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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