east par_ --e _cc_•red dow•nt.ar_s into the stror::a. --is ~e c' grc.._.. .:as ..._._ c:•.ar_c-_-.stic o` .r:e 1:.'-„ ha::•isters, ..rere as .-.e casz- as co.- car_,. .rith o ::t 3'. .:. -. ~. -..._ __ ..tern asscc:atec . .... ,:c :ar.cec ..,.:.:ea- c•. a~ -cr.. a:__ :e__ r.es:= ceep in t:e st-or-:a, ....toric .- Cc2^ Cel._, a.^.: a:2-..=tan:es Of DOssl--:e 1~?'mD^.a::.: 1.^.•:asio:. zL-s:ec - ess...- ... ear :asive ca- -:or•:a. ., ..._:an...:_ an ar.--•._.. . n.s :-ar_- •.t•as icund n 19"', of t .._ an-r.•.a:s :- 1=. .c ...._, a: _ ... .... • - c: .^e ::az:sters :aoles :_n_ I'apilioma formation - Sma:: scuar::ous papillo:::as, s:milar :o those occurring irn the trachea and bronchi, Nvere fo;:::d in the laryngeal epithelium of 25°,'0 of srr.c::e- exposed 87.20 aai:::als and in 15-, of the 1ti . 16 hamsters (colur:-.: :x, Tables XXII: and XXI%'). Occasionally there was pseudoepitl:elic-n- atous r;:peraiasia at the base of oapillo.^_zas. A few iarvnYe_ o: s::zoke-exoosed hamsters also s: owec chrc•.:c ir`lasrLMat;crn and/or seuar::ous metaoiasia o: ..._ ccus E:ar.ds icc'.-.:M~_ns x and x:). Chronic i-2iarrmatior GCCI:r rec, - i5- crR HN 0424, 91

however, also i n the control an ir::ais. The mild chror.:c .. :am. matio : w•'r.ic: occurred in the "sharr.-smoked" and in the cage-'r.eic control animals might be associated with slight epithelial thicker.- ing, but never with franic leukoplakia, pseudoeoitheliomatous hype:•- plasia or papiiloma fcrmation. There was no significant differe::ce between "sha^:-smoked" and cage-held animals in this respect. In a number of animals, the larynx was not exa^:- ined histolcgically, but was transplanted into the cheek pouc: oi otner : az::ste:s oi :: e same inbred line, .n order to detect Doss:- ble :_a'_igr.ant Lotent:a:. Lz th :s manner, the laryrYes frcr.: seve:: sn oke-expcsec ._:.a^ ste:_ (four after _-90 weeks, and three after r: ore t:.ar. °~ weeks o: smcke exposure) were transplanted into new i-7. ic hamsters. In acc:tion, 29 lar}'r-Nes from ha::ste:- ._ _ t: e..-. 2 0 _.^e (:er: a:ter -_ ?C- wee'r.s and 19 after more than •.vee:.= c: e::Dcs..:e) .:ere t:ar.-piantec ....c new• c-.'_C. :ar::s:e:•s. _ .r.a_:~ :e :ar. =tie. -:om " s:.ar s:r.o:;ed" cont:ol n a:r.s:er_ anc seven -.~.-n ca_ee ..-ic cor...=. animals, a11 e: the b'.2C 1:..d an d a:- a:te. .....re . nan -_ •x•ee;s ..::cer exce-.:-:ertai :ondit?c: _. «•ere .-ar.sc :r:eC ....c r.e.%- ST.=.. r.ar.:sters. Thi: exceri^:ent ., '.a": - :~:nett D2r:ent G: t:e _:c^: . ~. . _ nL.:..::e:s an _ . ~: . _. t.'.Jse -:o.^.:.tCe C ~ . 20 s::a:.. - e:c exa^-.~.n°= ..._.:_ ca'.:•. -n :S 'e•:ea:cd ~c:.. c'ea- ... s:._.-. c?i:=:e n: es and s~ nt e">_c.- -c s:no::e exoosur_. ..._c:oo :a_es .: bc..- s:ra:r.s c: ar..r.:a:s car. :o:... s:::a_. :._.. -s ... .: e :,a:en_. -:.a. _..._ ter.cer.c• is r:uc- ...:,-e .. :'. :: r.ar.:-:e: _ a::_ .s acce::..:atec b•, sr:.o::e ..n bc:.n st:a:..s. _....s, c-_r:pe_ r-a:.-cc::ages •.::e seen in 9-Tc e: t^_ _- :e : _ a'tee s roke ex:)osLre, b:t ..n or.lv .- ar.= .- • cf .-, two control g:oups of this strain (column vi, Table XXVI). S::a:a: clumps were seen ir. t::e 15. 16 line of hamsters, but more :a:ei.:, i. e. in 53•"'o of the srnoke-exaosed ar.i^:als and irn from zero to c-; in the two control groups (volurr.n vi, Table XXV). There were ^:orozoloeicai as well as numerica'l differences in ciur^pi ne among t::e various g:oups. In all ham- sters cf the ::. 16 line and irn the control animals of the 87.20 li-e, t::e c:•_^:ps cons:sted o: cc-r.pa:atively few, loosely packed. small :: acroD^aees co ntairar.e dark brown or blackish oiement. L^n the s^:oke-exposec 87. 20 animals, clumps were both muc^ larger and more abundant ir. the lung. More and larger macro- 11""TP Lr TR H{ ''f 0424922

phages were aggregated irn the clump and were freeuentiy mixed with a few po:•:mcr?!:onuclear leukocytes. Pigment, instead of being dark, was pale gold in color. These cells gave the Prussia n blue reaction for iron. The rare smoke-exposed 87. 20 hamsters which did net con tain such clumps were usually animals which were found dead in their cages of some intercurrent disease. Ectooic bone forr.:ation - About 2Q1~- cf :: e'.•,:ngs from 13. 16 hamsters contained small foci of bone :n t: e p~i:.onary parenchyma (columr. vii, Table XXV). T:.es: ..ere not associa:ed with ar.•; type of cellular re- action or an•: other ob~-ious disease process. Bone was found in '2°0 of the caee- ei_ controls of the 67. 20 line, in 2of the "sham-srrcked controls and i:: 12~'o of the sraoke-exposed ani- ^:als of a:a: ..ne icoi_..... v: , T able XX`: : he sign :icance ,,. .:is ossi::ca::c- ._ ...,' apparent. Adenor:^atosis - This ter:~ was used tc ___ --a:r :::eta?:asia o: flat a:'.'colar epit :eliu:r. ...._ columnar e? .r.e-.-... o_ the cronc::;elar •?e. There «•as a..ign si_ ..-ican: rence ..n t:at adeno~atosis occ•.:rred irn neari}• .,. ..._ -amste-_ o: . :e t 7.=. ..: - sr.:d:ec, out ... ._:- t::an ~-. ~i .:.e a n.:::ais cf t: _.. io lir.e. Tne r-o:ess was r.D: ~_Jvto:.s. _....,.....~ DCt was an aze-re:ateL c.^.aac° .......... .... __.. _ ..ti.tc..'.= ~.\- = - -ra,..c ... ° . : ai: C`os _Loa- eC ac..:e o- ...rcr.. cr.e•_........._ cr conta:nec ^:etasta:.c .,....,,r of some ::. , car_.r.c::-.a, or leuker:.ic =aere e:e c_id oe as....,__ .., stra:n cr t., t :;._....~ .:c and x. :ables XXV a _ ....~':;. Trachea a::c onc::: - -::e grea: majority of trac: eas and bror.chi from animals o: all groups anc of both strains s=owed no detectable abnormalities. : few ve-: small patches of ^:ild souamous metaplasia were seer. in these passages, slightly more frequently irn the smoke-exposed than in the control ani: :als (columns vi, Tables XXVII through XXX,. Son:e rare scuamo;:s paoillor:as were detected at these sices in smoke-exposed animals, i. e. in 4~o of the trac :eas r*; .,.. of ha*::sters cf t: e ..,. 16 line, and in 2Pc of the tracheas and in 2': c. the bronchi cf : a:r.sters of the 87 . 2C line, but none were seen :.n an•: ef the contrc: a::irr.als (column vii, Tables XXVII through XXX;. ... N W 1 i- ~~?F.1T'P t-~,r ?'c. 0 h~j ~'~'.~ v.' /p R / 1 I / •...' II 2/ ..J^ 3

ali instances pap:::~3:::as .rere histoioeicali}• completely benigr-. Heads - A section of nasophar}'nx with adjacent str,:cr.:res in the head was examined in 971i0 of the 15. 16 hamsters and 91-;o of the animals of the 87.20 strain. The only pathology related to smoke exposure was found in two 15. 16 animals. One developed a large adenoid-cystic tumor of the naso- pharynx, probabl•: derived from a salivary or mucous gland, after 71 weeks of exposure to s^:oke. The other had a large sarcomatc•.:s tumor w•r.ict::::2tastas:zed to the l::ngs after 73 w•eeks of treatment. Both hamsters were ?o•,:nd dead i.^- their cages. \o other naso- o:ar•:n_eeai :_... ._ ~ere see :..n a.^.)- group (see coi'a:r_ s x and x:, Table s .l-XX: and -:XXX). •.c•as much ne nspecifi: pauoiog}' i.^n both strains. O:r.zivitis, cepos... piaeue between tootr and e•.:r : associated with r::ore :.. _ess c..-_..:c ..~lar:-.matio:., was seer. i.^n approx:r:ate: zOC- of a11 an-:-a:s (see :ables XXXV and XXX'%'I) and was noted e•: en in the -....:a: _.,.... _is. Dental caries a nd t:rorr.oosis c: the 'ar¢e veir.s o' tne :_ad were seen somewhat r^cre freeuent:v :.. ..~... sters e: the -:. .__..._ _-an -.. _::ose of t: e _-. ZIC strai... Some a-.. :::ais r.ac ..- t^e eve deEenera::crn of the £iGbe, oo::.--:c _-e e.'es oeca,_se t: _ collars the ar.'::'.a:s Spcradic pathoioey not included :.n the nie cons:=:e^ __ a._ •. a~ :a_ azscesses ..c decaved teeth, .:.ree CaSi•_ ..- a:~7ar:L. a--.:..... -Csts. rare ...-:a:::ed saii.ar}• giands anG ..'.e case _- d': s7.i.=:a :•- JC::e asso:lateC V.':t'. a Dara- _._ r ..:.,.. ;ear O 1': '_: . c: t::e near-_ -r_... =• -_ .^.a:r.s:er_ _-._ .°ron: those of the _i.20 strat.. -'rer_ ex a r ec ...stc. .__. , s r' cr as CPO c: t: e hearts fror:: n a:n sters oi the .:. ._ _.ne showed r:-:yocardial deger.erat:o n, as oc- posed tc oni}' 2.-7: c' the hearts from animals of the 67. 2C stra:n (coiurr.r, vi, Tables XXXIII and XXXIV). The myocardial degenera- tion consisted of :r;:oi}'sis of muscle fibers with, irn the 15. 16 ani- mals, an abundant i ~iltration of lymphocytes. This was seen most frecuent:}' in t: e 15. 16 hamsters exoosed to smoke ar.d rr.a;: represent a soec::ic strain-linked effect of smoking. (;nfort-,:nate:~:, not er.oue:^n s'r.a:::-s:::o::ed 15. 16 anir::als were available for stud.: to exclude a n_-nsoec:ac effect e_' stress. _..e c•::er cardiac pat::olog}' observed w-as rr- ral t!:ror::bos: ,..:tec in''3^~ o~ the hearts of or.e strain (15. 16) and -18- ~n~j- P pr"°-, mF,~~ ~ ~ ~~R ~~~N 04'24 94,

Adrenals - The adrenals were st-,:diec ^:icroscopicaliy :n 9210 oi the 15. 16 hamsters and in 861,0 of the ani- mals of the 87. 20 strain (Tables XLI and XLII). The most frequent change observed was proliferation of the cells of the adrenal cap- sule. This is the type o: proliferation which has been reported as "AB cell hyper?lasia" in various strains of mice. A strair. differ- ence in this phenomenon was noted (as has been seen in mice also). AB cell hyperplasia was about twice as common in 15. 16 hamsters as in 87. 20 animals. Amyloidosis was seen in both strains, and again, was more crevalent ir. the 15. 16 harr:sters. Both strains develooed adrena': cort:cal adenornas and a few carcinomas. In rare instances r::etastascs fron: these adrenal carcinor::as were :ound in the lun s. FiistoDatholocical findin¢s in untr eate_. vc,:ne cor.trc: an::-a:s - S:x ra: :sters frcrn each of t::e two ir.brec strains nad oeen sacr:::cec w:e.^n their body weig5ts tivere i0S _- grams, i. e. w: e:: t`eir a¢es were comparable to those of t:e =-- iarnsters at , ne 5ez: n.:ang of :he ch ror.ic i:.ialation .s:ere w•ere no abnor:-a1 :::.croscopic :indin¢s or. their livers, spleens, kidne.•-, acren a'-s. -earts, 1•..r.ss, bronc: .. tracheas, :a~.-~%es .r heac_, u:... ce ._..c..i fe: exceptier.s: In the yo4n_ control ha:r.sters cf the cne an.:ra:ec:oJic cor._ .:.rr::a:icr. .-. :.e lu :car._ .: e-e -e tnree ar.irra'.s :r.... girE :.. =wc _-, c..ntro'_ har::_-_rs oi n_ .7. 2;. li::e also ..~_ Eiro itis, and t•a•o .._:.ers e\.:.:b:teC an .. ......^:JCrtanC ..'.e:. :es . .. e. C ne w :t: s. .._:ar:'..^".aticn an_ .Ge ct :_ 7 t.t an ... ;_. re.^._.. ..- :...... 7cC ...- ~.QL)La~es. DSC'_S J:C' , The results presented here n clearl.: demonstrate that inbred strai:.s c: the Syr:an goldei. harnster are eminently well suited for the srsdy of the biologic effects resulti ng from in- halation of whole cirarette smoke. A. Effects of srnoke i: zalatior. on vario::s sites or cr¢ar.s oi t::e hamste r Proloneed smoke inhalaticrn b•: hamsters fror.: t.c•c inbred strains producez~ at least 13 distinct and clearl,: defined pathologic and/cr ph•: sioiogic changes in the experirr.er.tal ar.in:a'.s. -.2 0 - rTr C~"~' ~'"`~~ 4~'~ ~'413~x"'

(:) _. .:.e larynx (a) The occurrence of advanced degrees of pseudoepitheiio:r:ato-us r.yperpiasia in the larynx, verging on, or actually reaching the stage of carcinoma, especially in the 15. lb line (19?0) and, to a:esser extent, in the 87.20 line (4°'o). (o) A hig : incidence of general pseudo- epitheiic:::atous ^yperpiasia c: the larynx, more marked in the 13_. 16 lir.e (4C1~-) .:ar. -.n the 67. 20 line (13o,•c). ~c) =:e aopearance oi laryr.gea: capi:icr:as in ect: s.rair.s ( :,.- •: e.:. ._ i: ne and 2-21~- in the 67 . 2"v line). (d) .. ._-ge ..,.n:oer c: ani^:ais ...t : iarv^Eea: :,c:: _..a.ns ... .te i5..,, 1:ne and ..n t::e • 2: _.nej• :.._ ... .:.ecC n.....n-.eaa were c:,.- iete: aosen. ... ..._ cz...-rc- a:....._--. _......._ _\.~'i :ez.'..~....._.- _. ~..~. s .-•a:.`:s ~~ . ..n tne ._.._ ...... C::z 27: _ _ ~. . .. . ...c . . .2....'~~ .....:.C in L..~. C7:.Lrv.1 a......... .c:: .:.etapias:a ..n tne -rac:.e:: .,a- ar. ..._rease= .r.:.a::-.s:_r_ ,,. ^e ~-.2;, line, as a re s:._. .. an- = . . : the cor..rc: an..-._-:s z .._~. ... (3) _z -r.e ;.ror.c::: Bronc :ial squa^:cus metiplasia was significantl}- increased o;: smoke i:alatio- in hamsters of the 8',.20 line (from C and -;"~ ir. tne cor.c-o1 ani: :ais to 21176 in the experimental ani:r.a:s;. ( -1) I-n t: e 1;:r.cs (a) Large cl;::rtos of oale golden r.:acrop::a_es aoaearec ... t: e-_. zs e: srn oi<e-excosed hamsters. Th?s phenon:enon «•as par.:c•.:lar'_,: str....r.e in tne c'.2C line of hamsters, where it .%as fot:r,d ... 52=~. o: t:^.e a n_r.:als iz :aiing smoke, .ahile only .- to -,. c: _21. Lx imf"r. i~iN 0'".•i`2'T ...RG

the control anw als exi:ibitec clumped macrophages which were much smaller and darker, however. In har:sters of the 15. 16 line, 53°'0 oi the s: :oice-exposed animals exhibited small clumped macroohages in the 1 ng, as oooosed to only 0 and 6% in the con- trols. This observation is a clear and uneouivocal indication that smoke has actually penetrated the lungs of the experimental ani- mals. (o) There was a considerable increase in l;:ng weic^tS caused oy smoke inhalation :r. hamsters of both in- bred lines. (5) ::: the r.asoo::ar•.r_x Twc r.asoo-.arrnEeal t-.:r.:ors were found ..n s:^oke- e: oosec : a r.s:_. o. ... ..r.e nor.e ... tne co...roi ... .: e ne-2., ka) Myocard:a: cege::era:ion sor.:e«•::a: :a:e^. ..n s:":oKr-ex70seC : ar.^sters c: t::e ... iu l:ne :.ar. ... .r. .:e-.- i ar.. ais _.:e n•.....~er o: c~....:. :.ams:ers .n r.e :,-. _C line u•as t;,o s:r.a'.i t.c draw ava.== conci::s:c- -. ... .-c ca:a ..n taa: _ .-ai... w:..= t:e :::\'oca'o:a: Ce'n°.n era:..... ~. ._ :_a-s:~-~. an ...crease ... the r.ear. ••teis:-ts ... ( 7 ) =,,.., •.t•eior ":s 5:':o'r.e ..^..^.ala::on markedly anc S:Q.^..-.Ca.^.t:. 'eC'..=-_ ..:.e GOd.: •.,,eights of .'.a::sters from both inbred lines. B. Differe nce in response of two inbred strains of hat::jters to smoke inhalatior. The two inbred lines of hamsters behaved oLite dif- ferently with regard to t.he biologic effects of smoke inhalatior.. Hamsters of the 15. 10 line appeared to respond more markedlJ to smoke ir_:alation tharn those of the other strain by producing more laryr.cea: pseudoepit:^.elior:atcus hyperplasia in general, as w•el: as rTP rnKiT~~F.n .~.('! '.. () () a c,rR MN t"..422-49°e"'

more of the malignant or oremali¢nant type. Nasopharyngeal t•,:- mors have only been oDserved in hamsters of the 15. 16 line. In addition, myocardial degeneration and increases in heart weights as consequences of smoke inhalation have been noted essentially in hamsters of the 15. lb line and not at all, or to a much lesser extent, irn those of the other line. On the other hand, smoke-exposed hamsters of the 87 .20 ir.bred line have shown more frequent and larger num- oers of ciL.^.:ped macrophages in the lungs, as well as a higher frequency o: scuaraous rnetaplasia in tne trachea and bronchi, tnan harr_ster s c: the 15. lo line. There was no oovious difference oetween these twc :a:nster iines ..n the response to smoke i naalation u•it•rh regard to ^e Gcc_r re-c_ oi t-ac heai a::d iaryncea_ zamillomas, cf lary'ngea: 1e,:kcola:.ia. _, tc the c^araes ... 1•.:ne or total oody ?esocrse hamsters to sr oke -:a:a: as a •:,n -- curat:crn of exoosure I _: e..__,:ence of , ne 1er.ct: of time r.ams:ers were exposed to smoke ir_Za'.at:orn is, cf co'.:rse, c: sor::e .^':?or:an ce on t;e phenor.:eza ooserved. Before cO w•eeks c: s:r.erce exp._re, , ne onl• .^.istoDathcie;:cal :eaor er.a GDSer.-_ were some :ar}nceai ie :koplakia and sor: e pulrnor.a-•v clu^:oe= ar.. r-.acrco::a?es i•:.n no more t-:arn app roxi:nate , one-tentn o~ the :::a:s ..n w:zic: .=ese renor.-:ena occ•-rrec d_-iag tne wncle excer. .. w i' weeks c exDosure :: smoke, a-e•A' O: :n_ tuc 'ary :eal aa~ .rac.^.eai papillor:as had ap?eared, as d:d the r.asop :aryngeai tLmcrs. A small number o: animals eti:.:bited pseudoeoit:;eliomatous hyperplasia in the larynx, and some mvc- cardial degeneration had appeared. The most important changes occurred really oni•; after 75 weeks of exposure and, in many instances, even only after 90 w•ee::s. By tr•at time, saontaneous mortalih• due to var:- ous causes, including in particular a:nyloido.is and some bronc :c- pneumor.ia, : ad consideraDly decreased the number of survivors, :::is n:ortali:: appears to be quite normal for the hamsters wit:• a naL life of approximately 80-90 weeks and compares favorably with the s~;rvival of other small laborator;: animals, irn partic,,:?a- mice and rats. -Z3- r~ r ~ •t ~n W T P r) r != CTf"~'. { f f -''{ 0""I' 22"°f' 9-„

ft D. L^.fluence of smoke inhalat:on ov hamsters on their mortalitti` Since smoke inhalation does not appear to reduce the lon¢evi:}- of hamsters of these two lines, greater numbers of animals were actually available for histologic studies in the ex- perimental than in the control groups. The design of future ex- oeriments will have to take this point into consideration, and larger numbers of ara.:,als will have to be included in the cor.trol groups. _. Si¢^:_:_a::ce of :r.e proper control eroups The practice o: maintaining simultaneous control Zrouos of :a:: sters i n this experiment, including a group of ani- ::als exoosed to sTMokit:g" conditions, was most val•aaole avoid~=e err o^eo•,:s conci-.:sior.s. Thus, adrenal AB-cell per- olasia occ•.:rred ::r.ere frecuently in sr.:oke-exposed tnan in cage- :eid cer.trol ar.:::•:als, ..n particular irn the 87,20 line. This :)het:or::e^c: c..uid possibiy be a stress effect, however, produced ov tne for c.-.'.e conditions neede^ for the exoosure of the anir_als o s:=-:o::e, certainiy no: a cor.seouer.ce of smoke ir.i:aiat:or., as is e%•:cencec b%• tne si::a:ar eff2cts seen ..n tne experir: enta. EroLp (exposLre to s:-oke) and in the control animals subjectec ...nditions (Taole XL:1, column vi). Li::e- %c•ise, tne =::ieen u*e:_ ::s of 5-.2C har.:sters exposed to smoke :.. halation tended to oe considerao:}• lower than those of the cace- r.elc cer..-c: animai_ c: t: e sa^:e stra .n i Lp to 90 weeks of trea:- :er._, _t were never =_:g^if:car.ay di_fere::t from the splee:: c_ ~-• 2•_ 4ar s:ers exccsec , sham-sa:ok:^g cond:aor•s (Tac:e }: -- :, o: tne :nrec hamster for smoke inhalatior s' ~es The re:::arkably high tolerance to the toxic e?fects of innalatiorn of cigarette smoke of ha-nster s in general, and of certain inbred hamster strains in particular, bears resemblar.ce to an equally high threshold of nicotine toxicity of hamsters, as compared to mice and rats (Table V, Figures 1 and 2). For th:s reason, the dose of cigarette smoke that can be safely withstood by the hamster is consideraol.: higher than t..at which can be ad- ministered to other rodents. Conseque ntly, a faster and/or more marked response to cigarette smoke inhalation is produced in tr.e hamster t:an in mice or rats. This advantage is further enhazcec by the use of inbred strains of hamsters, where one or the otn er -2g- rTP C"' T R N N 0 4 d 2- 4'~~

of tne responses is augr.ented and sharpened, suc:: as t::e indLc:ic-. o}• smoke i.^..:aiatiorn of neoplastic or pre neo?iastic pseudoepithelio:na- tous iyperplasia ir. the lar,vrK of the hamsters of the 15. lb line, t-.e occurrence of r::yocardial degeneration in hamsters of t'r.e :5. lo l:ne, or the formation of clumps of rr:acropha¢es in the lungs of hamsters o: the 8. 20 li ne. el'~~: YY' I R N/ / 0422500

AP Y Dail~: i-= alation of high doses of smoke from Kenn:c:,-;r reference c:_arettes ov male animals of two inbred strains of Svr:an golde.^n har::sters for frorr: 40 to over 90 weeks produced the follow. ing pathologic and/or p: ysiologic changes: (1) \eopiastic and/or preneoplastic pseudoepitl;e'-i- o:natous :^.}•-~erpiasia of the larvnx in up to 19?0 of the animals and ::-.iider _„-..._ of the same oaenomenor. i: uo to 4O7o of the hamsters. :amster=. ('_) _ar• r.gea: papilior.:as in '.:p tc 2-"c c: tne ani^:ais. (3) :.ar. -geai ieukopiakia irn from -C to 53='- o: t::e _.._-eas?s ... .: e .req%:er- of occurrence .." sc'-= i: .-._ rac nea. ..ronc::" ar.,. '.-,::is. k:~) -..e ~ea-ar.ce o: larse c;;.r-:ps o: r.-.acrc=:a;-: __ :- a ...~ : __ ...:.: :_ ... - o: ....s -cr. ... " -.._ r f :.. ..r ...crease ... -.Z. cj .=:-=_se_ ... near- a nc v.•:•~~._. (i0) =.emt.r:.aD:e decreases ... ooc•: wE:_ .ts. At least 75 weeks of exposure to smoke iti.alaticn •s as needed to e-*:cit most c: these responses. :he ooservation of cl•,::::-s of rnacroo: a2es in t: e 1,.:ngs of the antmals after smoke exposure ::ear:•: shows that smoke act•.:a:'.;: penetrated their l-,;ngs durine inhalaticn. There were obvious strain differences in the resoc-.se to smoke ..~: alation oe:w'een t::e t•.t•o inbred hamster lines st'.:diec. The aoove findings, togetner .rith a remarkably ., tolerance of tne han:ster to smoke inhalation and to nicotine, ma::E t::is species in genera:, and inbred strains of hamsters in partic'.::ar, an excellent and most suitaole experimental animal for the sr,:d;; c: tne biologic effects ef cigarette smoke i:.'^.alation. -2b- ., , r-'TP ~TR NN 042-t-501

Table I Chror.ic Inhalation of Cigarette Smoke in Two Inbred Lines of Hamsters \umber of Animals per Grouo Group I L^brec Ider.tiii- i iine oi * i Ex0ose~# Shan:-Smo~ed ~ Cage-Hei~ cation i ~ Hatnscers II to Smoke Controls ! Controi_" I i i ,_ I f v\ C i D 5 .2C G~ I eG` = cG : Males, .~,e;s::t at be¢:nninc. .x:c= ca: ;,er...:e -;a.;s ea:.. %tee~, ior eiz:.......; te- eac.' r.r e, each ....n.:te c_ e::=cs::r- .:.::sistir.e of ..,-c seco::ds ..: s::.oke generat:en. .: seconds c_ actual smoke expos•.:re anc .. sec- cnds o: eY?os::re to ires: a:r; smoke was genera:e= -re::: ::en- c. :.1 re'e rer.ce c:-arettes, and s::.oke cor.cen.:a:ion ... haied :-ne was :~.2~'0 o: that egress: _ a: t::e ..., ~t: en.. ..- a ci2are::e. _ reated esact:v '.;:;e experimental animals, excert t::at no ciga- rettes were in serted into the smoking machines. '- Received no treatment but v.•ore felt collars. = Seventeen lots of six hamsters; animals of each lot were housed in the same ca¢e and were treated simultaneously. ; Tere lots oi six :a^:sters; animals of each lot were housed irn the same cage and were treated simultaneously. ~':en lots o: six ::ar::sters; animals of each lot were housed in the sar::e cage. rTP r?f^T= r':r1a (-11 p Cj CTR I-IN 0~-~*22502

T able II Date Schedule oi Staggered Beginning of Experiment , \ur.:ber of Animals Started on Experimental Conditions' Group A I Group B Group D Grouo C 11/2i/i0 ! 11 8 I I 6 I I 12/2 j iS 12/:S 6 I .3 I „ ~ I 12/2~ lA ~ _ c I I _'- 12 ; . ~ .' • . , ~ . ~ . i 6 f Total I 144 (:02) i 60 I 60 6 ; o :2 12 Group E ~ Group F 6 1 1-~ (102) I 60 j 60 t SmoKe exposure, exposure to shamg smoking con- ditions, cr caee hcid:r.s. '~ T: ese lots o: animals were to replace previously started lots oi hamsters which had been lost due to carbon rr.onoxide poisoning during smoke ex- posure. ~-Nurr.ber oi hamsters not including the replacement animals (see second footnote above). (''T C.~ ~!7N4.~r ^('rr', rlq(1 C T R M N 04 22 =5 0~'

i Table III TOXICITY (LD_ p) OF INTRAPERITONEALLY I\JECTE~D NICOTINE TARTRATE Test Animals ~ O Statistical Significance (p-Values oi Differences between Species or Straias Soecies Inb:ec - gl~g) \ I I 1•iamsters ic R t i I St a I e a s i l 2. -(67.20 I RB 182. 62 i Mice' 0.01 G.Ci i 0.0: 0.C11 0.01 C: i i I ~ I I ^ Ra:_ ~ - I C. C: ~ 0. 0: 0. :. 0. = : ar::_ter,; ~ . : i Q. ~ i 0.C5i C•.G= ..C~~ i 1 B I _-. ~ G. C: - ! - I I C. C r:. C•= ~ G. C= ; i ~2. ?~. C. C: ... 05 ; G. c C. .. C. 0. ' C. C•c G= ' I , r C:a'_e s r.: e r CD- :• r, a1e s, _-.-ee r.:on.::s .,.... - Czar'_:. CD, males, s?t ,t•ee:cs c:c. ? D:_ :a :=z_rs. _:c... =F.i.: CC. 5 ar r7.arbc-, \;air.e, rna:es, t :ree r.:c:::::s oi=. i TP r-rF~-pP fQr-R= CTR MN 0 4 2-50 4

i Table IV INHALATION TOXICITY OF WHOLE CIGARETTE SMOKE * IN RATS AND FOUR I\ ERED F-LAMSTER LI\ ES =est: aimals ~ Statist:cai Sieniiicance c: i S-u:- -' Di:ie:er.ces between Species and St:aias T.^-e' I lir. . =c' :~c. Harr.ster_ S:)e_ies Rats i 5'.'G ~ : - ~=z= =====_ I C.O- - l-• -.') . I<C • CQO.5 i<V. ooo?i <,. GocC <. coo-; 5) <0. coo: ~<C. 0005: <. coo_ <, co: <C. oooz .oo~ -;) <0. 0 0 0: ; <C. c005! i =i <.OOC? <.•:,:c~ ~ - -,.... _._..__.. . - --_. _::Cc .. _-:_:tE-, .r•.D. _CC--2J E -- - _ - . ~ ... _ '.i'^: _ ~. secc:•cs s~:c::2 es- __:~ , ._. _ lLtec) c•..--::s eac : -_ sccc^_ c; -:e. S a :~c:c ._• oare=aeses. tT m • - r 1 :r ~~.. r r 1 'r {s , I L T R M N 04~." =5705

Table V RA\KL\G OF NiICE, RATS AND FIVE INBRED HAMSTER LINES IN ORDE 1 OF DECREASING TOXICITY TO I. P. NICOTINE AND/OR I\:iALAT_IO~ C= CIGARETTE SMOKE T est Animals Toxicity * i Ir.brez .ti^.a:a:icr. c: Whole C'.gare::e S:::oke I { Intr I of ape Nic rito or. nea: Ir.;ection ne : a: t-ate j7ecles L: ne _ D , - -~ (: ...._tc S) 1= =! `= M:ce =a^:ster s ~ --ache:s ..._.ca:_ ....:e-ences i n t's.e r.:eas..:er:en.: bet..e=.. ...., =_os o: t: at are r.o: sta- _..~__ =:s :... ar... r'r,P rnHTPnr"T'`-, CTR HN 0420-=506

Taole VI Body «'eishts of Hamsters Sacrificed for iiistoloeic Studies :-ength ~ tat istical Significance of of ~ ='-rr.e ( In- on ibred = *t Body Vv'ei_e :ts of i-iar^sters i (.Cra:rs) i I Diff ioi T iore I erences wo Grou d Line, between Means ps of Same L^- (t-Values)' ::pe: a r .::ental ; ster onci- ' Li ne Sr. oke Expcsed E ''•S^a:r-Sr.:o-~d" ! Caee-rield G xpt:. ~ roup I :xptl. I Group iCo ntroi :io:,s Group ; C:.ntroi G-oup ~ Control Group ii Ve rsus i V ersus I V ersu : ; e e e•: s.i ; Grou?, Contr.,- .) ; (Control 2) !Ccn trol ;Control' iCor.troi (a) i (ol (c) l~) (e) I _) (^) - -c:. C (c. 2. -j 3C ~ c. _ -i.45 v- 7_ Cz. 7 (-.=3/'i /0) - ~ - -_ cr 1122 -i .:2. . (22. C , .~j -. ~. 3 (F. 32 / 1 ) - - >Q0 ~. i25 , _-, _ i.~. c (i.6., _) .:-. . ( .'-/-) ; -3.- (^.'.2/c) 7.'= r.6- ~ . ~ =.-7_ ::-.: CC/") _-.. .=) S i - -_-c~ i.2 ( 1,.G5 /22 ) .- ( C/o) ~135.4 (1-.69 ~17) . . .. = l .. l 3 / 3V) 12) / 144. r (1 .. 02 ~ 1 ) . .. 2 ci ~- ~ ~ . only an: .a:s sac.:.iced for ..istclceic ?8.. :all._s• s:anCarC C°_ :at:Ons in Dare::..^.eses be:..re ..._ slant: z• r b_r_ o: ani~:als :n Dare nt::eses a:ter _._ s lar.t. Statistical sieniiicance is at the 117,'o con'.dence level (p ; C. 0:), unless the value is set ir. Darent.heses to indicate sier.ificance at the 5~'o coniide::ce level only (p S 0. 05). When ne t-%alue is given, difference is not statisticall,r significant. L^.sufiicient data. r H TP Q r"r- ~ (1(i1~,, (~',qa CTR MN 0~-~~~t ~"

TABLE '%'I: Day \u: n*:)er BLOOD CARBOXYY.EVIOGLOBI\ LEVELS IN 15.16 HAh1S T ERS EXPOSED TO SMOKE INHALATION FROM KENTUCKY 1R1 CIGARETTES T i.-:ze of Day ~ Blood Carboxvherr:o_gic'bir. L-evelsT ~~ . ~ in Six Animals : Sr•.:oke _x?osure I C]lecotion~ I 1 2 ~ 3 :0 a..-.. C.'_ . _ ( _..._._ .') :'_ L 13 (Weeicenc) :..~~ a..... '_:~0 .... '_.~.,-2:cC ~..... ~ ~ . ~ , ~ a..... '0.:: ~ C.3- . i . I . I . . --l a. .. .. C.:= - .l:C~ ..._. 2..... . _ ...~1i-_.=.. .... _ . : - . ~ :3 i, a..... i (•. C :Oc-?C a..... - D.'.. ..:3 V-2^C r 10:00- 10:20 a. r::. 2:30-2.5 0 0..... I _n - :.o . -.-. a..... 0. C. GC ,:. I _ :00 D. n:. 9:30 a..... 0. 18 0. 32 I 0.'9 C. 1~ ' C. C * For carbo~,'r.emoglobin determinations. * P.eiativc amou=ts ia Der cent of total hemoglobir.; averages of triolicate determinations. ** Pretreatrr.ent levels. '- Tw•o 20-:r.inute exoosures or. each of these days. = AU animals survived. C ^~r !~' /F 1'RP-Zr !R•_~~tr1 ~ (1QL, "~R -HN 04,~,~j~~8

TABLE V11I BLOOD CARBOXYHE1v1OGLOBI:~i LEVELS I\ 87.20 'rIAZ.2STERS EXPOSED TO SMOKE INHALATIO\ FROM KENTUCKY 1R1 CIGARETTES ~ i i :i_^ze of Day II Blood Carboxyhemo¢ic~ci:: i.eveist Dav \ --r ::ce r in Six Animais i Smoke Es:)os; re I Blood ~ li i I 3 . ~ I ( Collection C. 0o i ~ . , ~ _ ! iC:00-:C:_C _ :C:3C a....• ; ' 3 3.' - ; 2:ZC . :3G ?:~ - ! C ..:OC-.C._. c..... , ,.=G a..... C... i .- I - ...3i,-_.=. : .... :3C ~..... ~ i - = l =~-==a:~/ =:3G-O.:G ..:l2., c..... _.~V-~.-. ..:. ~ t ~:~ ~..... 3:C'0 0. ••.. 129.6 1 i C.- C• ~c I ~ ,::C 0. 83 - ~ 1 i For carbc:ryherr.oglobin determina:ions. Relative amounts in percent of total hemoglobin; avera¢es of triplicate deterr:-::::ations. Pretreatment levels. Died a fe•x• hours after smoke e.:;)os•.:re. Died during the holiday. Died durine : :e weekend. -17 r~ C~°R H~~ 0 4 ~ ~ -0 9--

: able Lt: CARBOXYH EMOGLOBI\ LEVELS I\ HAMSTERS AFTER PROLONGED EXPOSURE TO CIGARETTE SVIOKE Inbred Grou? No. of 4 No. of Blood Carboxyhem oglobinT l Harnste r Desic- Weeks of I Anima s Line nation Exposure* I Tested I Mean Value ` Range i :3 :. ~ C•.6 i C.7 - 0.~ 0. 1_ 3. 2- , `. I G. C• - C. _ 6 -. = - - ' ~ C. l . _ 0.0 - C. _ exposure. ....ce a ca}•. :e cc•: s a w•ee:.. I:. :)e: c_..- of te:al 'r.er.:oc-lebin, deterr.:in ec on a\_oncay morning beiore smoke ex- oosure . CTR f f f'f 042510

'abie X :r.brec vro;:o -ar ste- Desi¢- _...... .:d::on SerLr : T ri_1.•cer:ce Levels in Both Inbred Lines oi Hamsters after 45 Weeks on Experimental or Control Conditions Serz:r^ :riglycerice ~ Statistical Signiiicance o: Diiierences ~:etween Levels (r::g/1C: m1) i Means of Two Gro::ps, t-Vai•_es* T StanCarC \:ear.s' De ::=tior. I .~.. C . i ....•.:~ CCi. I f Cro ~:?;D ~ CroL? bi~. i i . ~ ° --~e_ .,- less .r.an _. - ...- t :, aosenc_ :_ stat:_.._I_ : .a.. _ . t::e cire :;:, ar.c .::ese va:::es are -ct e:::e-e= ... ..•_ ta :.,. ca-._ ee.. .c_. _crc cs o: t :e sa:r.e .-brec ..ne .._ _ :. H. C a-_ C.. ._- *.:e ._. ._ :;-_ ar._ C•r__c_ ~. ~. - a.... _ r __- ..._ _'. ~. ....~. rx ~a. `:,n,-C: a'..^.'.a-= A'.tflol:t felt collars o: 11,7 _~ grams e.OC K'elC n.. r-. r% •q- r-% CTR MN 042-511

i Table XI Vi:-us Profiles in 15. 16 Harr.sters aite- z5 Weeks on Exoerir.:entai Conditionsfi Virus Profile in Individual Hamsters* ^,rouo ~ i Desig- ~ Treat:: ent cf A::-:r.als ! nation i , $: :OiCt- Lx:)CseC ~ Saa: :a^noic_._ Cc:a-.- ,~ r Lz Seru m ~ Ln Splee n He:r:ag tion-L^ ?es glutiza- zibition t"* ~ Comple ment Test Fixat ion HALV - i ! Anticen ANT AC I ~ ... ! Ses:da: ' \i.A c I m. HV I _-C~S~ F;CV 2 _- 1 r . , =l :_: ~ - I - T- , I - - T- , - C, a: I - .:_. .._C• , _ .,:C r v . C , - ~ - ~ . .:~ a. ac a: ac C , - - C ~ ; - v . ~ .:T~. . .. ~ _ . - i . . J ac i iac C _C, . ...._.3:ec i Cor.-rc: ac __ ::50 1:10 - Tr Tr i C -~ „ 1:20 _ 0 i ~ T- ~ 1:80 1:40 _ ~I 0 i „ ~I 1:so 1:20 . ! 0 ; , ii 1:40 1:40 _ N C I~ t Except animals of Grou? CC which had not pre..ous1y been unde- an}• experimental conditions. ~ Laci: line represents one azf-:.al. GDV li, I''.2o3, 1:, po1Vo:-^-a, .`iV:.:, ::RV, 1•:-1, SN5 and Ectromelia tests were negative ..n all instances. CTR HN 042512

j Table XII Virus Profiles in 87. 20 Hamsters afte? 45 Weeks on Experimental Conditions * Virus Profile in Individual Hamsters I ~ ;1roi:o ~ Desie- Treatment o: Anir•als ::ation • In Serum I In Soleen i. ~ 2 I- j SerdaiT %:.Ad.1MHVILC`1~RCV1 ~ ~ i I ~- i j Hemagglutina- 1tion-Inhibition Complement Fixation ~ iLAL~'- ~ Test** Test Ij Anti¢e:: ~ D Smoke Exoosec ~ .:20 ~ i:20 ~~ 1:?^ ~ 1:?: i ~ :20 .. ~i • . ..-J ; .._.. ~ :2G .:2C :Tl :=0 . .. ..=C ' .._. ..-: ...G . .::G .. . ...~ I A\ T AC ~ Tr C j- - Tr , . T - , 1:Y0 1:z0 1:20 1 :20 1:40 1:40 1:2 0 ac ac ac 1 T- - ~ l jTr _ _ ac ~i 0 ~ ' tc l' 1 r T r ; 0 ~' ar . ' 7 r j .. 0 ; 3 -- IO * Except animals of Grouo F F which had not previously been under any ex- perimental concitions. * Eac: line represents one ar.imal. ** GDVII, Rec 3, 'r:, Polyoma, %`-%'\:. 1•:rV, SV5 and Ectro:nelia tes:s were neeativc in all instances. C1 :20 1:-~}0 1:40 ., `i 1:80 i T. 1 ~ 0 C Tr C Tr Tr G ri' i1 ! 1 HT'f i-.i 1 T". (1. ~~ i l 1(~ CTR IMIN 04251~'~

Table XIII Organ Orgar. Weights ... 15. '.6 Ir.brec r'-arnster s after 45 Weeks o:: ixoeriz•.:entai or Control Conditior.s Group Desig- naticn I Gro::p n i Grou:) C I: rou~ CC? I --: E aac trachea I A : - o _. G ; 3. c ~ C CC. _~ -ear. ' B ! C CC' c c. - 3 _ ` C CC~ -. -- ; Sp'.eer. ~ra:.. L C l.. ~ Statistical Significance of Differences Between Mear.s o: Two Groups, t. 'v'alues* lGroup A i -.3 i C T CC ~ 1=. SIt 2. 6 I I Crean Weights (r.:g) ` Standa rd \:eans :Ikviations of Means ~ ~ I : 2_ a . ~ - . .7. 2" ~ 3 - A ~ 77, ~ 19 B 3 C 9- I 14 CC1 A I 981 ' 43 B ~ 852 i 119 C, 4 928 I 97 CC , 5Q- . i 5 7 5. 3 _.6 Pituitary- A ( 3.21 1.16 ~ B Z.o' 0.52 ~ C ~ 3. 7 1. 53 CCfi 2.2I 0. 39 Continued on next page. L..r T R - i f i 7 0 4 2a...-' i. 4

.abie XIIIContinuec 'ootr.ote s ~ t-': alues o: Iess than 2. 7 indicate the absence of statistical si¢nificar.ce of the difference, and these vaiues are not entered in the table. an.r.:als without felt ccl'.ars of '-a:.`._ \i'e:t ::ss T >= n ~'~ - ~ i^'. ~ ~, ~' t. l'~', L~ ~ (' 1 CTR if-IN 0422515

i a A k Table XIV Organ «'eig'r.ts in 87.20 L^.bred Hamsters after 45 Weeks on Exoerirr_ental or Control Conditions ~ " ...~ i ' m ! S Continued on next page. W QII • ) W i h O Statistical Significance of Differences * Grouo rgan ts (i: ig e g Between Means of Two Groups, t-Values Organ Desi¢- ~ ~ Standard nation i \4ea ns I Deviations of Mean s Group D Grouo E I Group F Group FF ± ....L:n£ a.^.d trachea D ! 1 ~ 4. ~ ? C ! - 229. ~ I - I 3.0 c') c Z. _. -eart D ~ - 8 3 i 2 3- •.r.+ .0. 7 3 9 - C.' : 2 2. _ 5, ~: ~~_ 0 - i::c.^.e•: s D ` >>_ -_• I 2.c _ Z•- r 1, .,i_'' F-' 2 ~ 12. i c D .:. _ ~ 2. .'. ~ ~ 23. 1 ~ _. :3 ~ 3• i i - - _ : 3. 8 I -. o C i - ~ - ~ . - - .= 3 G.99 I 3.2 ! - i S?ie e D ~ 62 E 96 I . 124 FF' ~ 68 15. 9 I 36.5 27.6 , 10.5 - 4.8 - - I I - Y• b - - 4.6 Brain D ~ 985 112.8 - - I - E 1, 04c 207. 6 - - I - F I 995 88 - - - FFT 897 45 - - - ~ : it•.:ita-. ~ D ~ 2.8 0.81 - - I E I .. 1 2. 32 - - F t ~ -1.9 1.94 - - FF 2. 5 1. 01 - - Lr TR Nf-'{ 0""'f' 2-a.•.} .L 6

Table XIV Contir.ued Footr.ote s ~ t-Values of less than 2. 7 indicate the absence of statistical significance of the difference, and these values are not entered in the table. ._...:al control an:r.:als without felt collars of 107 - 3 grarr.s body weights. ti 1°r.'~ !% ri~.JTf-~r~`~'~' (1~(1~ i (1d CTR HN 04'2"=*I-(-'-"

Table XV i a'elative Liver Weights * of Hamsters Sacrificed for Histologic Studies :.engtn I Statistical Significance of of I ( I * **t II Differences betweenMeans -irr.e In- P•ela:i•:° Liver Wei¢hts of Hamsters ( of Two Gr oups of Same In- on bred (Milligrarr:s) bred breci Line, (t-Values)' eri = :: Harn . p - - ::-.ental ster i Exptl. =xpt1. I Co^t=a} ;:ondi- L ine Smoi;e E xocsed ! "Sha: :-Sr :oiced'' Cage-Held Grouo Group ::ons Gr ou! Control Group Control Group ' `:ersus Versus e ;: 22 Ks) (Exptl. '_~-__o) f (Cor.troi :) (Control 2 ) , Control : Cor.trol2 ~ Co .rol °) (b) (~) i (C) I (e) =2 0C (.2 7: (3.- ?) :v--5 " 5 750 (.0.0, -) - ( - /=) 4-10 _.'.'.-) (5i5, ~.7) 7VV „ 5 000 ! '...._:) =7 3 " ( io05 c) I I 391C (332/0) '' (2. ~c) (2. 0.0) - ( - /0) i - ~ -_,0 (72?/11)~ _ ~ . i ~ObC (i-ioi i_) - _ -_ 6C 3020 (2--/ 7) ~ =010 (429 /6) (_. i;0) ---0 -!7C (=o2=0=G f335 1"i .700 =4sC (Sc o) ~ ~cC ( oo/.-) l-; '' -~=0 (^-3 _ 530C 5760 (2430/-) - I d1v:CeC Cy individual body vPeiC :ts (i.. £ra...5, _. .- anir::ais sacr:iiced for histoloeic s:.:dies, ..... ..s aa::::als that cied spontaneously. tN1ean val•_es; standard deviations in parentheses before the slar.t; nunbers of animals in parentheses after the slant. Statistical significance is at the 1°•o confidence level (p S 0. 01), unless t:e t-value is set in parentheses to indicate signLficance at the 5;, confidence level only (p 5 0. 05). When no t-value is given, cif.ferer.ce is not statistically significar.t. ~ Ir.su!`icient data. m S r ~..n W r M t r'F115 f"'nWi'GI 'r'?r Irr n r1{g i (, , t7 Cf R i i N tir/ '""f` 2"5.t. 8

I a Table \%'I Re:a:ive Spleen Weights ' of Hamsters Sacrificed for Histologic Studies . ~ _engtn t of ~ i _ ime ` In- on I bred =x~eri-i Ham- ' R.ela:e-lee :'~Veights; of Ha:nsters" + i.%:iili_ra^:s ~:ental ~ ster =on... ~ Line Srnorce L~?csec ' S:a:n-Sr.:cr.ed' c: s ~ Gr o~_ Con:roi G=cuz ee.•s Ex^:i. G---? -on.r_. - /t 11 Statistical S`.Lnificance of jDifferences be:ween Means of Two Gro::z;s of Same ln- bred Line, :-Values'•' EXptl• I Lx0-1. iCor.re: Ca£e-i-ield I Group I G., .; o . I ersus Contro- Group Versus i'~ e:- s u s I iCont ' rc_ . Con.rol 2 Con::-- 1 C a -b ic d' I ei i._.:t 7S.2'iS.5 - - 8 13.4-~. ~ 12 ..2 '3:.=. "..:. : Ci.2 1!1.; i/C1 , - I ....t ._.~'o'll - 1 -. 8 23 :2 - I -„ -- „ .3_ =Y•r"''.c•' - 3.' lB.On,6 .l-". - . : 5. 5 '_:. 6 3 . i..E. 3 c. :?s~ .... 3;.Zc11 ..c_..... .:r.:a:s sac:•ificed icr ..is:oic£:c s:sdie<_, ..c. ... cludi^ an :r.:a:s .::a: d:ed s:)cntaneo;:sl%•. +`Iean values; s:andard dev?c::ons in _arent:^•eses before the slar•:: nu.-nbers o-' ;::?r_:als in parentheses after the slant. Statistical sizai:icance is a: t::e 1?o cor-fidence level ip s 0. 01 , unless the t-•~aiue is set in parentheses to indicate significance at the S'', cc--:'.dence level only (p s 0. 0>~. 'A'hen no t-value is given, differe=ce is no: statistically significant. : Ir.suificien: ca:a. ~("'I- " 'P1+4r1~nP r ~'+..^ ( * ( ;r C - ~~1-R N N 0 42 Ei; .1. 9'

a 1 Table XVII * ?.eiat:ve Kidne}• N%eights o: Hamsters Sacri:iced for Histologic Studies i _engtn I c: I cn e bred ~ * **t Relati•:e -.-_ze}• Wei¢its of Hamsters (M?lligran:s) --.ental ster _i.-.e al (~) 11 Smoke Exacs_--: `:rCil.. (::xz tl. Gr„__ ~ :ar:-S^:o::e~ ~o: .r~l ,Cont. _- :) (c) (6) 2 _- i ---. C -5-,,. -, c. .') - l /=) C tatistical Signiiicance of Fifferences between I'Means ~Of Two Groups of Same In- ', bred Line, (t-': alues) i iiExptl- i Expt_. Cage-Held ;iGroup ; Gro•.:p Control G-o4o ''Versus i Vers••s Control : Vers;:s (Cor,trc: '-) C,ontrol : , Contrcl - Cor.troi 2 i (e) i ~3 ~. C ,;:.. ~c/6). .__~. 7 (:33.41i1) ., •_.....;... . , (f) ' (a! , (:'.I I ?. 33) ('_. ~3) I -.; c (,? !'. ' z. _ (3 . _ . "j ~C 3i1') - i:c~. ~3- . -=vo. _ _. . ~ 3 ~. . 3 ~l -,.. G 3. 31'' ) - i ..^5....:tt ... ~ :._. .... .~2C . .-^.C:.-_.:al bCC ....... .c_ - ..1. K 't ..:.__.. an.^=als sac:.:iceC :0:• rlstolocZc f:,:Q:eS, ..,,. .- _ c1L`C: n_ a^.=-.a.s .:at died spontaneo;:sly. fiMean val::es: star.dard deviations in parentheses before the slant; numbers o: animals in parentheses after tne slant. Statistical si_n;-:icance is a: the 1;o confidence level (p 5 0.01), unless tne t-values is set in parentheses to indicate s:gnificance at the 5=c cotiidence level only (p 5 0. 05). When no t-value is eiven, difference is not statisticallv sign-ficant. L^.s;uficier.: dat.a. 1 r'1 ~ C.^ L+,; N C r"1 f ' C T R H N 0 4 2- 5-5, .~0

t i able XVIII Rela:ive Adrenal «'eights' of Hamsters Sacrificed for Histologic Studies! ~ ezgti•r of _r e i In- Relative :._r enal :': eie^ts¢ of Hamsters'~ ~~ on I bred llicra-as, ~xoeri-i I:a^ - ~ . :-:ental I ster or.d'. _iae S noi e se_ S :a:: .:.:-:^::ed' :cns Group ; ~o ..;.. Group ee=a i =xn:i. Gro• - Ccn.-... _ j Statistical S:_nificance of i'Differences 'ce:w•een Means ,of Two Grcu_s cf Satne in- bred Line, :-Values ' Exptl. ~ E~_-:. I Co-~1 Cage-i:eld Gro;.t? ~ er_ .s ersus Con.rol Grou_ ~'ersus ': - ~ Cc-.rai ,Con.rcl 2 Cc, trc't ~ Ccn.-_- ~ a: i b c d~ I ~e~ ~ -. c^ ~ ~.._ ;3.2 '._ :.:c _ ~ 1_.c 3.S2'c I..~ ~ - ~ - .'S.'. 13. 0c c - I - . ~ ~'1 ~ .. ?... 7_ -.- _ _ j 5 -.'_,- i ' 3 ' '5 ''f ' 13." z.6S': 3. 4. ~ - 'c. c. ,- . .c. ..5.1 .::.03 1_ , - 30. b 1-.3c : - ; - ~ :c:a: ;,. ._ a=-_n%a:s •.vere d:souali:ied and .ne:r adrenal •:Pc .-c--: s:a::s:ic, cecause oi :he oreser.c ece larze acre-._: •..•...,r s, r arg •-- .- size -. _... 13.5 to 2oS- ra. These 13 r.a^:s:e-_ .r.c:Lded three am= a:s exYosed to smoke inaala:iorn al: ci .r.e c-.: ..r.e ,.ive ani_:-a:s er)csecto sham-s.T.olCing conditions i:•xc 1=.._ and three E7. 2: , and fi•:e cage-held control animals (tw•o 15. 16 an_ three 87. 20:. = Absolute w•eigh:s, divided by individual body weights (in grams i, multiplied b}• i CIG. Including onl-,• a-.i^:als sacrificed for histolo¢ic studies, not includ:=: animals that died soontaneousiv. y •:~:ean values; s:ardard devia:ions in parentheses before the slant; nu:nber s of ani:^als in parentheses after the slant. = Statistical stgn::icazce is at the 11C confidence level (a 5 0. 011, unless the t-value is set in parentheses to indicate significance at the 57 confidence level onl}• (p s 0. 051. When no t-value is giver., difference is not statisticaliv sienificant. t Insufficient data. m ("TP n P C T R M N 0 42" ZE5 22 1

:•able 1::?: Re:a:ive 's:eart «'eie^:s~~ oz Harnsters Sacriiiced for Histoio¢ic Studies _engt^ ! Statistical Si?niiicance of oi • Diiierences between Means -.^:e .. :.elat. -e : ear. . eig'r.:s cf ta:-isters'" ` of :v-•o Grcu~s of Sar:e ..n oz i brec a:^a r ea Line, ..-~'alues ' =:-:er.tal ' s:e: Ex?ti. i ; nc:- _.::e S-no:;e Ex=_ se= _:& ... S:c-:ec ~ Ca2e-: elc Gr ouc I Cr, c. : ^= Cro . • C cn.~ c. G. _= Con:roi Gr cuc_ '~ ersus '•.-ersus j~ e. sus Cor... c'. i Cc.... .' ICo.... .: eeics -. . ;.c n:rc. 2 ~ - a c, c =_=.= ?=.= - ~ - Y 3 .,.... .. -- - - -` • - - I.~~ - S- .. - • - -- - - -' -- I---•. - ~- • - _:e :.:c• .•e: ... --a~.s , cr.'.•.- sacri?:cec s:udies. ...,. ...- cludi :z an'.:ra1s •ca: c:ed sflor.:ar.eousiv. ' Mean values: s:andard deviations in parentheses before the siar.:: nu:nbers of a n:r.:a:s in parentheses after the s:ar.:. Sa:is:ical s:_ n--`.cance is a: the 1-_ confidence level ;p s C. 01', u_iess :.^.e t-vaiue is set in 7are.^.:::eses to inc..ca:e si¢.^.::icance at the __ ccn:idence level or.l•< c s 0. 05.. When no .-value is ei:en, ci:iereace :s nc: s:atis:icall•: sier.iricaz:. - -.^.su:.-.ien• dc•a. 1 ~ ~ ~ T~ E.rT R H N 0 4 2- E522

:ablP XX Rela:ive iung <<'ei¢hts' of Hamsters Sacri:iced for Histologic Studies 1 er.gt: I Statistical S'.gni:icance of Differences '-e:•w een '.:ea: s G, ' ~ ' . _ =::ze In- «"ei¢hts o° Hamsters Rela:i-,•e _::n¢ of Two Crcu~s of Same _..- i _ on ~ ored (`:illi¢rarns • breo' iir.e, •:-Values'~= _ _ • _- -a::•1^ ...er`al i scer _•r.e :ee::s. S:noitie Ex=) cse~ S :arn-S:::c::rd Grou? Ccn:rol Group =sp:1. Gr., . 1 Con:r c: . Ca¢e-::eld Control GrouD Cor.:rol 2 1 al c c d I eI , ' I 1-,... :__ - il1240. 3-.,. c '_: c''-.31i~_. 2 c. 12 :3 . 2. 233.1 1S 3.:1 '12' ? 11: -_. 2.:~.. :_ 33:.. 1182. r'; 12-..3 r2 E fi.~ ::2 -. 1 -i~ 1 ._00.~ i: b 3, 5 E 6 Exp:i. Grou? 1 I xc. I , G= rcu::o ontrs... . ,. ersus • Versus ' o-s• ~s ~ ; 1 Control . :n.. , ( Con-ro1= ~ . I .'. .. i i 2._53 .'..'., ~ - 3. 2: . i'_ `10: . o '2=2. C i - .-_..~i-:c cr. a_.rna:, sacr...ce= for L:s:oie_:c a:::rr.als c'.ec soo-:aneousl.. +\:ear. values: standard devia:icns in oar=ntheses before the siar.:; numbers of ani:::als in parentheses after the slant. = Sta:is:ical sigr.i:icance is a: :>•.e 11~, coniidence level (p s 0. 011, :nless the t-value is se: ir. narentheses to indicate si¢nificance at the 5r confidence level orl,.• io s 0. 051. When no t-t•alue is given, difference is not sta:isticall}• sigr.ificant. _ ?nsu:••..ien, cata. r^nt-,fTP, 0 r"''`z ^ .n c.. i 1 r% _ L .rf1~°~'. f f ~ 1< "~ 0"", ~„ ~.. .~.. •JE

Table XXI Grouz After 40-oC Weeks i Numbers of Ar.ir-:als Studied Histopathologically * \umbers of Hamsters Studied Histopathologicall~: A:ter l After I After 00- 7_7 75-90 i Over W eeis I Weeks i 90 Weeks ^otal Numbers i ~e- ce::. ~ 7 (1? i C a ' (::) 31 (-) L i 29 (Y} _0 (2) . i ; 8- (22) ~ -2 ('C; . -0 ( ) -: C _ - CC~ c iC) ~ ._ - ( , - 7 - (; i - (C) I - - ~ , c (Cl =o (-i 6 (C~ :J . .'.: i _3 (5) ; 5; (:=) :2: C3 (c, ; 3-T ('C) •- ~ =ozi .._::.bers, ncludin¢ a:::r:.a1 s . :at --ere sacr::iced anc animals that diec s pon:aneoLsiv; t: e•.,._: bers cf the la:- -_- alon e are s:JVJn in 7arentaeses. T L-n perce.^.t of tt:e initial numbers of ani- =-.als (see Table I), referring to tne total numbers only. L^::ial controls; studied s:multaneousl}• with experimental and control anir.:a:s a:ter =C to 60 weeks on experimental conditions. S ~ .-,-/+0i a, r'n I I-f* P•l ."1 r~ T r .4 .-1 jr- 1 4 t Cr/ R / I ! 1 042524

Table XXII Cause of Spontaneous Deat: of Harnsters As Established from Histopathologic Studies Cause of Death Nu mbers of Animals That D ied S pontaneously Crouo A I Grouo B Grouo C I G roup D I Gro uD E I Grou F( All - + - ~ ~ p Cr ~ oup: :.er.ai a^:} _oidosis I _ i ~ 3 2 1-: ~ r or-c :oor.e;..- :onia i I , 2 Cac:^exia . .:ola::Eioca:-c'.nor: a i ~ 1 t ~ t --,-:r.o:^.osar cor.:a = 1 : 5 Carciac 2 _ ~-euice:r:ia i i Cere'--ral t:ro: -.,z~;is : - `:asoo :ar ;eal t-r--::or 2 ':assi:e acrenai :_:::c~ _ _ :,fedia=ten ...5 . - I - - , X= I ( 3 :11 ca,.~ ses 22 , 10 I 1i I lo i ; i; * Probable cause. ** In tissue examined. 1 Tf"', 1-1 i..r 7 R M 1 7 0 4 2 E } .IL. { 1~

Ih Table X 1:I? His:oiogical --indings in the Larynx of the 1 :5. 16 Inbred Line of Hamsters Numbers of Animals` ExDeri- ~ i reatmen: . ^ mental ~' ~ f ; ~ ~ PEH' ~ j S ::amster s C ondi- ~ o. o \o. o. Suggest.. C•r.rcr.:c qua- ; ~m~, iar- *,0r Leuico- Tota~ i nQ ~ Papil-, ~^:ous ti o^ s A'~to~- ~ .ra e s plai<ia l ?EL' j - ar ci C lor ia ( ~". _ n:a: : c I \Se: a- (\ o . o: • ~ s s ec. . ! Sacied I ~ 1 ~ no ^ '!a I . . ) I t)las:a ~~ee:s ~ I . . _--:JKe-7\~osec 60- 7~ 3 - I i 2 ~ 2 _ --c r ` ' 1 ' - . e O~ I 2- ~ _ Sub:c:ai - J 1a72Z-J::17YcC I I _oI`:'•C:° ... ~ 6 0 - ?, C SB~: .c. ~ ~= - C C C '~ ` - 1 _ -_ i ~ ' O.e: = i3 ~ i 3 - - - ~ - = ~ Sub:o:al 40 f63? ~ 1T i6S' C i0) 0 f01 C 0 ~ 0 G~ : otal per strain Ii 166 ` 109 [66] I 46 i=2) 19 (17) 119 (171 9(8) I 7!6) 2-1 (2? , 6'7: Group A. Group B. • Group C. = r igures in brackets represent num::ers of organs studied histologically irn percent of numbers of animals autopsied: 100 figures in parentheses represen: numbers of histologic observations in percent of numbers of organs studied: ~ 100 1~• t:rou¢h xi~!iivj; the sums cf the fieures in parentheses may exceed i':, r~^ ~ because there ma~• have been more than one obser~•ation per crgar.. r ~ NPD = No Pathologic Diagnosis. t1n b lasia Pseudoe itheliomatous i: er • W . p p } ? N c Length Time on ~~ ~ r-) f , CTR HN 0422=52-6

1h :•ab!e \.l":%' His:oioeical Find:ngs in re Larvnx of the E'. 20 Inbred L iae of Hamsters _ : eat: :en_ c: ' ength c= Time on' Exoeri- i; mental Numbers of Animals' I rDr ~ L euc:o~ --7ar::sters ~ Condi- ~ :ions ; (\ o. oi ,,. „ee:cs' i 0 _\ x' I iG I 2 I .' I 2 . - 13' I ' - _ ~ :'015:... i ' - - 1 ....... I I i . . i - - I - I ~ C': e. - C ~ . . ? ~ .. { ' .+ , I 1 C - _ I . 1 - 1 - ~ 1 . I ~ I - ( - I - - ~ i Over `• . . ~ Subtota' 48 { 3 ° oll _ I 311-OIL 0 0) 1 C (0) 0 101 I C C' i C- i21 I _; ~ : otai per s:r a.. ~ 1 1~~ I11-~(6-?) . 61 (3T) y24(21; 6 (5! 2/2 11 11G' ~ 12 i'.1 ~~ , Group D. Grcun E. Group F. ~icures in bracke:s represent numbers e: creans studied histoloeicall•; oercen: oi nu ibe: s of an.r ls au:opsied: 1 C0 (i~ i); figures in parentheses represent n;:m~ers of ^is:oloric observations '.n oercent of nurnbers cf or¢an s s:uc:ed: 1,^ t•: ;hrouc : .: e sums of ..^.e fi€ures in parentheses may exceed '.OC, ~ because there :na.• have been more than one observation per organ. L11 Ir E \PD = No Pathologic Diaer.osis. Pseudoeoitheliorna:ous hyoerolasia. W N -J C~`°~ ~~I N 0 4 2 522 7

Table 1\%' iis:oiogical Findings in the Lungs of the 15. 16 Inbred Line of Hamsters ir.: e or. . Experi- . -eatment of No. f ' ! ::an:ste- s :ne.^.:a: of , ung ~± o y Clw^n?ed ( Ader.o- i._ i v1e:a- or. s, Conci:i .:-:a :._ ls :~PD Sar:pies~ Macro- one na- : c \o. o_ :._.o?siec , j pcaoes :osis ~ -,. - ....,. I '.~'ee~:s~ ! S:.:ciec i ~ - :c: osec' ~ -' -_ ~ c . .. I ~ ` ~!c I ? i_ _ 1 i G e- 13 c 2 ~ j_ 4 ~ c ~ S^ a::ec ~ ~ C~ i ,^.~•e: ~ Su:._:a: 2 = - I - az e-= ~ ~ '--c- - ~ - I ' ~ - 1 ~ - • - - 7 2 - , -D I 3-~IE iz- i I . ~:al ?er s:rain . _o I 1 30 (9C'll 5~ (39 ~ ~ 42 (21 ~ I 31 (21) 5 (3 ; I ! - i6 , ' C i- I Leng:h o:'; Numbers of Animals' T , 'r NPD = \o Pat^ologic Dia?no:is. Group A. Group B. Gr ouo C. F igures in brackets re?: esent numbers of organs s'idied histologically in ?ercen: of numbers of aaimals autoosied: l01'~ iivi/!iiil; figures in parentheses re?resen: nuMbers of :astcloEic o*--servations ir. oercen: of nu.-nbers of organs stu--:'.ea: 100 i•: :..roue~ ..r.e sums of .: e figures irn paren:heses :-ia}• excee_ because there r ay have zoeen more t::an one observa:ion per organ. Lr f R N f'i 0 ""'f' 2' E52 8

I ; able XXV: ::is:c;ogical Findings in the Lungs of the 87. 2~ Inbred Line of Hamsters T ir::e on I i Ex?er. I l T r ea`-~"'e"-- c= ' ' i mentai o' \o• oa " lu.-r:?ed Ade.^.o- , ~fe:a- riar::s:er s or.di:.,.a C ..: ~ ai s i i'u"g ~ °~ ~ \:aro . - Bone :^za- i T^: - ja - s:a:ic ~ -n?ies S a. ( ~ ma::c_ ~ ' pnaEes I :os s _..._- .. ; , . . ' :uc'.ec } J 1 I ~ e e : s + ~ + I I x -...:.:e-- . sec Z~,_- ::ver ~'_....a~ .. 2. I ~.:e C J- J • a-- I ., ._ '" I i i .. _ _ _ • I ~ ~ ~ - _ I ? ( ve - ! D: - a: 7 _ 2. I I 1_ ~C - - i I 3 -c 1. ?: i I _' ~4 ,l.~ ~2 - 2: :.:al ce- strai.. 17 ~ I 17q(100~ 2e (1=l 12: (68, 41 (23) 81 r.l: G i i i. ~ Tene:h c: \urnbers of Anima:s` - . Gro . D • GroL'7 - Gr cup z "iE'.:res in bracke:s repr esen: .^.um':~ers c: organs studied histologically ir_ percer.t cf rs r.':-er s of aair:zals au:o?sied: 100 figur es in parentheses r eprese.^.t nL^:. ~er s of histoiogic observa:ior.s in percent of numbers of orgar•s studied: ?0: (v :hr cug^ x', ?v'; .:^.e s:._^:s cf the figures irn par er.tLeses may exceed because there ^:a•: have -een more than one observation per organ. Ir `:?D _ No Patholozic Diae.^.osis. T"ra ..~ r. i I~ IA .'1 TS T- i,i - . I - L.1 1 R ! i) 7 0 42 E i ~9.

: able XX%'1? ciis:ological Findings in the Trachea of the 1:. 16 inbred Line of Hamsters Treatment c: ::ar:^s:ers Length of Numbers of Ani:nals' ''-" :m e o n I E:cperi- :-:enta: No. of I No. of Scua..ous ,a Ccndi:ic::s Ani:nais ~racheas N ?De \:eta- ?aoi'-:. ::•: No. o: Studied I p:asia l -.':ee::s . f :0- : ~ -- - ( Over 5~~. - , -'' 1c 2 z - r Sc c SS~ . . ~ ~ I ~ ~ ~ - 0 0. S :a^-:- S:::o::ec Ccn:: =:s' - ~- C V . J _ ai 1 r . 42 y '. ,:3i I f . ; ? I / _ :r.:-__s i -. -- c - -__ 4 - 1- ~ Over =: 13 8 Subtotal _ 40 33 (83) 28 r8511 2 (6 0 : otal per strain 166 I 148 [89) 131 (89) 9 (61 3 .1 2, I 7 5 ) - Group :.. ` Group B. ~ Group C. ~ L"igures in brackets represent nur^bers of organs studied 'r.istologicall} :r- perce^: c: nu.:.bers c: animals autopsied: lOC rigur es in parer.: :eses represent -.-,-:5ers of histologi: cbservations _.n percent of numbers c: .,rgans studied: 100 iv thr oug^ :he sums of the figures in parentheses rna}• exceed 100, because :':ere r:za}• have been more than one observation per organ. • NPD = No Pathologic D:aenosis. .~% -F r-, - ... r r r• 1=. I-~ l. 4. .- n ,- It !. ^•, C "r R M N 0 4, 2- E: 1-23" (")

I I I Ik A I Table ?:\VI:? :iistological Findings in the Trachea of the 87. 20 inbred Line of Hamsters _ rea::rier.t cf t Experi ~ i i r:ea:a'. ~o. c: I No. of Scuar.cus I Chronic Liar:s:ers I r Cczci._c-_ .....:::a:s i -rac'r.eas \PD Meta- ~ ?apiii c:::a :afla:::- t :~o • C: ~u:OJS. 2c i 3.udied i ?ias~.a i n^m ati o rn ee::s , -. ~ ~ :.. ~r:o::e-~tip.sec -0-c0 ~ c ~ E i - f - 10 I - e S._ .c_a. ~!1 -. S::a^:-S^:c:-: ec I i~. e- ~ 2 '_ i ' I - r I e~. ~ll 1 ~.3 . i ~ - ~•.•. C __ -_- _ ~ - - - i 2 ~ 20 2o - ~ - - O•: e: ~0 b 1 8 S 1 Subto:al =S 47 [981 46 (98) 0 (0; 010 1 2 : c:al per strain 179 I 173 (97) 147 (85) 22 (13) 2;1 1 4 i2' ~ Group D. Group E. Group F. ~ Fi2ures in bracre:s re-resen: nu:^:bers of or¢ans studied histologicaily in percent cf •..^:ber s of animals au:cosiec: '.00 figures irn parentheses r cpr eser.t r.•-::-izers oi tisto'.ogic cbser~•a:ions in percent of nu_-nbers of organs studied: 100 !.• throueh v:ii i/(iv); the sums of the figures in parentheses may exceed 100, because t::ere n:ay have been more than one observation oer organ. ~ NPD = No Pathologic Diagnosis. ,a YP1 .~l.l IYf '~ .tei w~mR _ i-enoth of Numbers of Anir-als li:: e o r. ! I 1 • 0422531

Table XXIX ::istoiogical Findings in the Bronchi of the 15. lo Inbred Line of Hamsters I _ enQ-h o: Tir:ze on Numbers of An?:nals` _ r ea:r.:ent o: Hamsters -xper:- r:entzi i No. of I \oN c: Sror.chia: r Scua^:ousi I Chrcnic Ccnd::icns :nimals \ PD e:a- I ?api:: r^ai Ir f: a^ - Samples ' ., . ;No. o: %V eek s Autc:~sied j S:udiec i plasia ; i I :n a: icr. :v I 11., ~- .- 3mo::e-Exoosec i - i -~-cr _ i I i C0- _ : lc I 1C i : .. 31 Jc i ?~ ~ I ~ P- ~ ?C .~.- 1 ; , : I I I 1 r S c v~eC ! ~ I ~cr. cis ! - -c~ ~ - ..~ ; 2~ i 2_l , - 1 i I I --- . 1 i I Over ~C 13 8 -1 ~ Subtotal -~0 33 [83] 29 (88) 3 (o) G '0 f 1 ;3 Total oer s:rz?n 'i 166 148 [89] 1133 (90) 14 (9) 1 C (0) I '. (1! ~ Group A. Group B. • Gr ouo C. ~_:gures ..n b-ac%ets represent nu^:ber s cf organs studied histolo¢ical'.•: :.. Dercen: of numbers oi animals au:opsied: iC0 (iv)/(iiii; figures in parentheses represent nu_:.bers of histoloEic observations in percent of nu:nber s of or gzns studied: 100 (v throug'7 the sums of the figures in parentheses may exceed 100", because there may have beer. more than one observatiorn per organ. r NPD = No Pathologic Diagnosis. ~ C1'1 W W N CTR MN 042-53-22

Table XXX r'is:ological Findings in the Bronchi of the ? 7 . 20 Inbred Line of Hamsters ?r ea:-r_er.: of I - =xpezi- ~ , :ne:ta: ' \ o. o f I N o. of I I ~ , Saua:nous; ~ ; Chro ^ ic a^:s:er s ' I Ccncor _s .: ni::,m al s ~ Bronchi a l ~~ < D < .. I \.eta- ~ ?ao::. :. . . ~ o:::a o:::a ~ '~fian:- ~\c. c: .=.u:o~sied i Sa:::oles i i lasia ~ i I ' ' Studiec i p ! :na: o: : ee:cs ~ ~ i I • f i I I :. ~ ~': • . Sr::oKe-_x00seC I l ~0-6c . ~3 I C I ' 1 ~ _ ' _ iii 0-,= '2 I ?' ~ I °' ' ~ I ' e r 31 -= ; I c - .. i Sa: _- i-2 -ji j• : ~I i S::an:- s: :o=:ec ~ ~ I I ~ 0::•. .,.5 I I - - r - . - - C~e- ° .? ~ 2 I C' :O:a: --- 10 C L I ~ . . ~a__ Z- -'-" ~ ~ ! ` I _ on - ~"v 20 ~ 17 1 _ ' I Cver 10 b 8 7 ! Subtotal 48 47 (98] 42 (89) 2(41 1 0 0 ! 3 c = otal per strain 179 I 176 [98] I 146(84) I 20 (11) 2" i = Grouo D. Grouo E. ~ Group F. .= igures in brackets r e?reser.t numbers of organs studied histologically : percent of nu_-r:bers c: a:.i:-ials autopsied: 100 (ivi/iiii); figures in paren:zeses represent nu:nbers cf histologic observations in percen: of numbers o~ orgFr•s studied: 100 (v through viii)/(ivf; the su.-ns of the figures in parentheses r~a~: exceed 100, because :^ere may have been more than one observation per organ. r NPD = No Pathologic Diagnosis. ~ength o.' . Numbers of Animals' Time on C T R M N 0 42 5373

i able }tiXX: -reat:::er•t of riar.a:ers Length of Time on ExDeri- t:s:olo¢ical _ ndings in :he Head of :he 1 ~. 1 o inbred Line of Hamsters mer.tal 'I N o. `. Condi- ); Aj,` ,~s tions ii Au:c:- ;tio. cf ~ s:e-Weeks' I' .-e-=xposec~ •:0-60 ) -" i u'.,e.o:a'. e-- ...L-•. "-5`.. ! ,:0-Cl. ( C0-7 : i 5-90 ~~ :c:al _ .._._ ~ -••-- -•= j - _~ I =- - , _ ,kO:•er ~C ISubtotalj Numbers oi Animals' '~, o. c: r Heads \?D I Caries~ Thro-nbi __.e_` `: :s S .. = I - 3 - I 13 ! ~ ' - ~! :.de^.o:d ~ \e'.ro- ::b.o- C-,•stic =urncr . . . I x'. ~ - 1 - i - ; - 2 3 ) 3 ' I 12 ! ~ C i. I " 1 i - i 1C. 1 1Y:.3 ~ Gin¢i- -1 - j 3 iT ) E 13 ~ 3 _ 3c `~01 1 11 (31)1 18 ~150) J:a. per strain 166 161[t970 l 65 (40) 60 (37) i De_¢en.: i E.•e i i - i 5:? ~- - I 5 121 C 3 6 (17) 24 (15) v (C, 2-1 (13} I 13 (8) ~ 1 (:. i L Group A. Group B. + Group C. -'ieur es in brackets renresen: numbers of organs studied histolo¢icall.• in percent c: numbers of ani:^a:s autopsied: lOG iiv i!;iii,; fi¢ures in paren::^.eses r epresent numbers of histolog'.c ebser•:a:ions in percen: of numbers oi organs s:ucied: 1;,0 iv ;arough the sur.•is of the figures in parentheses may excee--: 10C, because there may have beea more than one observation per organ. r \PD = No Pathoiogic Dia¢nosis. T~ ~ r . 006 1 ~ ( CI `R IIN 0 42 53 4

lk Tal:le X\X'-: Lene7h of: i Time on ''; Experi- ~ =rea:rner.: ,,. ! ..,er.:al ::a^^s:ers ! Condi- i :ions \o. o: 'A•eeks His:oloeical Fincin¢s in :he Head of the 87.2~ Inbred Line of Hamsters /l Numbers of Animals` I ~ - ` o. i \o. o:i r~sl : eads ` . ?Dr 5:..: ecl sied ~ i ~ ~ ~ ::~:o::e-~.~osec' 00 ; _ - - 7. ' ---~- - ~-- , .. ~ - i - ma i i ; Su'ce:o:a'. _3 10'1 i :z ~.: ~..._. :-...-_.? 2 - ~ ..•: er 2 - I 2~ _„ - i - C. i -' ~ - - I C•:er ~^ j Sub:o:al 1 z: I ~?0 42 '88J G^ ^ Si - i I ~ . ~ 1c, (-1:5) / 17 1401 - i I I \euro- Dezer ibro- Car:es 'i C}•s::c ' "ye I Tu_nor Isarco- j j . i 2 3 (7) 1 (2) i ~ , I cta: per s:rair. 17~ 163(o1] I 7E 148) 62 (38) 13 (8) 1? (o ( 0 1~: I "0 'Q G r oup D. Gr oup E. Grout . . _ res :r. brackets represent numbers of organs studied 'nis:olo¢ica'-1~• i.^n oercer.: -7 nur:~ers of animals autoosied: 100 (i•::i(iii); ficures in oaren:::eses reoresen: ••::r::ber s o: h is:olo¢ic obser~•ations in ?erce.^.: oi r.urabers cf organs s:udied: 1G0 !%• through xiilii~• ; the sums o' the figures irn parentheses ma}• exceed 10C, because there ma~• have been more than one observation oer or¢an. NPD = No Pathoiocic Diagnosis. ~'T ~ ~n [..1T P P C T R M N 0 42 E- ~'~S-

. h i able XXXIII ..isto;ozical i:nc:::_2s in the Heart ci the 1:. 16 Inbred e ine of Harr.sters I I Lengt•rh of ! Time on : Numbers of Ar.imals° - r eat::•ien: o. ~ Expe r i- menta? \o. c: ~ \o. of i I Muscle i M ::a::zs:ers ~ Cond:.:ons ,\c. ;,: . .V ee s V A :? n.r =r.a:s . s:_ y ;. ' ~ ! :iearts i '.~ PD' ` De¢en- ~ ur rca..,- :1 S:ud:ed I I era:'.on i I , ,-i, .- : _•.:=::e-= ^sec , ~ Ove- Sub: ::a: - • '-- '- ~ ~ - _-.a:: 5 r:-:_.: e= r , • ~ - - cG- _ ~ r , . - ! . i . ! O-,•er , ' ~ , -...:a: Su - _ i• • _ - 1 >> i 3•:er ~. .. Sub:=:a: -_ 5 120; j 6(7, 5! ~ 2 ,25 -o:a: per s:ra_.. I l06 i 40 (2,] 1 10 (25 1 I 28 ( 70 1 c 23~ Group A. Group B. Group C. = i¢ures in cracl:ets represent n•_r::bers of organs studied histoloeicall•: :r percent of nur:•~bers of animals autopsied: 100 figures in c. parentheses re=resent r.um~--ers of histolc_:c observations in percent numbers o: orea-s s:udied: throug^ .:it::e sur:•is of the figures in pare.^.:aeses r^a}' exceed 100, because there :nav have been more than or.e observation per organ. ~ \?D = No ?athciog'-c D:agr.osis. ~ ,-.. TP r*l r ~C, r T C T R I'Ll' N 0 422 53 6

Table \XX:N' :astoloeical Fi•r•dines in the Heart of the 87.20 Inbred Line of Hamsters ~ Ler•gtn of ; Numbers of Animals` I ~ Ti^:e on i I i ~ I t Es~eri- I 'rea-:^•ier.: o` i , I rr.en:a'. No• ot `o. of Muscle riar••:sters I ~ ials r?ear:s 1PD Deter•- Conci:>ons An m j \c. c: -yu:epsied ~ Studied ` eratior. j '.t-e e K s i I Mura1 : !:r.,•:.o'- S: nci:e-zx^osed; ~ -: _ I 31 S•`'r c •L; lc - ` - S ::a^-•-_--cs.ezi I 1 - i ( - I ~_e = ' ~ 0 - ,: 6 ~ 3 ! 3 ~ S.:o:48 18 (? 81 I 14 '78~ 3 ;!" Total per strain j ! 79 37 [21] 27 (73. 9 (241 7 !:, Group D. Grouo r. Group F. = igures in brac^:e:s represent numbers of organs studied histologically in percent c: r.u.:.'--ers of animals autopsied: 100 (ivi!!iii:; figures in parentheses re.cr esen: numbers of histologic observations in percer•: cf orEans s:udied: I 30 1%, throug:: vii-/(ivl; the sums of the figures ::: parentheses may exceed 100, because there -nap have been more t`an one observation per crgan. r NPD = No Pathologic Diagnosis. PA CI R I I I i W+ I imv M~ tiw^ 7

Table XXXV Histological Findings in the Liver of the 15. 16 Inbred Line of Hamsters I 1 Length o: i~ Numbers uf Animals Ti.^.ze on I ~ I Cystic ~ C: ol- ~ i Experi- No. oi No. of T r eat^:ent o- , ~ . Hype r- a ngic- , menta. : r.:rrals ! Livers NPD Arr-yloic i i Ut: er -ar: sters ( ! ~ ~ plasia :arci- ! Concit; cns °.' toosiec : S..:died ! i Dycts - ra ~ (\o. o: ; V~ee:c S) i i ~ , ~ . - i - ~ - - ~ - OKE-L\7C52C . •i0-oC, i. Cve r °C -° ! - '- ; .. ' JL"c_ --_ .- (-7) 1 - (1oj G (. . . _. _ . ., C :a.,,,_.^•.._.'J Cor..-_•: ,. ' S•.. -C, cC e..'~.tG: ? ( ') I , (2_ .~ 2 ..-r c - 3 ~ -~ - i,. ;' ~ _ i i i 2 1 ( . ' . I I I Ov er 9C ii '_3 6 ~ ` Su ttotal ii 40 1 17 [43; 8 (47) 2 (12) ( 1 (6) i (29) otal per strain I, 166 1 60 (36] 27(45) 9 (15) 110(17) , : 115) ~ 13 (22) i- _ * Group ... ** Grou-- E. + Gro:. C. Figures in brackets represent -^.umbers of organs studied r histologically ir_ percent of numbers of animals autopsied: 100 (iv)/(iii); figures in parentheses represent numbers c: }:isto'.ogic observations irn percent of r.u:nbers of organs studied: 100 (v through ix)/(iv;; the sums of the figures in parentheses may exceed 100, because there may have bee : more tha : one observation per organ. NPD = No Patholocic Diagnosis. '~'"r,_` ~ ~T V ri:l :r ' rl~ f ~ *r , i Lr Tf °-. Hf 'i S..d "'r 2538

Table XXXVI ::istoloEical Findin¢s in the Liver of the 87.20 Inbred Line of Hamsters Length of ;; Numbers of Animals Time on : ~ Fxp r•etrai_- I T reat:::en: o: ~ -:e , No. cf ~\o• of I Cystic I Caoi- ~ :iyper- i a :eic• ~ r:arrstEr_ ; Cond::ions Ar.i^:als Livers \PDr ?.rr•}'loid Other (\o. c' j_~•.:to?siec. Studied i I ~ plasia card:- i Ducts ~ noZ:a j itieek5) ~ (iV) ("i) i (vii) (~-..) (:xl ~ , =c:•.-z_xD: sec -J-o~ '_ ~0 . 5•. _t cta r • NPD = 1`o Patholoeic Diagnosis. ' ~ -,..~... ..... - •:E _ ....G^.. __a ., (C) ~ C' • : er ~l T otal oer strain I I 1i c =b • - - '~ 'C . i -- - i • ~ • 5 (- (3) _ 5-1 (60) 1 6(7) i 6(9) *r. 1-19 ' ') I 33:69J I 4 (12)I 1(3) I 23(70)i =(i2) 2(6) 0o l50] 1 23 (26) 1 5(6) Group D. Group E. Grou;) F. _;ures in brackets represent nur.:oers of organs studied histologically in percent of numbers ci animals autopsied: 100 (iv)/(iii); figures in pa-ent:eses =epresent numbers of histologic observations in percent of nuznbers o: o-,ans studied: 100 Ov througa ix)/(iv); t: e sur.:s of the figures in parentheses ^:a•: exceed 100, because there may have oee:: more t:an one observatiorn per organ. c"i°R M N 0 4 2'_ S_ W, 3 ~

Table XXXVII ..is:olo¢icai r ir.din¢s in r.e Spleen c: the :5. 16 Inbred Line of t•ia:-isters Treat:nen: of :iam s:er s Length of I \umbers of Anirnals' . i-:•ie or. ~ ~xperi- I No. of ( ~oN of I Leuke- ^•ien•.al C3 nd:t:ons` An.rnals I Spleens \PDr '"e:nan- I ^:ia No. cf "Au:o~siec! S:udiec i gio: :a i L}•m?::o- i I r.•ia ~I-ees' . i ~ i :... : ioic 10 ::.e r ~.•ii .... ~ ... -C_ - --. - 1 - S::a-:. ._... _.•.e: I I i a~z--e:= --.... _._ ; -- -- - _ -: I I c3 To:al per s:-a:.. li 166 ~40 (241 28 i701 1 1..~ i- iic, ~ _ 8. Group A. Group B. ' G:. C. _-= res ..n brackets represent numbers of organs studied his:olo¢ical:-: .- :ercen: of ru..:.bers of animals a•.::opsied: : ~O iiv: iii1, figures ir. par er.::^.eses reoresent na.-r.bers of nistolo¢ic obser vations in percent of nu:nber s c. organs s:uc:ec. 10^ (,.• t^-cu¢^ ix i~ .re sLms of the f:sures in paren:heses may exceed because there I7:a\• ::a.•e beer. more than cr.e observa::or. _er organ. r \ PD = \c oa;l:olo::c Diaenosis. r~T : r) t-., CTR HN 042540

Table XXXVIII ;-:istological Find'.r.gs in the Spleen of the 87. ? 0 Inbred :ine of Hamsters I i Length of ' e on -i Tr eatmer-: of Ha.^.:s:ers ~ ~ :r. = xperi- -^ental Ccac'.tioas No. of als I No. o: '.1"eeks Autc--sied I I - - . - , - ,- S::•:o::e-~xpcsec ~ -':' 6C I- I .3 - ~ - ~ - ~ - .36,11 3. i'_,. ;.0 ~ 0 I C' ) ~ 0 0' S^a::•i-5... ::e_ ; I Con.: 0:_ -- i Numbers of Anir.:als` Leuke- I No. of Heman- mia, i Soieens \PD~ ' ?r::•;loic i Other gioma i :vmpho-` ~ S:udied ;In a I ~ I I ~ ~ i , ~ ----- ~ i _- - ~- I - I - 1 - I - I I ~ ~ I 1 I . .. . i _ , . i - 6_ v e-: eic: i ' _ - - := .= t i ~ - . ~ ' - : ~ ; ~ i 3, . . . = - i ~ ~ i , I i - -~- `otal per a.in stn 179 I 78 f441 68 (87) 1 3 (4) 1(11 i 1(1, i 1 5+6, , Group D. Grouo E. Group : igures in b: ac::ets represent r.u^:'--ers of organs studied histologically in percent of numbers of animals au:opsied: 100 (iv!/(iii); figures in parentheses represent nu.-nbers of :as:ologic observa:ions in percent of numbers of orga::s studied: 1CC' (v through ;:: e s•.::ns of the figures ir. parentheses r: ay excee-- 1QC, because t::ere ^:a-.• : ave beer. more :::an one observation zer orLan. r `: =D = No Pathologic Diagr.osis. L.r Ri R i f N 0422541

Table XXXLX Treatment of Hamsters istoiosica: Findings in the Kidneys of the :5. 1c Inbred Line cf Hamsters W e e k s i Ler.g:^ of \'umbers of Animals` l ime on Experi- No. of I r.:ental No. o? , Ccndi:ions Anir::ais Kidr.ey I Sam?les N PD A^:.•'.c'.c r Calc.un: \o* of :~u:opsied I Studied ! i .. ... ~ :t•. i ~ V I S^~o::e-Exoosec' ~ -_-__ - . ~ ~ O•v e- 2 = Su'o•_:a: _- S :ar.:-S:nc=:ec C on:'e:..o r ~ - • 1 : ~ ~ 24 2 I : I ~? 't'_' ; 1' I 3 ~c:a'. :er s:rair. vey:J:G: 166 ? 9E ,3y) I 13 !13 ~ 84 A r NPD = No DiaEnosis. Group A. Group B. Group C. Figures in brackets represen: numbers of organs studied histoiogica:'-~: in percent of nu:z=er s of animals autopsied: 100 figures in parentheses represent nur: bers of histolo¢ic observations in percent of numbers of organs s:udied: 100 (~• tl:rouehv'_-*)//iv.; the sums of the figures in ?aren:heses r:•ia}• exceed '• 0-., because there may have been mor e than one observa:ion per crga-. n,rrrr CTR MN 042', ~'2

Table X L 1?istoiogical Findings in the Kidneys of the S7. 20 Inbred Line of Hamsters I Length of ~: Numbers of Anir :als` Treatment of Hamster s Time on i Experi- mental I Conditions ~ ~\o- of' ~ «'eei s ' o. of Animals Autopsied i dneV Samples StuCied I r NPD { I A :r.}•:cid ~ ~ ~ alcium ; i, 1 ivr i Ivi S:~ o;ce- Exoos ed" I 10 -__ c. 2= ! .3 7 i Over c: 3: .3 1 .? ~ Subtota: F- ~ Y= .='. '-~ - Szam-S:^:oiced ' ~ Cor..rc'.s' " '- - - ! - ~ Su~e._:a: S I ?~ - ;aee--elc . ~ _C;--: :3 ~ 13 ; 2 - : ' 2C ; O•: er " . Subt~:ai E 48 35 ;-3' ~ Tc:al per strain 179 ( 108 (60] , 28 (26, ~ 75 rcc ~ ' i6~ ~° Group D. #= Group E. ' Group F. ~ Figures in braci:ets represent r.umbers of orgnns studied :istologica:!% : in percent of numbers of animals autoosied: 100 (ivl/(iiii; figures '.n parentheses represent numbers of histologic observations in percen: of numbers of or¢ans studied: 100 !v throug^ .-ii)/iiv); the sums c= ::e figures irn parentheses may exceed 1CC, because there rz:ay'.:a•:e cee:: more than one observation per organ. Ir NPD = No Pathologic Dia¢nosis. ~ ~~ -r ~~ n a ! I 'r ~ F%r.,.,.I CTR HN 04~"5-423

I ' Table XLI , 1 1-iistological Findings in the Adrenals of the 15. lo inbred Line of Hamsters Numbers.of Animals' Time on Experi- i I I T r eatment of i mental No. of No. of AB-Cell f:ams:ers , ..als Condi:icns Ani P.drenal ~ \PD Hyper- Adeno- Amyloid Carc:- ti i\o- u:o?s o.° ied Sam?1es plasia i ma norna «'ee::s I S:udied 14 .. . i ivii S-:o=:e- ~s?osec I - - ( - If . ~ 28 :' __ 12 O~•er 2_ :2 10 3 I Sub:o:a'c 31 1431 i 34 143, S^ar -Sr c~ec ' ~ I { I I c c I I ~ ~ I ~ , _ : ^ ~ Sub:o:a: -- 17 ~-1. I 3- 3• c = •y ( 4J-C~ I I i L iC ? C : - ~ O~•er ~., :3 i c = 1 i 2 i- ~ S1J.J•a: -. 33 ;63) / i6 t45~ 11 (33, 3 otal per s:rain ~ 166 153 (92) ( 63 (41) 62 (411 I 15 (1~ I 21 (14; I ~ Group A. -- Gr ouo B. ~ Group C. : Figur es ir. brackets represent numbers of organs studied histologicallN • in ?ercen: of numbers of animals autopsied: 100 (iv)/(iii); figures in parentheses represent numbers of histologic observations in percent of numbers c' organs s:adied: 100 (v through ix!/ (iv); the su::zs of the figures in parentheses ^:a~~ exceed 100, because there may have been more than one observatiorn per organ. ' :~?D = No Pathologic Diagnosis. II Length of ( ~n~~~ ~°PQ ~^~=. nt~.. ; ri ~ ,..,' TR MN 4°`~ 4iw 54)

/ Table }: LII Histological Findings in the Adrenals of the 87. 20 Inbred Line of Hamsters ! i Length of ` Numbers of Animals~w Time on ~ 'reatment of Experi- 11: No. of mental No. of AB-Cell 's:a::zsters Adrenal ~ Adeno-i Carci- Conditions ' Animals NPD Hyper- Amyloid I Sa-- "'a I no' a nples plasia !No. of '?.utoosied ` I ~ Sc udi ed I eeks~ ! I ~V e eks ~ .~ ~ (11) fiii' I tv) 1 /vl :e-Ex?osec~ YO-cC ! I ! 60- 7: 23 `? 1 13 I I 6 1 I I r3-on 2: ' i4 2 ~ 1 ~ = ( Over °^ 3 1 +i i 4 3 ~ Subtotal 57 i 56 (641 _1 2: 1 3!:, e.1~ C 0 S .a: :-S^n G;ec I '5-6V - 1 c0-7~ ~ 1 i 1 I j 2 ? ~- ' Ov er 9_ ' 2 "BCI =a e .-:eic I I ~ v = -0-6C 7 ~ 5 ` 1 - 1 ~- 1.i ' :3 I 12 j 1 - ~: - Gver ~C S b 4 J Subtotal 48 ( 42 (88) 29 (69' 3(7) 2 i ~ _.: ~ 3' iotal per strain f! 179 133 [74] 78 (59) 26 (20) 8 (6) 17 113; i 6:' Group D. Group E. Group F. Figures in brackets represent numbers of organs studied histologically in percent of numbers of animals autopsied: 100 (iv,/(iii); figures in parea:ieses represent numbers of histologic observations in percent of numbers of org.a ns studied: 100 (v through ix!/(iv); the sums of the figures in parentheses rr.a}• exceed 100, because there may have been more than one observation per organ. r NPD = No Pathologic Diagnosis. 1 rTP P+ ''° h 'Pr', nr. CTR . t1N 0f' 2-545

Le¢end to Fi¢ure i Lethal response to intraoeritoneally ir.jected nicotine tartrate in males of four inbred lines of Syrian golden hamsters, in males oi one noninbred hamster line (RB), in male CD rats and i n male CD- 1 Charles F.iver mice. Le¢er.d to Figure 2 Survi~•al ti:r.e ,,. males from four lines o° Svriar. ¢ciden :amsters and o: maie CD-rats of t::e same body aei¢^ts d r:-_ ex- pos~re to w•^ole, ires: s^:o::e .ro: :!R1 Y.en tucic;: referezce c:garet:es. :esenc to F ieLres 3 and = ~5scissa: `:_...cer of .reeicc on exoerir:.er.ta: ccndi.:cns ~s^:oke e~pos~re, expos::re tc s~a::.-smokir.g conditions. or ca¢e- ic:.c!; t^e a~:z-:als w•ere ap?roximate'.}• '.3 weeks c: a_e a: t=e .._ gi ~.~ _ (C wee::s). Ordina:e: ::L::::~er oi s::r ::~•ors ea~ressec .n ~er:_... o: ..._ n-:::~er ci sa.~a:_ ... eac: ~cu~ at t::e bee:..ni::c c: .r.e ex=".- er•-• -.-.•. ....._.. r.ac oeen s•,._.ra.:'.ed:ne ..,:r::ber o: a::.mai_ .r.a: r.ac d:e= ac_?de~t~il~. ~e. -..,..: :ar_,... r.:onoxice poiso.... -_~.. _ e:. =-'= c~ a_.._= sa~.._.ce ( a::er -_, cG ~+'eEt.-. ct~ ? _e -- .- - :e•-res _ _- ~bsc:ssa: `• :oer ei .vee}u c:. e>:oer.:~:e-ta'. ct __.._-._ s-_. -_:cx_..r_. e~• cs:.re tc s: a s~ok:r.g ccnd:.:or.s. :.. css -.-._ Ordiza:e• ..~:eraze boC)' w'elg~:ts ... grarr.s, as detE~_ r:~ined w•eeklv; the values at 91 and 92 wee;cs are those of Table 1'i, i. e. they represent average .veights oi all animals sacrificed after r:-:ore than 90 weeks on experimental conditions. m ^ ?` ~ r~ !"~ c..~'F ~ ,'? ~' 'r` =. r'':,''; ~ i ~ ~ ~,. `~ ~ ~~~ ~~~ ~~~ ~

Toxicity of Ir,trc~,eritonec~!~ :,eritonec~!~: injected Nicotine Tartrate Ct: , 20 - :-;C_ HAMSTERS RATS ~ E b IO 2C Z„ 50 I00 200 500 9 GOSE , mg / kg 2OCY WEIGHT ) I .:g,_re 1 -.1 .~r~I r" rA ~1(-.-w ,.- ^ ,~ 4 ^ A L.r i f"'o f 1 f"f '~.d "'f' 25`"f' 7

/ , I ~ ~ 300- T OXICITY OF WHOLE CIGARETTE SMOKE ! z 2c0- - I - .:. , 0 R E 67.20 62 62 15.16 RATS HAMSTER,S ~ ure 2 m Fi g r r T P rn~T~~r~~ n ~~t~~48

MORTX-ITY OF l1(igR~.~ E'CPQSED TU 5,40KE FRoM ,e,rNTWKY RFI~R~NCc C'/ A~.R 17"r C l R l)~: AJVO O~ UN~71'~OS~ D C'o OL lV/ G! ~ ~ ' 9 , ~ NTR ~ , ILS . l00 ~«-............. ~ ~ ; ~j ~•~....«.• H-~~~`~ ~u.• • .-~:• •~.~-•.•.• ~ • ~- '•-~-o~- I ~ . ' y4NAM -± 5M0KEb" \-•-. ; .~ CONTjQOL j4JVIlNRLS -'~* • • • •~ ° 6o Af 0 4o 6-RQuP / UNTREATED, CAGE-NEL~°°°`•~ 0- , GQNTROL. RN/MALS FiGURE 3 I i r 6f1£SlH3.l.

,MoRTi4~irk : dF ~Nr~R~~ , H,On~sTERS i C' ,~ l~ ~ . EX~oS~ to: ;SMoff6 j FROM KENTUC'~CY '• Rc~R~" cE. C i R 1. ) ~ i9XD ~ l~ I UNEX?o~ i CoAU rRo4- /9NlM~L S~ , ! . i ~-.-.-.. ...a , , I G`/6~R!E i , , ' ~ HAM 1 SMOI~D" ~ ~o~lrRpt A1~iM9 ; ~6.~,rolv ~ E~ VNrR~ArV, CA6E ME~~ Co,vrRqc. APMAcS c G RovP F, ~ 4io.: i l4UM EK, 0 FIGURE lf- OSfSlil].L yl -+ 0, ! i ` 1

i TV , 6'-F ; SoPY I Wg1&y rs, ,or-: ,/5.S /6 HAA~fST~R~ ' G!h, t OL j c'qivDlT/ ,S S~oKE i9NU DuRU~!G CONTI l ~ ~ I I ~ I f~0 , , ~ j~. `'cA WL OE1.D~ ANiMA ~---~-t-- , ~ , "Fx"D ; C!~R lN6 ; ANlMALS EXPOSEO Tv cl6/mETriF S1No*6- _.. _ .~ , , ~ , . , ~: ' S' . I b I i.7D ; I;.2 t ~ 30 I 35' ' 4s0 JV (J M f3CR OF WEE~KS Fi<>uR E ~ , I ' . , , < A lS~Stl13_l.

R I L a `.1i / Z~_-, ~ SIIIJi.