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` a / 1 5IO-RE SEARCH INSTITL;TE, i.v:_==,,-•.--c_) I C _;;vA':ENUE ETTS ~'di '/ e1<'e-3t Dr. F.oSe r: C. I-iocket: _=-ssociate Sc:enti:ic Direc:or The Co,:=cil :or =otacco ?•esearc: - :. S.A. 11C :ast 5 3 :1_, Street N e : York, N ew Y c r k 1CCZZ Je_r B __. -'lease --=- __close_ the origi a_ c_ c_-.:za1 rep:_: cc _.e s..:c;- eatitled ": :: i>:panded Sr_=-; o- ::ouse 5..... ~x?os-.:re _ ~':a_:e S=. oke Co^.densate Co::_:ared ._ S:::r. = a:n _: : cu cc:'__ recaL, : have :nade se:eral :i^:es ._ •__.... rrports c: ._.:s s:ua,:, -'_e last :i: :e __n ry i_:ter t: y:L _ a..-ar; _ , . -.. o_` :is re:-r: are be'.-; •_... , a •- r. =ioyt, Dr. Gar,_..__ an- Dr. Lisar.t:. ?• ,:s ....._ t: _ res:.___ ._ -- _.:eres_ ._ .ne gener-l pub_._ c:- -- a =::a= oubiicat:o:: ..• scienti___ > cz h:.:,e tt:a: Tilr C~ nca f~r _coac_ suc:~ ca!icn. A corresponding s:_cy o- -air:es_ ::.._. ._ s:_~ ; - _'css. o.._ •oL: w•i:: ge: an ...Cerirr. repor. C : =-c,;et_ of this year. WitY besc,personal regards, I a-: Very yo•,:rs, .:51~_f. -'_ ?eter _;:~er:.Se:d, Pi:.D. D'.r e:... r o: _'.esearc.. FL:?.:_ Eac:osurc IWD[PLNDGNT-N, ON•PPJF(; OPCRATCD ERC_USIVCL'• FOR Sc1ENTlF:c •tiD E7U=.TI_-V._ P_FP~;SCi CTR CaHTRRCT cz) 005325 11225066 CTR NN 042 351

3io-Resea-ch L^stitute, Lnc~roc:ated 9 Corr--nercial Aver.ue Cambridge, hfassacn_se::s Q' :=: ~ r'- Fee JUfV8 19'3 ; Y 1~3J ~J ZrJ Y J ~zJ ,~" Fir.al Repor: o.-. Contract CTR-70,'2 A Project E:t:t:ed xpanaed Stud-,• o: ',`_ouse Sb::.. ~ _..:e :_ . : o:e Fresh Smoke Cor'_ensa:_ Co^:pared to Sx:a :. T:=a _ ' ----- -.. - . . S. r.. Dated May, 19'? Prepared by Pe te r Be r:Se ld, P:.. D. 1.r CTR CONTRACTS 405~2~ 11225067 CT R M N 0 4 2` 3 'x2

i /k C•.:rre::: concez:s of :_-e biologic e.°fects of :oba:c~ smo:<e emanate from two principal sources, narr.ely (1) f:or.: in- vest:;ations on the statistical association of certain clinical man i- festa:ions in human populations with smoking, where cause and e:- fect can never be ascertained with absol;::e certain::.•, and (2) !:c: studies in vivo ar.d in vi:r~ on the ir-'luence of tobacco smoke cr smoke oroducts in exoerimental ar.imals. In the latter case, t-e ci:c-=-^'is:a::ces prevailing ir. the human smoker can n ever be sat:s:a:- tc:i:y sim•.:iateC, because of the differences in species anc i n the :.a tu-e c: the target tiss.:es, and because of the stress to t:ae ata=Ia:s res.a:i:._ from their .r.t•o:~n :ary exnosu:e .o toxic c: :ritant ::a._- ria:_. L^.ves:? ¢a:ions in :..e latter dom_ir. have bro•_,:: c:... one c: the most incri..•.r.atir.g and seemingly delete:ious r~ani.:=sz- t:_n= ca_sec by tnbaccc smoke, ..e. ...e aopea:ar.ce of c•.::cnec•_, pao?::o.:as a::= carcino: ias ::...._e as a co :seq:ae=ce of re:e:.:ec aco-::a::oae of =_: oke con_ensate :o t^e st:r of t:ese ar.i::za. The res_.ts of the prese..: wcr:< s:ron,iy sc¢_as: that w._ _a._•noFer.'c e°_iect o: tcbac_o smoke condensate .- -.._--_ s::'--. s ca•.:se-, at -ea_: to a la:R~ PL::, by ar. a-:icac:. ee~. ,.:.c-. .:es : ci¢a:e:ti s:r.cc... .-.•as __::de:.secd by .r:-.pLr, ..._n: C:- rrc::.. t: e s:<: :_. .::ice smoke a::= s'.a .•ican:;v feu•e- oaoil'.or:as and carcinor:a=_ ::e:e cause_ t- --ea-, _:=e!e ~._ ce :sate, ,...•' .:•a.^, . .:_ .._....':5._._:.. __. _..~\.~~. ..._ Gen e:a_ -=s:. n ..- ex:- r•..._r.. - ..._ _ .:...•. c: •..- expe^::::enta: •^'ia- is s'.z•x•n i.. _abie :. ::iice - I^ a;i, i, O1 : fe na;e C: F1 -- ~• ce;•:ec -rc:.^. T:e Jac:<scn Laboratory in Bar l:aroc:, "a::.e. C-n their arrival, t.`:e animals were from four to seven wc^ks of age. T: ey were distributed into six groups (Groups A throug:. F) in s.:ct ca- a rr.an ner that both the mean body weights and the bo'_y weight tri!;•.::ion in eac: of t:e six gro•,:ps were t::e same, as shok- in T a- ble 2. Animals were ho_sed in disposable p:astic boaes, covere,_2 with well-fit::cg, v.•i:e-screen tops. At t~ie begi:.r.in_ cf r* ..r ~ ~ CTf; CONTRRCTS 00530217 11225068 C T R I •N 0- 4 2 3 E5~"....

/k .:-e experir::ent, there ccere ten ::•:ice per cage in Gro--:)s A, 5 and C, a-.c either eight or six mice per cage in Grouos D, E an d F (see bclow•). Cage mates were selected so that there was the least pos- sible body weight difierer.tial amo-:g the a nimals in any one cage, in orcer to avoid do:aneering bebavior of large animals. In this x.ar.- ner, fighting among the mice was mi-•W ized, thereby prolonging their life- span. In addition, the inte rfe rence of wounds, inflicted bv fighting, with the appearance of skin lesions, caused by smoke ._=c.e nsates, was eliminated. As rr.ice died i.^n the course of t-._ ex=er:rnent, the n::mber of animals per cage was generally allowed :c decrease accordinglv. Only on two eccasions were rr.ice rea:•- :a nzed, -.: orde- to reduce t:he r.•.:r•:ber of cages, w:ereby animals, ..cich : ad :..,: been in the same cage at the beginning of the e:•peri- _. , were s•.:bsec,uentiy housed together. These two exceptions in Groups D, E and F after -. and 93 weeks of treatment, -•=s;eat those t:-iec, the -Ur-:e:s of ca;es irn eac: of tyes: _.ce gr:,cps vvere reduced f:rs: fro:n 1 to 12 (se_er. to eight rrice =z: cazej, a nd then fro~: :2 to e'.ght (five to eigh: rr..ce : er .-age' these , cDerat•..ns r_s__:e_ in _-ic.easec ~.crts'_i San-i-ce:, DeodDr served as ..._ be-_~c:n: rnatc- a_. C„cs and beddinc •.rere c: anvzd •:+eekly. The mi:e were kept :: Ous:. %•_..tila::d a nd :e::pe:_._re-_~ aed roo::: f-'c- "_ =).;•:-•) a:.tor::at:call;, cen.reLlec f:_cresce n. :bht (7:.. a. ••.. tc : o. T. ) wit: cu: any o::tside light. The anirr.ais ..:e:%•ec _ _r t::e p•_: pos_ of a'-: mice •xe-- . _ c. .=.:a'_-'. ca_- a:red at the beg?r :i: g- tc.e experi-ent acc :.a: ::zi_!. .a.t:,cec to r::ari. .:e No of G:o••. :.. _z.•_ C.;-as ._r•^ovec :r,,... ._._ an:...a: exce: a:ter sc _<- ..___s Zeatc cr sacrifice. _M:ce of r. E a: c. wer_ _ a...zr~ laboratory, four tirnes a just lon_ encug: to b^ ea_ :Se:. .. . ns:ant s:: ::<e ... :de :sate, „_._rwise ... were lCCD: ... :__ ar.:r 1 rco:r• in tc:e same r.:a.nner as the mice o_'Gro_ps A, E a~c C. Sha%;ng of mice - All an•r.sals, except a few r:-.ice kept as untreated and unshaved controls (not shown in Table I), were shaved by means of electric shavers on the'-ower parts of their backs. This was dcne immediately before the first applica- tien of test materia:s ar..'. periodical:y thereafter, usually twice a «•ee<, :. e. on Monday or "Wear.esday before treatment. Ln order 1 cTR coHTRRCTS 005326, 11225069 ~T R M N 0 4 2"" 3'53 -Ar

to prev_c: cor.ta.-ni:ation of the air wit: *-nouse hair, shaving :r_s carried out under well ventilated hoods. Application of the test solutions - Administration of precondensed smoke condensate (suspended in acetone), of lanoli= disso:c•ed in acetone, and of the vehicle alone (acetone) was r:ade fre^: disposable plastic 1m1 Tuberculin syringes without needles. The desired volume was delivered to each mouse by means o_' a Ea=ni::on repeating dispenser, which allows to mete out exactly 0. 0Z mi of liquid, or entire multiples of that quantity. Smoke condensates were ac.-:?r.istered to the an:-:.a:s un der a hood. All test materials were applied to rccently s;eve: mice on an approximatcly 0.6 to one-square inch area (7/8 to c,'S in ch in diameter) in the sacral region of the lower back, adjacea: to :he root c` the ._'?. Frecondensed s:noice condensa:e was prera:ed:=_... i:e nt-.:_:;: re:=re nce cigarettes by the b:elov iab=rat:r:es = Sprir.gfi_ld. t'irgiaia. This test material was shipped to cur ra- torics Lnder rc:rige:•aticn, was kcpt in a freezer and was use_ v.•::^in ..._• •;.•eeks of arr:c•a:. It ontai ned (•1~1v.) of r.c.^.aqueo_s s:r.o'Ke c,,..___sa:e, 4?"c w-a:er a nc 46°'c aceto :en A total cf :60 m¢ nonac,:e- o.:s condensate were, thus, applied per ^:_,:se per week, ad=in:s:er__ .. e~~al oorticns, each given on a different dav oi the w•ee:•:. __. _-pose of slow1% • hab::.:ating the ani:::als tc the ecu:e to:ci: _:fec:s con cai ::= i:, tne s:no•r.e condensate. „- one-_°o•_r:_ c-` :.. a.... ,.nt, :. e. =C mg, r:ere give-n du:ing the -•rst ueek, an.a tn .. - was gracnaay a-c regularly increased d•_r: :nc• subse .•.:e_: 1_ «=e: s the finai dose of 1oC mg was L^stant smoke con--'e=sate was a:sc , r eYared ~ar.._cc; re-eren ce cigarettes. A s:ighaie a.; :coria:e:: ...=c_..=_ -, .~_ reverse s^-:okiag machine (U. S. Pate n. No. -:?6, 11 use_ for t!:is p,.:rpose. Twelve cigarettes were smo•r.ec stn:u=-.e- o.:sl}• in an upright position, with the use of a ccnvenient:y a•?_ened (four inches) plastic piston (or cover) and an appropriate rn o:at:e base holding 12 cigarettes. One two-second puff was genera:e'_ from all 12 cigarettes every 60 seconds. The exposure chamber was re- placed by a small plastic mixing chamber of approzimately 6C ml volu:ne, leading to a ten-inch long plastic hose at the end of •.v'o:ch was a metallic orifice of 1/16 inch diameter. The latter was : e:= at a 1/4 inch distance f:om the mouse skin for smoke exposure. While -3- CTR CONTRRCT9 005329 11225070 r*7 CTR HN 042355

proc_c?ng the smoke, 3° m1 c: ai= were allowed to Dass t: rough each of the 12 cigarettes d,_rir.g every two-second puf.`, and when the 420 ml of smoke, thus generated, passed taro::g: the o-iSice, smoke condensate was deposited or, the mouse skin beneath. The amounts of in sta nt smoke conden sa:e, produced in t: is maaaer, and determined by alloacing the tar to condense on :i!xr oaper instead of mouse skin, were on the average 157.4 r-:g tar f:e-: 1.', cigarettes, or 13. 1 mg tar per cigarette. The amounts c: smoke condensate resulting from this manioulation were verified seve-a: tir:es in t: e ce•.:rse o_° the study. Taking into account tnat 13. 1^:g of tar were g°r.erated from r.i:.e outfs of ea: : cigaret:e, _:n:•ked to a ~...t lengt: of 30 it follcws that our pro:edure y:elds 431~, of the total amount of tar }zziowrr to be delivered from a 1: 1 F:en:tac}-; reference cigarette, as determined bv standard =asure::.e-a (J. F. 3enner, Proc. Tobacco and I:ealti: Con- -ernnce, iexingtcn, KentUck}•, Fe_.;:a-y, 1970, p. 3G). T'r.t ..•a:er cor.ten: of t.as ...stan : smoke ccnder.sa:=, as ~.ea=ured with Kar_ : isc^er reage.. , was 4. 65~ of the totr.'_ %ti•eight o: the .._densa:e, as op_osed :: 7.417, for :he cold-t-a= o:econde ns-z= smoke condensate. Care was :a•r.en ea:.-:::o;:se, trea:ec ...... ;--- star.: s^:oke condensate, receive--' (a; the same cuanti:y o_ sr.o:<e can '= nsa:_ _a._- day, as any ot:.e: ..n the grouP and (b) ccnden satc cc-. •:d _- _... an ccca: -.•.::nbc- c_' t__.. s: ccr.c, et-, , •::: (:::oke :rc... .: _ i:.-- was rejected) c.. - a perioc of _ne ..eek. :;.is was ac:ie•.•ed ir. At eac•rh sessi~r., ::.o%~se sa:e_ of p; :s c-'smoke from 12 ._;a-e:tes, Ibe;_ were e:.:e- tl~~: second and -....:h p•..ffs, :he third puifs, .^e four_n a nd se•:en ti: puffs, or the :ift : an d s:xth pu:_~s, as see n in Ta~1e L' .-. na= been established in preli:ainarv ea: eriments that eac:: cf t-ese four two-puff co:nbinatic.is fro^n 12 cigarettes always yielded ap- proximately eq•-ai quantities of smoke cc.^.densate, ranging from 35 to40rng. For practical p;:rposes, two smok:ng r.^.ac::°.nes, ooerated si:nul:aneous:y by two perscr.s, were used. L' each srroic- ing machine, two sets of 12 cigaret:es were smoked alternately at cTR coNTRRCTS 11225071 CTR ~'~h~~ 042356

30-se=enz :ntervals, so that the time intervals between twc p, `a re^:ain ed 60 seconds :or each cigarette in all instances. Since instant s:noke condensate was administered to the test a niznais c:z four days of each week, perfect rar.domization of the smoke con- densate application was accon:plished by employing a rotating se- cuence c_` exposure to the four di•'fere.^.t two-puff ccr nbinatior.s on each of the four days of the week. The sequence shown in Tab:e r.I is t:at used on the first day of each week. Or. the second trea:- *-r:en : day of the sa:r.e week, t^.e secuence irn which t~le four cages ca=e up fo= sr:•toke exposure, was shifted from 1, 2, 3, 4(fe: puSfs 2, 4 ar.c 5, respective2y, see figures in parentheses) t_ 2, 3, ,:. Oz tr_ third day c_° the week, the see,uence of cages was changed to 3 , 1, 2; oa the fourth and last treat^;en t day cf the week, it was fu:t :er modified to 4, 1, Z3;.n:il it reverted :a t: e original sequence of 1, 2, 3, = on the f:rs: day of the fo1::,c::=; J: .. In t:•is -:o 254' -..":e cou:_ be t=ea:.._ .:.: -.atan- s:r.cke ccnder.sate ir. o.^.E da ' or 4S0 r::_e _culo be trea:ea ic•,::• :..-es a«•eek on f:ve weef_ay s. I: was ad•ra nta¢eous to l:c-:se t: e a:.::::a:s nur•.:be: s of eit: er s:x or eight ~er :age, ac_ to =z:e er..-c r.-?::=--es =-- =ages .a eaci: Fro•_;. Fo. :cis --_- .--.:a! r.•_...c--5 of rnice :c- caz•.. Grc•_--_< D, _ an= _!se_ were _ig3: (i : eight cages; and six (ir. eEg^t caces). „ages oc;th sa r.:ice were used, e n'.y cne sToktng rr_aci:ine was neede: 'c° seco:._ rc __ ...ne - :s li.. ..._ _7:rer ...ne -_::es of .J.a_ ..- :ns:ar.t s7:".crce _..:.densa_ _ :ec cn "--_ bac.. .,: ea:'r. ...:-_- =. week was Ln c=ce: t: t;:ate 26 n.:::a;s :Z~' tze .c..._ _Le=-_ ;. 5...~-.r :...;..: _ i. t-.e sTc'[° ccnden9a:e. _n!,' -n 7:'^€ 7C instant _:..?~. u•ere ac-..:a:s:,!re.- to ea:: an.n:a_ the firs:_.._ ~.._ -an.. was s:_•x•: and increased by c:..__..-.. ...~r_ ;r.c na d;._.zg t: c f:rs: 14 weeks of .reat: :en: .., exa_tly the =a--." :ra=e: as for the preconder.sed s:roi<e con densate, unt'.1 t: e a no~a: of 160 mg condensate per mouse per week was reacn e_ d_-- ing tye fifteenth week; this level was main tained for t:e re:::a:nder of t::e exoeriment. Lanolin - A 5°•0 (w/v) solution in acetone was From C. C8 to 0. 32 r:- of that solutior. were adr.:inistered per mcuse per week, b;~ a sc^edu:e of gradually increasin g dosages the first 15 weeks, which is identical w';G•^. that follcwed for the azo_:can tion of the suspension of pre:ondeased smoke condensate in Gro_? A. -5- cTR caNTRRCTS1 oa"50333 T 11225072 CTR NN 0423SEr

L-. Crc•.:n E the lanolirn t:eatr.:ent preceded t:e apolicaticn :.: ia- s:ar.t sr: c:<e condensate by less than 30 ^:... _tes. Vehicle (Acetone) - Frc:-i 0. 03 to C. 32 r::: of ace- tone, t:^.e vehicle of the cold-trap-precondensea sr.noke condensa:e, u•ere ad...! :istered per racuse per week, by a schedule of gradcaliy increas;ng dosages d•.;ring the first 15 weeks of the expe::mect. This sc-eduie was identical wit:. those followed for the applicat:orn of prcc-~r._c :sed smoke condensate s::spension ir. Group:_ and of lar.cLr. sc:,:tion in Groups B and E. L-. Group F, the ad.:.inist:s- tion of ace:ore followed the treatment .:•ith instant s:noke con_en- sa:e W::::n ~= rnin::tes. Body we:ghts of mice - After weighin g ail anir~.-a:s incividualiy at t^.e tirr.e of distributing then^= into six gro•_Ds at t:e De3i:_ in_ ._ -..e exC)eri_-nen:, collective wetghts of aa a.:-::als .z- _~;:i:._ ..._ s;.e cage were determ'ned at r: onth'_ inte:•:als. ..z . ,::sible papi'o-:as were r.et included _.n th~_e ce_~c- .:ve w•e:g :.ngs, and :het: Nc•e'.g!as were de:er::iined separa:elv. From ::zese data, mont:.ly average bod;: weights were co^:pu:e= a_. cf each gro•_p, with a:.d v.•it'--out ;:ap::lo:::a<_. ~e?:nitior. e: = aa~il:e-a bt t:e c-:-er~.a e: ;ress -soec :o: 5ki.. ...egL:ar:ties c: less thar. 1.._.. ..n c!a-:e::-, ctser•re--~ on •r:ee::ly :.s pections were aisregarded. Lesions o: . rnrr. c- :-:cr~, . _: c: less ...an 2 .-.-.. in dia_:.ete: •4.•ere recor_ec as :ab:_ :lc as, , ro•:ided .r.a: t.ese lesicns s•_:ia:e -: t:e s::'.:. a.he^:. Les:c.^.s o: _.._.. o. ..._... be estabiiS'_.. Ja~illo:t:as; ....:e•_ , c...= _ esion =a_. reached or exceede= a d:ar.:et:r c_ 2 thc• t:^:c c- .._ rea_ -c ~ _.rst acoea:ance Ras ass.:^:e_ to be :aa: %c: en it :.ad firs: !.-__n c:a:ne:e:. Once a ies:on had bee_ accep:e_ as an esta'--:._; __ alc-a, its host con::nued to be ccnsidered as a rtiouee a _:c^. , e er. i: the lesior. reg-essea s_Sseque:,t'-y'.. L.c_ ~._c or «as sacri:iced. Mice witc: established papilioTas we:e sac:. -easor : _, :iced fcr _.:stologic studies for on e of the foJoa•ing three if t^e lesion had reached a diameter of o::e-ha.L inch, or (b) i.` the :_ sion had not regressed during the six months following its first dete:- tion, even though it had not reached a one-haL" inch diameter, or (c; if t:ere was reason to believe fro::•1 the general appeara nce that a:e lesion had become ^.zalignant. Multiple papilior.-.as ccc-u:red not infreq•-~enay. :... CTf~ CaNTf~RCTS 0Q~~~~ 11225073. V rl u/ 1 . 1  1 I 046v 3sV

a /k account of thc n~:zbers of an i:nals so affected and of the nLmber: of lesior.s per anima: observed was made at termiratio n of the ex- periment. For an evalcation of the diiferences in biologic respcr.se between animals o: the different groups, no distinction was made, however, bet•wee: animais with single or multiple lesions. The "ef=ective number" of mice - In accordance •-rtit~h a sugses:ion made by Bing^am and Falk (E. Bi_gha.^.: and H. L. Falic, Src :. ::nvirc Health 19:778-783, 19:9), the results of t.e papa:or..a incidence are expressed in terms of the nur: bers o-' ---:=e with estabi:s :ec a3p:Lor.:a= in oercent of the fleffect:ve nu:nbers" c= anir:a:s in ex:s:cr.ce e: the same time, rat.':er than i:. oercen: o: the numbers of s-_-:: vor s. The "effective n ur.:zer" cf ar.imais is defined as t: e of survivors wit's.out papillcr••as az a given time, pl•.:s the :o:a: r.u:::oer of animals with establis: ed papillomas, dead cr z.1:•:e, a. ::e sa~e time. The use cf the not?or. of an "e:_'ec_ :ive nur--e:•" c: a-.ir5 s:s satisf:es the assu ptica that t:e c_... _- of :..--= a:a, reac:: at any ,.:^.e, =_t may never e..- ceec _ nc _G ; ..._.... i~g:c studies - An?*:.a'-s u•::h papi:ioma_, e:-- __c. 1'.c r c:.e of the three reaso ns g:ven above, - ~e-`ore ter::..:at•::. :• .-e _.x:_r. ..._..: anc we: = s•_:_`wel: preser -vec, •.vere kep :.:. ne :ra_ :o: alceh~ d: _._ :.: _ pz:nologic sr-d:es of t=e treated area c: ._--...._.?on, ..c. after .0: .:•:e::s c: s:.... «•-._ sa_..:iced. The _res:... exciseZ s:.... -. ...- ..e a:cc a.ea _- -__ : _-. ra: was st-etc: eZ . :e- a s-.a1: -:e bca-d and __-c<= -..-..ec.atelv ir.:o Garno•;1> sc:_tio n. r-.1: skir. __.•s es were .ak.e:% c_. c. _::s fixative after ic r:....`s, curir.o ..:..c: _.r.:e tiss::cs :ac ____::e se:^itranspare-.:. Sic:- lesior.s . __c be CetecteC a: _~ac1e _rOts whic : woG:C :a•:e c: ie~•N:se e5Z37e= at:enticn; histe_c,:c sect;enswere cut t:rough suc: soots cr any o-her lesions. Ln t:e absence of any visible les:ons, skin sez_ tions of treated areas were raade at random, and all skin spec:-:.ens were transferred : nto alcohol and were stained with hematozy'_:: an: eosi:. T:.is was also carried out on the skin san:::es ke^: in fDr:na:- dehvde fro:a an:r.•a:s that had diec spo_ taneous'yo or that had bee-n sacri:iced earlier. Gon:o-ete a. :opsies were perfcrr.eZ r_ all ::ice -?- CTR CCHTRACTS 005252153 11225074 CTR HN 04~'3'vr9

a / h sac-i:iced on ter: :iaatic= _: t:^.e st•.d;: or ear:ier, as a•_i-i as .,•. ani-:zls t::at ha_ died spontaneously, Frovided t::at the :at:e- .ce: e su_'_'icientiy well preserved. 1?istoiegic sections were :~:ace o_ a:: grossly a!~no_-r::a'_ tissucs. _=.ES_LTS The i:.fo:rr.ation, forthcoming _'ro: : a study c: s:= -)::;- ccn_°r.sa:e application to the skin of experi.-ne :'-al zr.i :zts, scc:. a_ the _ rese-*_ invest:£at:on, can basically be d:v:ded into _°o_r czte- gories. _:ese are (i) acute and chronic toxic r.:an::esczaons, __ .-_.::ar r c-tal: y and char.ges in body weight o: t.e tes: aca.:.z:: ; (2) aoservat:crn of gress:y visible s<:r lesions i n the anir::a:s, .r. c:uci^; a.^n e:-al'.:aaor of both the extent and the rate of appeara:.., of s•.:ch :-sicns; (?) '~istc~a:^c:o;ic stvdies on s-..: sec:ions f-.,..• ce t_eate= arez c: the independently of t:e ._..-.- .n g: syster.::t effec:s. zs de:ected by gross .-- a_. ^s; his:a?a:ho:ogic examina:-.,- of ..._ toxiei:v e: ice - Co::.der. _ the .:..~ _ , r...._.. , ...~ ..-=-~1: , c_` ...• -..ice a:._. : :ox_c::: _: tr.e test :::a:e-ials, :•eze '-ca- in all six grc : _ __ee _ _- _.._ :_.._ _; ,%'-.-. a fe.r r•-:i::c7 ex.ept'._^s, ..._-_ %'--e-- n_ s: =etweer. :o•_- gr:-= _......__: - .- ---___ .,.t'r. A, L, E zn_ : ; ~cc .._._ c. %e•it: cold-.-ac-?recr:-nse= -..•c.._ __nsa:e; e:.:...,..__ z.- on e time a sc-r.e•x•hat !a;-`.e: ..._..,_... :.: s__- t: e.:: ee ...::e:• groups, i. e. Set•.vee : t::e ..~a ~... a..= _.- :_.._:. .c=_.e c_ t:ea:.-._.. . That timr pr:iod :. _.._..- ,,- t: e-a~--a:u^: a: cerr:aaent dose lev-_ ci _es: ...__. : appears 1 te:y, t'r.e-efo:e, t:at the ele•.a:e= ...:..z'.:`. __ :_ .-.-:.e - n Crcuo A at that moment was caused by an a:•::e .,...- e`_ec: o-` r.icoti-e in the orecondensed smoke condensate. -c obo,:s az;ae tcx::i:y was observed Ln any of the other grot:ps. An:m:.ls in the two control groups (Groups B a:'_ C; showed a somewhat lower mortality, than those in the ot=e- A s•.:~cen drop in survivors :r. Group B(controi gro-.p, treated •aa:•n lanola only) at 22 weeks was the result of accidentzl conditions and was •__re'_ated tc :.._ trea::ner.: with tes: materia:. I: thus a? pears CTR coNTRACTS 005334' 11225075 {.wI' I R /MI1 I 04236,0

that t: ere exists a very slight systemic, chron ic tcxic e:fect c: sr:zoke con_ensate applied to mouse skin, as ;•_dged from the sur- vival of the test anima?s. The average body weights of t:e mice steadily ir.- crease_ in all groups (see Figure 3). As early as four weeks af:er the beginning of treat:-ier.t with test materials, the animals of t:e two contrc: groups (Groups B and C) were slightly heavier tt:an those c: t_.e four other groups, and t:.:s difference became pro- gressively more marked during the re:-iainder of the experiment. The bod; w:i¢l:ts cl the ani:aals of all groups leveled off towarc the e nc o: the oroiect. Because of the extrerr:elv ciose s:milar;.-: of the average body weights of the rr.::e in Groups A, D, E and F at any givea t;rae, t_.e data of these four gro::ps are represented as a sir.gle line i.n Figure 3. For the same reason, the data of Gro•.=s -5 a-.c C.:•e7e cozr:'=ined into one --..-, It is apparent :ror-n the results in Figure 3 that t: °_ s: :a: ._, s:,nifi:ant chronic toxic _..ect of smoke condC nsa:c aC at:atic n to t: e skin of mice n:an-'es:ec itself also by slight:: red•,::ez. G._. =e body we:g: ts. T: is systemic toxici. : was exac::•: sa-.e. •s::et*-er the smoke condensa:e had been co::ected ir. cc:_ .-a?s, s~ •-ra1 c.:s to several u•ecks r tc tne ex_ eri--:• en:a1 an:mals, or was dire:tly ceposite-' or. the skir :_ t: e....c- _r ~... fresh smoke. a1s •Nitn --ross-; vis:b1= pap:::o-: as exhib: zc•ara_e :-= %.ei;-:s -A•:.cl:.,ere, __r ai. _rac...al -?oses•: _.- sa..._ as .:...s_ =e:er:r.ined si....ataneo_si in ...ice o_' _._ sa:r._ SrL= .;_- t skin lesions. Conse,_. ::1;, it is -.cs: , psc:`_r.-.a f:_-:::at~on was cornected .v::-., or fol:cv:ei tec.: ::ei;_e.•iat:ng signif:cantly frer.: t.e ..:cans. lesions %:sible by gross e:;a:r.i natior. Papillornas - The cumulative n_:nbers of anir:a:s wit: grossly visible skin lesions in the treated area at the ti:r.e c: teru••.i.-iat:on of the experiment are listed in Table IV for all six groups. In accordance with our definition of a papi'_'.oma (see atove), these da:a were divided into several categories depending on (1) whether the lesion was an established or a questionable oapiLlo=---; (2) whether or n ot the lesion had regressed (partial:y or comple:eiy) and : ad n c: reappeared and (3) whether the : ost animal was dead cr r^ ~ "~ r cTR c(]NTRRCTS 00~3435 11225076 CTR ~`°~N 4~~ G'~~ ~

alive at t^e time :.f terr..ination of the e.:periz:ent. It is apparent from these data t:.at the incidence of pap:L'om-as (estab:ished papillorrsas, colL.:.r. ii) is considerabl;: higher in the group of :nice treated with cold-trap.precondensed smoke condensate (Group A) than :,-t any of t:.e groups of anirr.a:s treated with instant smoke condensate (Groups D, E, or F). These dif;eren :es are highly significant by statistical criteria, i. e. at coriide^ce levels of p S 0. 01. ydr^-r.istration of lanolir. to t: e treated skin area of t ne test a nir.:als, j;:st prior to the applicat:-na of :nstar.t s:r.o:ce con :er.sate, did not alter the response of the ani:: a:s, as seen from a comparison of the papillo: :a incidence in Groups D and E (Ta- ble :V, coiurr.rn ii). On the ot :er hand, application of acetor.e, the -•e:::cle .,: co:d-trap-precondensed smoke condensate, to de- posited .ns:a::' s^:c:,.e condensate on ...- ski.n o: the test ar..-na:_. sligl:t:: increased the papilloma ....-__n ce, i. e. _'ro=, 17. &._ -.. ?=: (compa: e f:b,:r _ s for Gro;:ps D and _ i:. cc! 1:-=. .: o: Table :V,. , Y-io•.%-ever, this _ifferer.ce is :ar from bei.^.a it shct::^ _=!sa be noted t: at the :ar:'.ioma i-c:dence .: _z.:. J, E' an:~ _ ..h:=.. •.c•as ca:.__- by apol:ca:io:. of ir.s:=n t s^:o::e condensa:e to rr_ice, is signi_i:an::y cifferent(ps :. 0-) frorr .: e abser.ce of papilloraa occurren ce _.n the two contrc: cr c ps .I Ea:: _ :.ate app°arar.ce - Cr:ronclog:c rec.rd- i: ..- t:^._ papi:io.^.za -..c.ce::ce, when ptot.__ ._ a -cordtna:_ . ..... c: aa ro!:abilitv scale (ior the papille::.a c_currence, on t::e cr__:.a:e. - s a lin.ear sca:e (time, on the abscissa), yie'._s a r.ear strai.. ;:or these two parameters (see = ig---e 1; Exa=.'-r.-_- tior_ c: these data s:ows that precondensed sz-.:ore condensate (Gro;:c :. nc: on'.-i causes a much higher incidence of s;c:n :rs:crs in t::e an:- r.:a:s than instant smoke conCen sate (Groups D, E and F), but ::at th° forr: er s:r.o:ce condensate (Group A) also affords a conspicuo:.sly steeper slope of the time-effect relationship than the latter kind of smoke condensa:e (Groups D and F); the data of Group E are sit ihr to t: cse of Gro•jY D and were omitted from F4-_-re 4 to preserve clar itv of exhibit. Regression of oaoillomas - The r.::a:Sers of an:.rrals -10- ~ ..C L!1 L11 C.n CTR CONTRACT S 005336 11225077 C~°R ~"'~~'`'~ +~~ ~ '~~ ~

a /h wit'rh regressed pap:licr.:as are shown i: colum.r. (ii?) of Table IV. When these figures are expressed in percent of the total numbers of mice in which papillomas ever appeared (figures in column (ii)), it becomes obvious that there were considerably fewer regressions in the a:.ir.:als treated with precondensed smoke con=ensate (GroupA), i. e. 21. 1Pa, than in two of the three groups o: animals treated K•it'r. instant sr :oke condensate (Groups E and F), i. e. 66. 7% and 61. 5"0, respectively. The regression rate in the third group of mice treated with insta nt smoke condensate (Group D) a•as about the same as that in Grou: A, i. e. 25. OTO. Questionable oaoillomas - Alt:^.o•_g!: r.:ost of the estab- lis'ned papiao:nas, listcd i.^n column Sii) of Table IV have, at one time or an:.ther, passed through the stage of a questionable oapilloma (for definition, see above), very few lesior.s have a::_allv remained a: this state for an len_ : of time, as seen fro:n the cata in colur_ s Civ) ar.e (v) of Table i:'. The decision as to .v:et::er or not such 1e- s:o::s were significar.t was left up to the outco:ne of the histODatLoloSic s- '_ies (see 5elcv,). Fer this reason, such histc::_:c studies were per::)r^:e_ on the skirn of a_1 surviving ani::ials with cuestic na~-:e or regressed c,uestion ab:e papillor: as, as well as on as many as p:.ss:- :,1e of t.._ mice that die? y.:e-^.zaturely, provided :at the sample -.as -z: lost ...rous : auto:.:s:.or cannibalism. Multiple paoillo: .as The time a: U•:ica the largest n. --ber of establ:she= papil:cr :as .vere actua:l;• in e:•aster.ce st:r:'.:1- __.e~ occ•_rred a: ter...:r.ation e: the exper.:r.er . e. in a.:,tal :_ =^ ar.i: :als in all groups corzb:-ed (see col ::~', in Table ':i, a:ti:ougl: a considerable n urr.ler oi iesicns : ad a:ready disaD=eared, e::'r.er because of sncnta neous regression (co-_: :(:•:) in Tab'-^ V), cr pre-^at•,:re death of the host an :mal (column (.ii) in Tab'_e `: ). Gi tce 40 r-..:ce with existing papiilornas at ter:ranaticr., 75=ro had sint:e _esions (cc:ur:•i:. (vi), Table V). All of the :n•.:lt:_'_e '.esior.s occ;:rrr'_ t:^e grcup o' anizr:a'_s treated with precondensed smoke conzensa:e (~roup A), and one of t:esc znice even exhibited three or more'_esions (columns (vii) and (viii)). Survivors without established papillomas at terrr.ina- tion - There can be no doubt that questionable papiliornas more fre- quently regressed than established papillotr.as. This may be due to the fact that many of the questionable lesions, small by definiti=n, could have been ca--sed by factors unrelated to the skin applicat?c^ o' smoke condensates, although no questionable papillomas have been CTR CONTRRCTS 00~1323? 11225078. C ~°R IIN 042363

observed in eithor of t-.,e control groups. The number oi s-:rvivo-s without established papillornas, as judged by gross vis::al cbse:•ra- tions at termination of the experiment, would then cons:it•,:te a::c:her meaningful criterion for the biologic activity of the various smoke condensates. These data can be derived from the sun:s of the f:g•.:res in columtts (iv), (v) and (vi) of Table IV, using the n•.-bers in parer.- theses, and can be expressed in percent of the correspor.ding effec- tive numbers of anir.:als (colusr• :(vii) in Table IV). Thus, only 12 o•,:: of ;3 animals, cr 14. 5°je, exhibited no papillor.:as in Group A, as cpccsed to 82.2?c, 8C.4cl, and 69.8;0, in Groups D, E and F, res?ec- :?•:e'_v (i00'o in each of the two control groups, Groups B and C). :iistonat:clo5ic studies c_° skin sect:c=s All an irnals surviving at te:rn i:.atio= of the experi- ..._-:.aere s=.:.r'.ficed and autopsied at that ti::.=, and histology of ..._ treated area o_' their sK:rn was s^_died. ; an i-als __ Gro•_? A, 124 o.' Groua B, '!- of Gro• _ C, 43 ..- Grc.:p D, 40 o: E, 4: of Gro•.:p F and 40 untreated and Lshaved con trol ani- ra:s, or a tctal of 4=E mice, were i.^.:estiga:ec irn t!as manner. L: ac~:t:oa, the sii- from a total of 60 anir`ais, that ha? died (sponz- : eocsl}• o= b;• sac: ifice) pr.or to :er^:ination, •:.as preserved :. - st_...es. Of these, 13 .:ere ....:e c..... ec:ablished pa: _ :c-as (existi n5 z~: regres:ed a: the time of dea::^.) and had bee= :-. ci:dec, t:erefere, irn the correspondi:~g effec::ve nu^:bers. T:e re:::ai.^.ir-2 -r2 a..;=als :.ac died spontaneo•,s:.: eit^e: «it^c_t g:css:v .... ia 1_s:c_., _. a: cuest:oaab:e _e_.n::s, ar.- t: , hac I-- : under the exne::r :eatal condi:ions for a: least iG wee'r.s, „_ ..-s.:.patl•.oicg'.c s..:dies revea:ed e:t_her a ca:c.no.:,a o: a:ao:1:c: a. _ .-._-ty, hist~,logic sec::o-s were orep_-re_ on ..._ ani: :als which c.ad d:ed after less than 90 weer.s of trea:-er-:; :_ : a=-i1c -as ~r c~.rcino:nas were detected histc_:;_ca'.•:y ...- r.es_ latter 5-s sections, an= they were excluded __:a:ions, since ?t :nigh•. `_e reasoned that lesicns could i.a•:e __..:ed :.n these ani: :als if they would have surviv--_: lon,e.. Histc=ogic studies o: the skin, th::s, incl•.:ded a grand tot.al of 565 anir_ ais out of an initial populatton of 976 rnice (inc:,:d:r.6 the 40 untreated and u nshaved controls), or $7. 9`0 of the total popu- la`_ion (32. 0% in Group A, where the initial mor:ality had been high; 67.5;o in Group B; 60. 0°,'o in Group C; 62.5% in Group D, 5:.4is :Z Gro•:p E, and 65. 2% ir. Grc•:.p F). There were a tota: of 33 ca: cino- rr.as, eight preir.vasive carcinomas, 49 papaomas, 89 eoide:z-.:a: -12- CTR CC~~~TE~ACT S (~Q5~3B 11225079 CTR HN 042-3641

Ih /1 and/or de:r: al ulcers, 185 instances ef hy'oerkera::s'.s, 2. 71 cases of epidermal hyperplasia, 265 sections with dermal i.•lamrnaticn, 249 skin samples with mast cell accumulation an d 158 animals with normal skin, including both derr:is and epidermis. µi ith the exception of the nora:alc.: c: s'r.in, these features almost always occurred in association with two, three, four or more of the other pher.oraena, as seen fro-: ::e resul:s in Tables Vi to XII. Th_s, skin sections with carc:::orn as o:ten ex- hibited also papillomas. Ulcers occurred alone, as wea as con- cc,:rre::tly with either carcinomas and!or papillo^:as. Epidermal liyoerplasia and, to some extent, ::yperkeratosis coasistently ac- companied a:l carcinomas, papillomas or ulcers (=ables 'o I, IX, % and XI), but were freq•,:entl,v found also witho•.:t t:ese :::o:e severe lesions, such as in partic;aar in the co n:rol g-oups (Ta'Dies V:1, V.^,I and X_T). Dermal i:.`lat-unaticn and, to a lesser deg:ee, mast cell acc•,:m%;iat'-ern were very frequent ..c_,..:ences ... a_1 groupe, even in the control animals. Carcinomas - These we-e clearly r:al:r- .._..t lcs:ons, and a'_^zos: all were e~:?er rcic sq•,:a:::o•_s cell carci ..omas. Frequently, one or several papi_c:::a: -;~ere seen in a::c:- .-„- to a car...no:zia crn t: e same sect:o-. Alr..cs: a"-` _: t.ese .es:o=: were accor.:panied b•; :narked eplder-:a1 h•;peroiasia ar.dio: hyper- <e-atosis. ': arious degrees o_' mast ce:: accu.:.:::a::o:: an_ derr.za: _'ia = a:ion were present in most instances. e:. :as:v A carcino^1as .nvas?ve carci.no:nas ^nen:ione_ above. .:.ere -_re -,..._ i~stance~ -- ' cseudoep:t:elioa,atcus hy?e-plas:a" ::erc d:st:r.- :s : frorr: very early invasive carc.no:r.as and .•_._.. ..._s: be class.- .._.4 as "; re:r.vaslve" or llq;:es:ionable" cazc... ..:~_. Paoiaomas -':hese were diainose: cr. basis o- localized presence of hyperplasia of the epithel:v.•ith hyperkera- tosis. They were lobulated tumors a:ising from t.e ep:~_er::.is ir. a localized, ciscrete area. Sometimes they were r^ultip'.e, and alr..•=s: alway-s were they accompanied by widespread eriderr.:a: hyperplas:a and/or hyperkeratosis, as well as by mast cel: acc•:=.:aation and dermal inflar:.mation. Ulcers - A cor.siderab:e r.u:nber of sections showed dermal and/or cpidermal ulceration s, mostly tsgether wit: eztens'.ve :3- CTk CC1NT~2ACT~ 005339 11225080 V f R i i f 3 042365

a /h epidermal hyperr'-asia, without'r.o•.vever ex~-.;bitir.g the char>_c:er- ist'-cs of a papilloma. Not infrequently, ulcers occurred concur- rently with either carcinomas or papillor-:as. A conspicuously large number of ulcers was seer. in the untreated and "uns: avea" control mice (see Table X1I). Since the animals were shaved aftcr sac-.i•fice, these lesion s were probably artifacts.. Hyperplasia and hyperkeratosis varied considerab:;: in intensity from onc sectior. to another and these graduations are not ;r.dicated i n the accompanying tables. It should be etnphasized, however, that the instances of hyperplasia and hyperkeratosis ob- serc•ed in the control animals, i. e. i n Groups B and C, and in t:e gro•.:p of untreatec and unshaved mice, were nearly exc:::sively o: the mild variety. Severe hyperplasia and/or hyperkeratosis o_- curre: in ani:nals treated with smoke condensate (Groups A, D, E aa_ F), either with or wit: eut s:rnultaneous carcinomas, papi:'_or as o- ulcers. De-r._a'. iaf:a::-~.-rLatic:. - =h:s _nomenon occLrr-c .: a great number of animals, both from : the ex; erir-.ental and con- t_01 grcups, and varien in severit;: c.er a w•ide range. .: v:as fc•.:n? t: uch less -reouer._'.v and was much w ilder in t:e trc•o ce-:-c: (Z-~:=_ps B and C) than rn the fo::- groups of animals treate= ....rn s^zo::e condensate (Groups A, D, Z' and F). How•e ver, t:er e•.,as a rather high incidence of dermal 1aFia:-L-riatior. :n the untreated and ,.r.s :aved" contrc: a::irn aa (Ta::e X*"), where this type of was -::)=e-a:e1}• seve-e in many ilsta:lces. Mas: cell acc_:^•_:atic.^n ranged :.n deiree -r on-. a e: e increase in the number of c:soe::ed ceas in the der mis :_ .ise existen ce of sing:e and even rr.ultiple, w•ell-defined nccuies :- c!•usters of :nast cells. Like dermal infla.:r.:a:ion, mast =e1: a: ._.. l;laticn was more freqsent and r::cre pronounc-'_ in a-.- ^:a'-s trea:e'_ with smoke condensate (Groups A, D, E ar._ _) :.._n in the control m:ce. Lz contrast to dermal infiar.^mation, mast cell accun:,aation was rare in the untreated and unshaved control ar.:- :nals, and whenever this phenomenon did occvr in the latter group of mice, it was very ::zild. Normal skin - Ar.ion5 all of the ani:nals treated witn smoke condensate and studied histologically (a total of 218 :r. Grcups A, D, E and F), there was only a single mouse with nor:::al ski :(in Group F, see Table XI). However, 92 out of 131 mice ir. Group B, -14- CTE2 CaHTRACTS 005,540 11225081 CTR MN 4~ ~~~"~ G~

a /h 54 out of 117 animals in Group C anc 11 of the 40 untreated control mice, i, e. a total of 1-5' oL:t of 263 control animals exhibited corr- pletel;: nor-•ial skin, oc:h with regard to the dermis and the ep:dermis. Y.istopatcologic confirmation of gross:y visible ::nd:nss - Out of a total of 53 animals irn all groups with grossly visible papi'_lor.:as, irn existence at the tir: e of death or sacrifice, as many as 43 r.^.ice, cr E1. K',, exhibited carcinomas and/or papillomas by aistopathological criteria. On 1v seven of Lhe 21 mice (33. 3%) with regressed, grossh• visible pa?t11cr-.as showed carcinomas or papillomas. This would in- c.icate tc:at in about one half of t:e instancos of regression of gr;,ssi•; Visible ?a??llcr::as (b:. 1 - 33. 3= 47.81N), there was complete caa:- pearance c: the lesions, as judged by histopathology. The number of a::ma'-: with questionable, grossly vis:- ble• pap::lor.:as, studie_ l:istolcgi_a:1}-, was srnall, .. -. only fc;:r -_..co ..:e - wi:-. :ionres-essf-r' q_es:ior.able . _-p:_lo.^.:as and 11 •.ci:h rey-esse--* tio na'cle papillcn:as in all gro',:?s. I:istopathologica'-a•, there ..==2 ... ( 5C'•1 and t: ree 177. 3T,) instan:es ef carcinomas er oa=alomas, re- so ec--re1~ _..ese ;gures are tco small to be ef a:, sig::'-icanc_: the a.^.-..._-_ grossly .is:*D._ skl_ -__.:n .. . .C :arc:..:-..._s 5a n.= s:x 7a.'+ ..~..-.ai ...t::OLa CarC..._...a_, ..E. a o' 1 lesions, were ..e:ected by h:s:oiogic stLdies. Wher. these ::g-_re: a:= co::oared to the nu•^-zbers o: :nice without v:sible les:on s i: .:.- exper.-.e-.tP.: (_-o'.:t)s (Gro•_:s A, D, E and i.e. a tota= ar..:::a'_s, the ....c_ g:cs: •.:-:bie lesicns , eld o-. _. _..- .:-cg:ca:iy c'e_-..-: oa...omP.- _.._%c: careir.o::.as. -iI:s f:g•_--- c•: e:=n ^:uc:: smaller :f t::e an=.als of tae cor.t-c. _roups ~ce=_ ...- c: in th?s cor.siderat:o.^.. It :h::s appears t=a: :::._e wit'-- quest:~-.ab:e a-.= :- :_ cre:_ed les;ons a: e 3ar more 1.*-ely to : avc a}:sto:ogical:•: de :: n- s:rab:e :arc;r.cr..a o- pao;llo: na ::-.arn anir:a:s wahou: an•: .:si_:e _..... lesio.^.. Numbers of mice with carcinomas or paoillomas by all means of observation - In e;ra:::a:ing the biologic response to ad- ministration of cigarette smoke cor.densate on the sxin of test tn i- mals, a decision has to be made, as to the definition of such biologic response. For this purpose, only the appearance of carc:r.c.:.a.s an =/ or papiaomas wil: be considered a resF'`nse to smoke condensate ap- plication. Irn order to cor:putc the total number of animals u•i::-. car- CTR CahtTRACTS 005*314 1 11225082 C~mR MN 4~~-~~6f'`

a /k c:nomas and/or papil:o: ;as by all means o! observation, it has tc be taken i ntc account that histooathologic sttdies on animals wit :- out grossly visible lesions were limited to three categories of mice, i. e. (1) aniznals s;:rvivir.g at termination of the experiment (10z weeks), (2) ani.-r:als studied ristologically which died prior to ter- mination, but w: ich had been exposed to the experimental conditiors long enoug:: to sufficiently minimize the probability that a lesion would have arisen in the event that the animal would have lived to termination ; this limit was set arbitrarily at 90 weeks, and (3) a::- :r-als which died prior to that 90-week limit, but where papillomas or carc?n-_:nas were d:tected histolog:cally. SnEr: a:: v,•:Lh erosslv cisioie lesicns that had never reached a 2 m.:m diameter (cuestionab:e papilio-nas by definition), or with regresse'_ ques:io:able papi:lo-:as had been shown above ..3 have a cor.sicerabi,; :':: er incidence of histopathologieally de- r.o: stra'cle car.--nor:as ar.d''c: rapi'_ioz:as, i. e. 50c'o and 2. 3°'~ r~s::ect?~•ei.•, ..._...-.icc a:;tnout grossiv v:sible lesions `:evcrtbe.ess, -..e cocsicer_tior.s concerr.:n_c the cor:putati7n o_° t^e total numbers of an::nals .ca:: car;._no:: as and/or papillomas arc tine sar-:e as .-, t::e caee o: .-..ce .;•i::..u: Erossly visibie lesions. ..-e n u:-:e-s c: all mice wit^ .as a- lo~:eal:; c-:,•.•e n car,:::._ ::i. and/or pa_'.lioa:as, w: er= Sress ha4 reve=led eitr.er r.o lesions or only q,:es- :..,-:abie :~a::Uc--:a ~c--:a~ ,==-esse_: or not regressed), were a~ae: io: eac~ g:.,.:?, a::~ ...:sc Lr_ sh:.... ..n colu:^.. able -.__. -_ t:ncse _~_res were added the au::bers o: a-.:- a1s wi:: "e=:~ss_y vis'~__ ca=:!'c ras, ..._e=e : e^t_;: ofec•-e::er _r no*_ t.-_se iesior.s rac :e~r~sse~, ar.i regardiess of .ehet: e• .. ..:.- t:e sro:s .:n_:ngs had bee.n con`in-::ed cy oa:..clog,:. : ce res_air.3 fig~res, ind::atir._ .i:e t::;:1 r.c rbers _. aYi:na:_ vriti: ca-_-r.o:-:a a-:/or oao ac::a b: a:. r.:ear.s o' c::ser :ati:,: , are s:own .., col,.=-n (iv) c: :at'-e X:il. T:.:s _-c- cedure appears because over alre o: the nonregressec, grossly observable papil:c.:,as could be con:irmed by histology, while only 53 of a total of 70 animals in this classification were available for histologic studies; the others had died prematurely. It remains unknown whether t:.e sarne proportion of ccnfir:nation prevailed in the 17 m:ce with grossly visible papillomas where no histologic studies could be performe,_4. Slt :cso:^. the proportion of histologic corfirrnaticn CTR caNTRRCTS 0053412 11225083 CTf"!it l I i i 0 "T .Q .. ~,.~ i,,..a 8

was coneiderabl.• s-laller -: animais regressed ~api:i ^ias (33°0) than in those with nonregressed papillomas, all of the m::e vviti: re;:ressed papiLornas were included in computing the totai numbers o: carcinomas and papillornas for the following two rea- sons: (1) In accordance with the definition, a papilloma is a non- malignant lesion, and the regression o: a papilio^:a does not i::- ply, therefore, that it has never existed. (2) As in the case o: mice with nonregressed, grossly visible papillomas, histopath:loEic studies on animals with regressed papillornas d:d not izcl;:de al: c: ti.e animals with such lesions, because of mortality prior to ter- mination of the experiment. For this reason, the exact n,.::nbe r o` mice with histological:y cor°irmed, regressed papaomas is no: lcnown. Since t: e figures in coi ..:-r. (i• ) of Table MI_ inc:_ce some ani:: als which had died before terminat:cn of the experi:::e-::: w:thout grossly visible oapaomas, :.`.e:r numbers are not in t:e effect_ve nu^: bers. To adjus'. _ne effect!ve nur::bers acc_-~- inglv, the n.:r_zbers of the corresp::.di:.z a-i-: _is have beer. co:-.- p_ted frc::: the figures on the las: l:r.es of Tac:es Vi to XI and a-.e shcv.z: :.. colu u: (vi) ..: Table XIII. _:^.e "ad?•_stec" effective .,ers in coiur.:n (vi'-) are, t=•.:s, o_-:zine_°. Finally, c_ .._^:5er_ __ a=.:rr.ais wit : carcino:::as andJor papi:io::•sas by al'l rr:ea ns of ctse-•._.tiens were expresse_ in cercer.t c: th= adjusted e:fective ;co'-um_n viii; =_ Table }~~). -hese z- res are, --. o•_r ooir.io-., .:._ z::o,t .^.:ca::. -,:1 expression o: the bi..-og?c acti'. :; of t: e var.,._- test .-.c.=-_als. =nei- -._^^-erical val~es and ._ .-_ rclative or---_ _:on :. are .,..:er _......=r to ..._ ecrr-=s.ond:-:g oc--_- _c .-. :: e bas:s of Eross %:sua: cose-<<tic:: a'-:)ne (see ::; ces .n par=.. :.. > >: col.:r :n (i:) .:. = ab':e I'.'). Carcir.ogenicity o: test :-:aterials a°c r.iali<r.~r.c :or.s - T: _. _ were a totai of 33 an?r:als w•:L^ ha:o_at:cla_t ._. Y-ove n, in vasivc carcinoma in the treated area of tic s::in. i:. tion, eight ^zice exhibited lesions which were c'.assif:ea by his:-- patno:egic criteria as preinvasive, ir.c:::di ng four cases in Grc-_= A. one each :n Groups D and E, and two in Group =; t:a latter w•er e not inc ;:ded in the follcwing considerations on the ma:ignan cy of tt.e le- sions. Of the 33 instances of invas:ve carcino-w, as many as '_ ( cc- curred in the group of animals treated with precondensed sme<e con- densate (Group A), :osr in Group D and one each in Groups E and F (see last two columns of Table XIV). When these figures are ex- pressed in percent ef t: e msmber of animals in which histologic -17- CTR CaNTRRCTS 005343 11225084 CTR HN 0~~~~69-

sn:dies of the skin were performed (see co'-;:a•in (vii), Table it becor.:es apparent that precondensed smoke condensate (Group A) is overwhelmingly and significantly (p«0.01) more carcinogenic than instant smoke condensate, Independently of whether the latter is administered with or without acetone (Groups D and F), or in con;: nction with lanolin (Group E). The differences in car cino- genicity arno ng the test materials of the latter three groups are :n sig nificant by statistical criteria. The maligna::cy of the skin lesions observed by _ross visual inspect:on was greatest in the case of noaregressed papiilo- : sas, i. e. 45. 3',', (see colurr.n (ii)) and decreased to 28. 6?•; i: or u•he:: t:.e pap:llc:::as 1:z3 regressed (column (iii)). Animals wi::h grossly visible quest:onaz:e and regressed questionable papillon:as ex'r.izited a very low incidenc_ of carcinomas (see columns (iv) and (v)). :ow- ever, two ins:a=ces of carcinoma were detected by histological s:.:d- ies in t:.s nin e surviv:n g m:ce without grossly v:sible les?cn:, s. Gr o•.:c A, nor.e of the 120 ani.•:~tals o: Groups D, D ar.c~ _:o:'r.- biaed, vvhicLh survived wi:hout visible skin lesions, showed a.;• signs of car c:nomas (see cci•::an (vi)). Even tv :en t-.e n•_^:bers of carci- nomas are ex:ressed in percent of the sums of histologicall;: --cn- firn•:ed s}::rn iesions, including invas:ve carcinc:nas, pre:nvas:ve or c::est:cn ab:e czr cino.:as and papilior..as, it is apparent t:.at :-econ- dcnse-_~ s:aoke condensate produced many more ma'_ig.^.an: 1=s:cns ne (62. 8~o in A) than .nstant smoke conden sate under P• o° e ccnditio n_ tes:e-e tr:::•i ::. 3 to 33% in Groups D, E and F, see colu::=. =aole ..:~';. A`ner::.a'_it:e_ cbser•:ed on autoosv ter-::.~:icn A total o: 452 anL-na:s were autopsied at of t: e expe::Yen:, .. e. after 104 weeks of treat:ne nt with cor.=e nsa:e or exoosti:re to control co nditions. At :ha: 2:' ob- ser.-ations of aberratiorn from the normal state were :ne::e in a:cz' of 171 mice, and t^ese aberrations ranged fror. slight discoio: a:ion s of an crgan tc the occurrence of tt:mors. All six groups of mice, as well as the group of un- treated and unshaved control animals were thus affected. It is note- worthy, however, that a markedly larger proportion of animals from the control groups than from the experimental groups were involved. As many as fron: 76.7 to 85.450 of the mice treated with either kind _18- CTR CdNTRRCTS 005344 11225085 CTR ~~~~ ~~1~~'70

of smoke condensate exhibitec no abnormal autopsy :in3:ngs (see Groups A, D, E and F, column (i:i) in Table XV), while only fro::: 46. 0:o 60. 0?'c ef the con trol anin:als showed no gross autopsy f_ n^- ings. There were altogether 27 instances of grossly visible tumors at various sites, as seen from columns (vi), (viii), (x), (xii), (xiv), .(xvi), (xvii) and (xviii) in Table XV. To these must be added three lur.g turr.ors fo•,:r•d in three animals which died after 70, 7: and 91 weeks, respectively. The nature of these 30 t•:.^zors, as ideat'. fied by histo?at::olosic studies, and their distribution among the :•ar- icus ana:;:r.ical sites and ar: ong the different experimental o- ccn - t=o= gro~: s of ani: :a.s, are shown in Table XVI. It is apparent t: at tumors occurred as frequentiy in the cont-o'- groups as in the experimental groups. There can be no dot:bt, t: er_:ore, .: a: none of these tu:.^•c-s was a co^seque^-e _- the .-ea:rn ent cf the ar.::r.als wi:: smoke co::den sate. O: t;cc 3~ mors detected on auto0sy, Z2 were malignant, five were and :.•=ee we=e n c tumors at all. Reticul'.:m cel: sarcomas sn_ ad•_r.cca-c-r.o:^as -,c•e_e cy far the mos:frequent eccurrences ar.:oz: the z::ali;nan cies. :h_ee o: the ta:mcrs (one .. Gr;;•.:o A an_ :•x-c in G-ccp D) and er.e r.::re case oc inflarn.-nat?c-: (in Group F were acco:r.:~anie_4 b.; invasive scua rnous cell carc'.no:::as ia t: e -:eeted a-ea c_ t: e a:c a..:sto:egica:ly defined pa=illor.a .- a a~r_:.._.... instan:e (Croup A). On -:,e o::._r tar._, •.._. _.cere a: east s:x cases of r.-aligrant k•ry.:ors -n ar.°:::als r„aced wa:: s~o}:e con_=asate (one of K•'--ich vrit : a subcu:ar.ee•_: f:bcosarcor::a beneath -'-.c treated area), where histopathology c: s:::r. d:_ not reveal any oap:Lor:as cr carcinomas. Fina:l::, :~ .: t:e 22 mal:_: a-t tumors o:cu: red in cer.:r:,: r.:.:e t:a: 'r.a_ never bee-. .:eated w:th s:ao:ce _,.::densate. No histologic studies were done on the e-'.argec irregular (pale) spleens, nor on the few irregular (pale or spotted) livers (see Table ?N). These observations, as well as a few cysts ir. the kidneys, ovaries or uterus and one instance of an enlarge'_ bladder, indicated i n Table XV, appear to be of no signiSicance. There were however, as many as 101 cases of hydrouterus which af:e=:ed f:or.: 25. C to 38. 7% of all control -~9- cTP CaNTRRCTS 005345 11225086 C w) I 1 I ) 7 1 0422371

atiitr.a'_s_, .c•hile the mice treated with smoke conde_sate exhabited this phencrnenon only in from 0 to 7. Ocro. Histologic studies cn hydrouterus were performed in eig :: samples, selected at rando:n from among the 101 cases at hand. Sevea out of these eight cases exhibited no obvious signs of obstruction. The one exception showe-' epithelial 'r_yperplasia of t: e vagina, possibly consisti.zg e-° a carci- noma, which did obstruct the lumen. i i i D:SC USSION The tumorigenic acti%:ty of cigarette smoke conder.- sate, ......a applied to tr.e secin of test ani.mals, has been recogn'tzed for abcut 20 years, and the earlier findings on this subject rnat:ee have been thoroughly reviewed (E. L. Wynder and D. Hof:man :, i- "ToSacco and Tobacco Smoke," Acade:z:ic Press, New York and iondc=, 1967, p. 177). Der:r•.al bioassays of tobaccc smoke c=n- de:isate in the mouse : a:e beern pe:•fc-r.:ed -.n a great number c: ._ C::= stances, and tae re_al:s of large scale eXC1er:^:ents o: this have been reported as recently as this year (•:,arg•.:s, J. I, Ga:•t, R. P. and Gori, G. r. Abstracts of Papers, S.Dc. r•.r.__-Mceti^g, Ne-• _ork, -Mzr_... 1973, - j. the ^:an•: fac:crs, predoa::::a =tly i-voived .n t: -e_==ess cf h nio:i_e-esis by co_ee^sate, the ^•sa:u:e: ni ^re; ar:nz t::e cc.^.densate :rom smoke appears to be of decisive ;...- _,O-ta..__ ..= i:s b:oio=:c activity. T1:•:s c:gare::e srr.oke cor.der- sate, ..... pre?are_ by two seoLra:E !zrboratorLes, ir.d--ced r..ornec-` differe... .-.-r:=b^_rs o: si:i-r. :esic :s, bc:-. pap:L:o:nas a:: carci-.c--a=, assa;ed under .__::t;cal con•_°i.:o-s, a nd on_ o_° t.`.e cc--_.s sates cc-itained three t;mes as m•zch ~er.a(a)~;-e^e as ...e ot: e:• (Orris, . Van Duu:en, B. ..., i:osak, A. .. Nelso:: . an= Sc hrr.i: F. L. , J• Na:1. Cancer ins: 2:55-, .958). Consida- able c.___:ences in biologic activity ba•:e`also been reported twee- c:garette smoke conde nsates prepared in the same la-,crz. tor;: cy di::ere: t procedures, atter these con_ensates w•ere kept for _:.eq•-a1 lengths of tir ne before a??:ying the:r- to n.ouse sk:- (7ay, T. D. , Brit. J. Cancer 21:~0, 1967). There can be r.e do;:b:, therefore, that artifacts are introduced during the combined processes of condensing the smoke in cold traps, mixing the cor.densate wit:, acetone, hea:ing the r:ixtLre under reduced pressure (at the relatively low tem- -20- cTR cOh{Tf2RCTS o05346 11225087 CTR MN 0423F2

peratures, i. e. 3C :~ 4:o C) in order to rer::cve most o: tc-e wate-, adiusting the concentrations o' nonaoLeous condensate and of water to ts.e desired levels an d, finally, storing the finished smoke con- der.sate-acetone-water mixture without causing phase separatio n. Davies tried to c:rcumvent these diSficulties by ao- plying to the dorsal sicin of mice instant smoke conde nsate pre: ared by impingement of fresh smoke on a capillary needle wit.h the use of a capillary press smoking machine (Davies, R. F., Brit. J. Can cer 1959). Comparison of the turr:orige:::c activity, of t:.is t, ' :pe c: smoke condensate wit.L that of a conventionally prepared, cold-trao ccndensate, did not result, however, irn a ny appre;.iable difference bet•«een the biologic eaects of these two t;:: es c: smoke cocden sate. With precondensed smoke co::'_ensate, Davies pro- duced R:.:.ors in 42, 7;0 of his animals after 56 weeks, whereas only 4.3' of the mice ek-:bited grossly visible papi?lo:nas under the con- ditions of o-ar own experi:ncr-t after t:e same of ta::c, a=-_~ -. tco:: as much as 82 weeks under these latter condit'-ozs be:orc :__, r-:ar.;: a-'-mals had developed sk`_:: lesions. The ar:cunts cf no-a-le- ous smcke ccn densate delivered to each animal per week were very s:-i?ar ..n both experiments, i.e. 150 mg ... Davies' case, L.._ :=,g .n our own experi:nent. The lower r r r:~enicih: of prec:n_ense-_~ smoke condensate :. _nc by us, as compare= to :::a: descr'-bed = -^ __ .:ight bo ascrioed tc difSerences in e.-.._. the to:acco,~ .:.e met: ods c_' preparing smoke condensate, or t: e s•.;scepti~Dility oi t:e exoeri:nenta: A1:ho-.:z: instant s:noke condensate was aL:,c_: as ..- rr.c-igenic as preccndensed srr.ofe ccndensace . n La-^es, ex:e:-mer.: (3-. 3 versus 42.7;; mice rocit : tt::nors at:er 5c. wee'<s), ins:a n. con- c=-.s-a:e exhibited sicnif:car.t!y less biologic act:%•it-; t:an p:e_en - ce::sed condensate in our owr. hands. T:?s -was true for t_-e n%=aers _` s:<.:z lesions o5served by gross visual inspe=:ior., for t:-e ra:= c: a: :earance of these grossly visible lesion s, ...r the numbers c- -..-.. ...:no-s identified by histopathologi: studies, icc:_dtag both ben ign a nd malignant tumors, as well as for the numbers of i.nvasive ce-m-a: or epidermal carcinomas. Our findings were not significantly influ- en ced by simultaneous administration of the vehicle of precondensed snzeke condensate (acetone) together with the instant smoke conden- sate, or by concomitant application of lanolin to facilitate absorption of li?ophilic material by the skin (see Table )CILI, column (viii)). Bct:. types of srr:oke condensate were admin:stere= in equal amounts, as _21- CTR coNTRRC?`S 0Q534~ 11225088 CTR HN 04~~~~~

I . /h ':vea in Lac•;esi as :r. our own exY-:i:.^.ent. The a:ac;:ats of water contained in both types of smoke condensate were almost the same in o;:r case, but were possibly higher :n Davies' precondensed than i.nn his instant condensate. Finally, the amounts of smoke conden - sate generated from each puSf of :res : s:noke were exactly the same in Davies' case (50 rng from 30 pu_°fs) and our cv'n experiment (40mg of co ndensate from two puffs of 12 simultzneousl/ s::zoked cigarettes, i. e. 4C mg fron: Z4 puffs). Unpublishec experiments, still in progress in our :abo-atcr.: at the ti_^'ie cf this writing, have uneoaivocally shown, a: least i: fe^za'_e hairless Y_RS/J r: ice, that instant smoke con- ciezsatr : asc.esses a:'e-y potent pre:n:ting activity; the instant sn:oke condensate vr<s prepared and a'_:ni.nistercd under the same condit:o: _ a s in the expe riment wit : CAF1 /J r'iice. At t:^.e sa: :e time, the >.:::zorigenic acttvity of instant s-noke condensate was ec::all•r HRS '- and Ce:F ~ r:::ce. For :e afere:::enti:)-e=• reasons, i: is assuz:e-~ _..at the c;_ierence in rs:Ysorigenicity i:e:ween :..° t.c': types of s-:-.oke cendensate is ca'ised, at least in part, by a paucity of instant sr.zokc conden;a:e; suclh an assu::otio:n is substar.:;a:~c by o;:r of a:rar:cec .: less steep slope :r. .: e re:a::on >ap bet•.t•een oapilloma appearance an d tirne :or the s:an: sr _~i;e con_e:.sa:e t`an :: the o_econ_e:.sec s-.c:<e cc-.- densate. This co nce?'. is i^ agree^•:ent wi:h Bere.^.b_,:r.:'s viek• -..._ =._cH.:nisr.: =: a t.'ro-stage carcinogenesis consisting c: a .. :. stc_ c' in-.iatic.. .r pr:ming ....n_:e a: :cun.. o: a.. __ ,5.:c:: as benz(a)pyrer.e er ci-.e:^.:lber.zan:n racene IYa J- a-_:-iast?red on a s: ` . cccasio.^n and \l': ic. a:one, is L.SL'if:Clea CD ca'.:se tllmor ._-.^..a:ton), .cJo\t'ed C~ a ._cor.c ste: i. e. -cm.,:ing by a ec;.a_cinoge=, suc.- a., an:hra:. . ;,rotoz. oi-, p5z)rbe1 esters, or ot:crs, admir.i..tered repea:e'_: , `-- ProdLCcs ...: or or:y very few _-nors when acol:ed ....:iato- fBerenbl•a.-r., I. and Shubik, ?., Br: J Cancer 1:3_=, Fina:iy, there is further support fcr c•:r .:r ~ot: esi~ that the weak t•,:r.:crigenic activity of instant smoke condensate is caused by low levels of carcinogen in this type of condensate; i. e. our observations on the high regression rate of papillor: as in ani:-ials treated with instant smoke condensate are consister.t with the generally accepted views of a two-stage carcinogenic system u'it : high levels of promotor and low levels of initiator. .72- r ti cTR cOHTRRcTS1 aos~a-8 11225089 CTR ~~N ~~~~ ~ 4

: or the in:eroretaticn of Davies' results, it is c.:-- ceivable that unintentional exposure of ::s mice to extremely s::ali quantities of an initiator, such as r: igz: have occurred if other a:i- mals had been treated with carcinogens ir. the same room, would have drastically increased the ski.^n t•smor incidence in his experi- ment. Both Davies' an_ our findings are in good agree-:en t with regard to a somewhat higher rate of mortalit}• in the gro•:ps of a=imals treated with precondensed smoke condensate than in thc« receiving instant smoke condensate. Our result< s,_ggest, therefore, that instant sr:c:<c condensate is considerably less tti:nnorigenic t: an equal amo;:n:s of precondensed smoke condensate, and t:zat this dissimilarity is c•.:e, at least in part, to corresponding differences irr the levels of i n::'-- at=rs of b=.or formation in these two types of smoke condensa:e. S-ch an :-:pot: esis .:•o;:1d ir-..p•sy that a n ajor portion cf LRiaatc-s or cas cinogens contained in col,_~, :p ap pre.c ndenseC sr^oke cer.ce: - sa:e v,r-s actvally not prod::ced'.r. the course of burning the tobacco, but was formed subseq•,:ently to .: e process c; smoking, -. e. __r'-r.; storage and/o: r: anip;:lat.cn o: the cc::ac.^.sa:e with solven: (ace:_ne). Any conclusions on biologic ef:ec:s o: tobacco smoke in the =•_n:: cf hu_^•ian smoke: s cn t:.e basis of t:.e t•:rnor:gen?: activ~,:%• o` = reccr- dense_ smoke condensate on r-..c,.se would :h•.:s be <_'.nce it is basec or. arn art:-`act. cor.den sate, by .a: a e~er proced::re :: r-.a;: ve : repared, co::ir. es to be a valuable tcc' fer the co:npar-.:ve evaiuati:n of sr.:oye c=n- ,._ nsate f:-on: different smoking p:•od•.:ct_, as ...ne, as th= cor.=_ are obrained and s:ored under identica: :cndi::ons. 5 _:.L\S.tiRY Two types of cigarette smoke condensate were tested for tumorigen:c effects by skin application to t.'~e shaved bacxs c` fe- male CAF1/J mice, in groups of from 100 to 200 animals. The first type was a precondensed smoke condensate; it was prepared in t*--e conventional manner in_cold traps and was applied to the test ar.in:als as a mixture with approximately eoual amounts of acetone, after re- ducing the water content of the riLCture to about 45c. The ot::er type -23- CTR CQNTRRCT9 005,349 11225090 CTR ~"~~~ 0~~~~~~~~

of ccnce: sate was obtainec by i: :p::.ge:: ent of :res : s: :oke on a: oriiice and was adr-:inistered to the mice as instant smoke con_en. sate wit n seconds after generating the smoke. Both types c: condensate were manufactured from the smoke of 1R1 Kentucin- reference cigarettes, both were ad.^•iinistered to the test animals at the rate of 160 mg of nonacuecus condensate per mouse per vree'rk and bot:: ccntained approximately equal quantities of water per non- aqt:eo-,:s condensate. After two years of ski n treatment, 85. 556 of the ani- mals treated with precondensed smoke condensate exhibited grossly visib:e p:?iilo::as, as opposed to only 17.8 tc 30.2°', of the rr_:ce in the grc,:Ds having rec.eived instant sr:eke cor.densate. No skin le- sions •.:o-L observec after the sa:::e length of ti--;e in control broups of nace not treated with smoke condensate. The wea'r, biolcg:c ef:ects of instant smoke cor._er.sate ,:•ere .._. ..._rcased b,. • si::zuataneo•_s aa4:r_inistra:ion to the an--.a:s .._ citn•_r the ve: icle o: prec_r.censed s:r.c:te condensate, or oc lancli-.. tatin= der:::al absorp:icz of lipopnilic substances. : here a:_ o was a: d:ffe-ence in the rate of papii:cr=.ca . appeara: ce beturoen t:e t,.v; ,tyoes o.° s:-:cke condensate, the -r;_:._er.se;. t.:?- r.aviog a..._._ ra?:d acticr. .aer. instan. co-^-=== -a--• Ins:a: __= o_` :v .-____ remiss'_.n of grossly ..=:5:_ .. _--.c: - _ _c-..-- ir. ... .ce...re..c.:_ witn ei:^er t•:pe of s:..:.._ co :- den sa.e, bLt .,;ere ...__.....ore frec_=•.- in an?...a1s .eceiv: .,^:ore co:.r.e::-te. =ne s<irn of a'-: ar.i:na:_•_•_rviv:^g t:t:r two years c: t_eat. ec: wa : sr:o<e condensate c: cor.tre: substances (ace:cne, lauoL•:.; v:as studied : tsSological:y, and so was the skin frc:r. : nan~ r:^ice :-: had died or had beer. sscrifi=ed ear'-'er. T: e o7se-•.at:c-.= thus rr.ade included in vasive squa::-ous cell carcinomas, some Yrein- vasivr carcinomas, papillomas, dermal or epider:ul ulcers, hyper- keratosis, hyperplasia, dermal infla::•unation and mast cell accu.^..•:- lation. Very frequently, several and sometimes even all of these pheno:: ena occurred simultaneousl-i. Carcinomas and/or papillomas were found in o1.'.'7o _24. CTR CONTRRCTS 005350 11225091 r CTR N~~ 0~:~~~~~

l a /1 of the an:rnals with r.o nregressed, grossly visiole papillo^:as, a nd in 33. 3i'o of the mice with completely regressed lesions, with zo significant di.fierences between the tu•c types of sr: o:ce condensate. Among the animals without grossly visible skin lesions, or F•ith questionable lesio ns, there were 24. 5rJe instances of carcinomas and/or papillomas in the group of mice treated with precondensed smoke condensate, but only E. 4% in mice that had received instant smoke condensate. No carcinomas or papiliornas were seen by histopathology i.^n any of tha control ani:nals. Malignancies in the treated area of the skin o:- curred in'42.9Q1o o: mice after adrnir.istration eE preconden seZ smoke cz):;: :sate, b%it only in 3.9',a cf the a.^.i^:als treated instant s:no:;e condensate. At tie same time, 6Z. 8ro of all histo- pathologicall proven tumors were malignant in mice treated with the former type, but only 25. 155 i.^n those treated with the latter type of s:noke condensate. Systemic eL=e::s o: chronic :o>::city cat:se--~ two types o` smoke concensate were :imitec :o siigaiy lower boc.;• weights an_ somew:.at higher r: crtaa:r o: the animals in e:-. peri:nental than in the control groups, without distinctic n 5eti eer. the hvo types o: smoke condensate. There also a•as s~:ae ac::t_ tor-ic ef:ect c_° the p=econdansed s:r.o}:e cor.de=sate, increas:--._ :e: :oeraril• :}:c r:or.a'_: cc ,::e animals ... -..- earlier -ase- c` the e:coerir..en:. -he coLsi'erabi of iLE.3... Cver preccn GC.^.seZ stn cke tcndensate -_ b__e :ed tc be c_- .. _.- ferznces in :..- levels cc carc...oxen=_ (initiator) ccr.:aine--~ ..n t:ese two types of smoke con A_:n eate. _: e increased a:r.o::nts af :_:- cinoge.^.s in conventiona:ly preparec, precondensed smo::e con- ce nsate, or w: a:ever else ca::ses its higher t•umori2enici--~•, ar!- the result o: an artifact .:•hich is unrelated tc the p=ocess rette s:'••:,:,f:n g and, hence, bears no relationship to ena that occur in the lur.3s of t=e h::^:a n smoker. _25_ CTR CGNTRRCTS 005.3.51 11225092 C T R I'll N 0 422 3 77 l.7 ~.: C

a /1 Gen era'_ Design o-' Z'xDeri-=en: II Materials Applied to Mice ,:r: oup No. c_ Ai:ce II Preco3- densed L^.stant Identi- Smoke p + fication per Group Smoke Condq n- s ate Conde- "sate Lanolin Acetone : i ~ 200 160 rng . i G. 32 tnl ( 0. 32 : _ i ; , t i, _.._ .._ I 0. 32 I ~ r:;t=es__c n ..:antities ?er -..__se per wee::• ** Ca :ae ski.^n c- the sha•:ed backs. ~ : ec irem 1P._ P•.__ren:e ciga:ettes. i- aceton e, :a pro::.s:e a5sorp- ticc cf 1i-~och:1?_ ...ater__? b•. .he sk? . * _ _ : -ed to the anir:ia:s ir.uned:a:el;: ^rior to the a:'._^.:inist_atiorn of :r.s:ant s:rioke condensa:c e ve ::c1e of precor.der.sed s--o:ce con- densate. . Applied to the animals immediately atter the adrniaistration of instant smokc condensate. ti CTR C0NTE2RCT5 oos3s2 11225093 C ~°~' H ~~ 4~~~ 2 ..~ r~"~

a /h Table 1: DistribLtio- of \2?ce into S:x Grcups Nu.-nber of Mice in Body Weig-'-ts ofMice (Grams) Total Popu- latioa Csrcu=) A Group B I G roLp C Group D G roup E Group F G All Six roups I:IIL'Sez' 13- 13. 99 ~ 2 2 ~ 1G_ 1L, O9 I ~ 15-15.99 1 16-16.99) 12 I= 17-i7.9o - 3 18-18.991 136 ', ~ 16 i6 I 1.' 1~-19.99~ 214 45 i 25 25 2: I 2-0 ?C'-2V.99~ 267 - ~' 57 I 32 32 ~ 32 I 2:. 21-21.991 1c2 3- ; 3-; i 34 -9 ~ 1 , I L9 I I . • 22 '2.991 I1c 1- ~ 1- I .:- I I I ~ ~ 23-25.99I 39 ~ ~ 7 ~ / ! I d - I i i : 3 2-:-24.991 12 1Z 2 --25. 991 I 1 I 1 =cta::::. 1C14 ZG: ~ 2uC ~ ZCc I i 1 9. 86 g c. c i lc. 9.' ~ 1 9. 97 ,c 9- I19. 5 ~ -4.90 -_ ~ CTR caNTRRCTS 005.3~3 11225094 CTR HN 04223F9

Table II? Expos_re of 32 Mice, Fioused i:. Four Cages, to Lstant Smoke Co=densate* Sr:oking SeqLence: Mouse Numbers (Cage Numbers i n Parentheses) Puf? ** Nu_:, be r Smoking Machine A Sr-.:o king Machine B First Set of 12 Cigarettes Second Set of 12 Cigarettest First Set of 12 Cigarettes Second Set of 12 Cigarettest 2 1 (1) - (1) 3 (1) 4 (1) 3 C 10 (2) 11 (2) 12 (2) l (3) 1S (3) 19 (3) 20 (3) (' ) 2 6 (4) 2 (-i 2 3 (4) O 2~ (4) 26 (4) 2 -. (Y; 26 (4) 7 ~ 17(3) 18 (3) 19 (3/ 20 (3) 6 9 (2) 10 (2) 1- (=) 12 (2) ~ ~ 1 (1) 2 (1) 3 (1) 4 (1) ~ G (1) (1/ 6 (:) 3 1= (~-) 11 (2) 15 (2, 16 (2) 't-1 (3) 22 (3) 23 f?i 24 (3) 5 29 (4) 3~ (4) 3: (4. 32 (4) ~ 29 (") 3 0 (4) 3 32 (=) 21 (3) ( 22 (3) 23 (3) 24 (3) 13 (:) 14 (2) 16 (2) 5 (1) 6 (:) 7 (1) 6 (:) la:•olving 6 X 12, or a total of 96 cigarettes, and lasting 2 X -~, or a tota: of 18 minutes (60 seconds for each Fuf.`). Of 12 si :zultaneously smoked cigarettes. t Smoked with a constant delay of exactly 30 seconds after the first set. t AU first puffs are rejected. This puff was started aSter all nine puffs, shown on the lines above, had been co:r.?leted. CTR CaHTRRCTS 005354 11225095 C~°R HN 0~~380

.iab:e I`. :Iurber of Animals with Cross'.y Detectable Skin ?.esions at Ti:ae of Terrnination of Exoeri:::ent I Numbers of Aniznalst I Gro: p 'Desig- nat:on I tit' i t.'I E stab- 1:shed PaPil- e. lo :zas W ith Re- ~* gressed Papil-, loma s With Que stio.^.- ableC Papil_ lomas# Wit'- R ** gressed Question- ableC Papil- lon:as, Witho::: Visible Lesions and Alive at :er- r:linatior. I ffect:v e Nsrnbers of Arii- ~ r.:a_ s' (i) (ii) (iii) - I (iv) _ I (v) i (vi) (' i) A 7. (S=. =°io) I n 1! 0 C 0 D 8 (1 i. 8r~) E 9 (1 "• 1 r 13 (3:.2c5) 15 C 0 2 6 6 4 (3) 2 (1; 0 0 0 I 0 0 2 (11 0 3 (.j 4 (0j . (o? 1 a '2= I 115 30 T At termination of experirnent; independentiy of whether or no: the ani- r:als were al:ve at tern.^.ination, ur-ess stated otherwise. ~?.egardless of «':et:.er or not t: e lesio ns had regressec (par::a'.:: _- corn plete:y). 5 Figures in parentheses i ndicate the va'.c-s .., percent of t::e e?iec:::e r.•.:.-nber. == Co=,plete regression. • Figures in column (iii) are izcl%ided ir. t:.ose of coleu r. (ii). - I .ncludi ng lesions which had never reached the req;:ire=.en`_ ..` arn es:::blished papilloma; figures not included in those of col,.:_.: _: igu=es in pare n:heses refer to survivcrs only. S (vii) = (ii) = (iv) (figures in parentheses) + (v) (figures in paren t~:eses) CTR caHTRRCTS 0Q5~5~; 11225096 CTR IIIIN 0402-238-1.

Ih /1 Ta^:e V Occurrence of Multiple Papillorr:as Tota1 Animals No. of No. of Survivore* with Existi^g G=ouo No, of ~ Wi.t: * Survivors Papillo-nas Desig- Ani_•nals Papillo- Sh'ith Re , ~ *r , ' Total No, of Lesions oer _*.:o:se a•io^ mas which gressec § i Papl-*t Died before Papillornas I No' 1 2 3 or r: cre lorr.as i Terrainatioa (-) ! I (ii) ! (iii) (iv) (v) (vi) (vii) (f:iii A Hi 34 6 I 31 21 9 1 D 2 I 2 1 5 5 C _ ~ 9 I c i 3 i G C : ; Q _ I I 13 I ' I 3 ~ ' 6 e. I q I G I * A: te.: ina:.,.: c: experiment. ~ Indepencen ::v c: u-:e:Zer or no: t:e a n'_raals were alive at te::r.:--.a- t:_ , a..; ._ca: -:ss of •N::et-'-.er o: nu: t::= lesions had or regressed paoi :Drr,as at :.r.-e of = Corr.:'_e:e :eoression. ... (v':) (v) CTR CCNTRRCTS 005356 11225097 C"T R H N 0 42~'~BE 2--

.Ta'~le V? Res'a:s oi His:opathologic Stl:dies in Gro_p A No. of Animalst Where Skin ; Histopathological Observatio=s Histology Was Studied and ~ T_ Where Gross Visual Ex- Ca Hy- In- vlast * arninatioa Had Established I ** and/ ETl- ~ Hype r- per- flam- CeL' I Nor- ' Ca Pap 4 ' kera- ~' C OL e r r I or Icer pla- rna - ! mal Ac . P RP Q RQ N3. i Pap tosis sia tion~ cum.l I I m 0 z a 0 F 8(7) ~ 7(4) ~ 4(4) 1(1) 1(1) 1(0) 2(2) 1(1) 10(8) 4(2) 1(1) 1(0) 41(31) 9(6) 3(3) 0 2(2) 3(2) 4(4) 9(8) 1 + + ( + I ~ + T ~ + !1 '{ ( T il I I ? 1 24 3 1 1 2 + + + + I + T + ~ + . T ~ T . T + T + + + + + 29 1 1 0 35 3 2 1 2 4 1 1 5 18 4 0 1 5 39 9 3 ~ 9 41 9 3 1 9 41 9 3 1 9 :A.:so .a•:t:: '-e : an;io:: a T + T '~~ro (vr.e+ Io::,sar:c: :a r: I b=c:arco: :a 41 9 3 1 9 CTR CaNTRRCTS 0053 J?` 11225098 CTR IIN 042383

Legends to Tab'-e V_ fNumbers in parent::eses refer to animals s::r~:ving at termization of experir^ent. * P = existing papilloma; RP = regressed pa:)illorna; Q= questionable papilloma; RQ = regressed questionable paoillo:na, NL = no visible lesions Der.=a1 or epiderr: al; squamous cell carcino:na, unless stated otherwise. 7 Derr:al or eoiderrr.al. 0 De-:--a1. s Derma: an'_ eo:derr.:al. -`:u.m:ers c: ani::zals (dea_ or alive at te:•r :,a:ion) svit::c•:: grossl.• visiblc papillomas (establis: ed), but histc'_o,-:cally proven carcinoma (including ques- t?en ab:e carcino::za) or papilloma (see sun: c: t:.e tl:ree lines directly abcve). ti,:rr.bcr of aC:.^..als sc:cied histologically oc•hich were n ct i.nclud ec i=. tae effective number s, i. e. wl-ici: 'r.a --' died without established papilicr.:a before ter: :i:.a- tion o: experiment: dif:erence between the f•igures a::c with _oa_entheses ir. the same col::rr= a:~c•:e. CTR CCNTRRCTS 005358 11225099 CTR HN 0412~`~8_41

/ k Ta=-: `.? R esalts of :istopatholog:: S:udies .n G rou? B r ? : No, of Anir.ialst Where Skir. Histology «'as St•, died a :d Where Gross Visual E:x- * arni^ation Had Es:ablished I P i RP I 0 0 : Eiistooat: ological Obserratio: s 1 r li yper- ke- a- tosis + Hy--' lz:. aer-I f_a:: - pla- ~ rnay sia l t:or. 1.ias: Cell Ac- C• curn. I Norf, =al" Ot-cr i i1:-;4 0 C 7 0 t i ~ 1 1 12 i 3 1 I 23 2: ~ 'r.ait: ' - -~- --:~ c2 I .r' CTR cONTRACTSzj 005359 11225100 CTR HN 0423SE5

Legends to Table VII t^iumbers in parentheses refer to anirr.als surviving at ter- :ni :ation of experiment. ~ P= existing papiLoma; RP = regressed papilloma; Q = questionable papilloma; RO = regressed questionable oapilloma; NL = no visible lesions. *fi Derma] or eoidermal; squamous cell carcino-^-•ia, urless stated otherwise. `. Derrnal or eoidermal. C Dermal. C Derrnal and eaidermal. :~~-:.ber of an:rnals (dead cr alive at term.i.nat:cn) wit^c%:- gro;sly visible papillornas (established), but ..:` histologically proven carcinoma (inclua:.zS ques- tionable carcinoma) or papillo:na (see su.:m o` t.e three lines directly above). :bzr oo ar.: :~als st•'!diec histologicall}- w: ic: v:ere -c: included in the efiectivc numbers, i. e. aicd without established papillorna before te::r.:na- .-_^ of exoeriment: dif`ere-^.ce between -°i¢:::=s ..'_: c_: and v:it1 oa:ent.eses in the saTE cc_.:-_ above. -, AL se•:en sn'rnals which died be:ore ter:nLl'r.ad 1--_-- icep: under the experir: e nta'_ conditic-s fo: at :__:. 90 weeks. -tr ... CTR CCh{TRACTS Qa~360 11225101 W ~I R HI , .r.' II' fr i~.' ~s^ V'

Ta~le V.ZI Res;:lts of I:istopathologic St,:cies in Group C No. of Animalst Where Skin i Histopa:hological Observations t d Hi l di d W S s o as e an ogy tu Where Gross Visual Ex- # ~ I Ca H~•per- i-iv- ' In- i Mast ' a:aination Had Estabiished *~ and/ UL pe:- flar.. , No r4 CeP RP ? Q Q Q NL ` PiiIJiJi Ca I Pap or Pa cer S ~ kera- tosis pla- - sia ma- tion2) ` Ac- 1„ ~a _ cum ! C:he- . I p . i ~1) i I ( + I i - - + ; - ~Wi:: iib-:: I sa. co^:a I I 1 ~ 1) i T T y ~(0) I ; I T ' + T _.(3) I + i ; _ + _ T 1 / v `O ) I I ~ I I I + I ~ ' + T ~ , . f + I ~ ~;3y I I- f f I ~ J s ` ar.. _ i*Es j ( .~ 1 C ~ ( ~ I ~ I I I • f ~ I , C : c~ 0 ~ 0 0 4 ~ , 1C 2c 49 1 34 ~ 54 j ~ ~ 1 1 0 0 C 1 L o 0 7 ~ CTR CCNTRRCTS 005361. 11225102 • CT R MN 042387

i . .k ...ege.._: to Table VLL: f\ur.-bers in oarent:^.eses refer to a^.irazls surviviag at ter- minatior. of exnerir.:ent. * P= existing paoillorna; RP = regressed papillorna; Q= questionable paoilloma; RQ = regressed ques- tionable papillorna; NL = no visible lesions. *~ De=r: al or epidermal, squa.mous cell carcinoma, ur.less stated otherwise. S Der:: al or epide-ma:. ~ De ^mai. ~ Derrr-al and epidermal. -::ur.:bers c: ar.ir.:a'__ (dead or alive at terr: ;aatio. ) wit=c;:: gross?y N•isibie pay:llomas (established), but a•ith histc- logicall.: p:c~en carci nos: a(includir.¢ cuestioaacle car- c'-a~:za) or parao.:.a (see s::r : of the t:r ee i:nes direc-_*; above). of studied h° stologicaLY which were not :: - ir. tr.e e:°ect:%•e n•.:--nbers, i. e. ha(i died cs: establis :ed papiLoma be:ore ter:z:inatio n of experiment: _ifference bet-::eea the figures w?tho.:: and with parect::eses ... the sa~_ c:)h:zr^ abo:•e. be-.... all i.:1 seven ........als, included r. this Table, .•..:c : Zie;, :errrd nat:on, hs: bee- ke?: under expe:::aer.tai coaci:ic-.s for at least 90 weeks. skin ::or: :i~e a=::r.a1s sacrificed a: ter-:ir.zaoc a-== ..:t suitable for histology. B cT[~ caHT~2ACTS ao5~61 11225103 CTR 1 l 1 1 1 07 d6... ..w L.a 8

Table D: Results o` 1-:istopathologic Studies in Group D No. of Animalst Where Skin i Histopatho'-ogical Observatio-a Histolog Was Studied an d y . Where Gross Visual Ex- I Ca Hy- In- Mast I * a_-nination Had Establis?:ed ** and/ t~l- H e!- yp per- flam- Cell - Nor Ca Pap S kera- OtF.er / ~ or cer Pla- Ma^ Ac. P / RP p R(~ ~L Pap tOSiS I sia tion" cur.- ~ 1~1) ~ Ii 1 + + + I . ~ + + i ~ ~) 2 3) T + I ~ ( ~ l2' + + / + ~ + T ~ I 1 p ) I I I t I 1 :G) I T + i + T T - T - I 1(li ~i I t + + ~ , j 1(C~ ~ < ' _d)(p) ~ ~ + < } i ~ l ( ' 2 (2) ~~ I+ + ( + ~ T i c) (3) + ; + ~ . C-2 (_-.o..E~ . I I I~ I I ' .'i7lt:: ..... ~. T I ~ ~ I .0) T . i . I (,)I ) 3 I c I 6 ' 1 5 ~ o ' ~y (1) i . j C I '_ 2 1 ? 2 i :. . ~ I C + 0 ~ 1 : ~ 0 1 I 1 : ~: C ~ h5361 ~I ' / 4 5 3 35 1 15 ! 45 I 6 -- ~ I l I , 6 ' I - --- o 0 9 I CTR CCHTRRCTS1 005363 11225104 C TR NN 042389

/1 T Legends to Table LX NLr: oers in parentheses refer to aaimals surviving a: ter- r: :aation of experiment. * P= existing papilloma; RP = regressed papilloma; Q= a_estionable papillot:•ia; RQ = regressed question- able papilloma; NL = no visib'-e lesions. Dermal or epidermal, squamous cell carcinoma, unless stated otherwisc. I Dermal or epiderrnal. ~ De:-=-a'_. ~ Dermal an: eoiderznal. `+ r be s of animals (dead or ali e at teraiinat:on) witao::: %:sib'_e papillc:-as (es:.ab'.is::ed), bot with aist~- lcgicai:}• prove^ carcir.oraa (ir.ciuding c,-estionable carci- .......__) or Yarillorr.a (see s_:rn o: the three 1?nes direc:l;: ab_vel. of an.rnals studied Iastc_:~ica:lp w:ich were _ot _.. .---= -~ :? efiective n,,:.rr.bers, .. e. .~ :. .. ha;: died .ri:.- eu: es:ablis'r.ed papilloma before ter:ninatio n of experiment: c`.f?eren ce between the figLres w•:tho•_: and with parent::eses t:-.e :a.. - cclu:r~ above. a ) Or•.= ar:i.:.a'- papilloaia died a:c-r ?? µ•eeks c_' treat--e,:. c; One ar,i:^-.a: u•::h regressec pap'.acr..a c-ed after 96 weeks c: trea::::e.... c) :?ll ani::•ials, .n cluded i.: t:.;s :ab:e, that died befcre terr.:i:.a- tion a:thout established papillo:na and exhibited no carci:.=-a or paoilloma on histology, had been under treatrnent for at least 90 weeks. d) One mouse died atter 24 weeks, the other after 28 weeks of treat:nent. CTR CCHTRRCTS 005,3641 11225105 . C l' R • •  • 0423 _/' 0

Tab__ X Resti::ts o: 1?:stopathologic Studies in Gro::p E No. c-' Anirnalst Whe re Skin Histopathological Observatio=s Histology «'as S W: ere Gross V arr.ination Had tudie isua Estc,..b d a.^.d l Ex- * lished * Ca and/ l- Hy pe r- H pe y- r- In- ila:n- Mast CeL' eor ' a ~ pap s k era- A ~ 1 Ot^e_ i p p Q I R Q ` 1:i. ~ or ~ Pap cer to sis si a ma~- tio^ c-C cu : ~ p I i 1 (1) I .~ T I + i ~(y) + T 1 + + ~ 1 l') . i 1a) 1 (0) 1 ~ + ~ I I (,j I 1 i L I ~ ! (1) la (.j . I . y ~ ~ + I + T ~ + ! . I . _a_ _s_. j-ce1'. Ca ~a1 (1 j ! ! ~ r 1 + T ~ + i- I O::e (or.e j ! I + ! I ~ i i I ( + al>~ -- ~' _os>r:... + ! 1 11 !T i .- I : ! C 1 1 ' 3 I ' ( 1 ! 0 0 1 ~ 2 2 ~ 2 ~ 0 (3o) I 1 1 1 2 ; 3 3 1 4 ~ 3 19 !1~ 0 CTR CONTRRCTS 005365 11225106 CTR M~~ 0~~:~",.~~ ~

~.ege=ds to Table X T Numcers in pareat-hes-s refer to ani::zals surviving at te:- r.:ination of expe riment. * P= existing papillor.ma; RP = regressed papillo:za; Q= questionable papi"oma; RQ = regressed questionable papilloma, NL = no v:sible lesions. ** Dermal or epider:;zal, scua:nous cell carcinon:a, unless stated otherwise. ~ Deri:zal or epiderznal. ~ Der.: al. (9 Dermal and eoider:nal. • Numbers of a:.a:als ('_eac c r alive at terrninat:o: - vcithocc grosslj •:isib:e papE::. as (establa~ed;, b,-: with logically proven carcinoTa (includi=g ques:ionable carci. norna) cr papillc:::a (ste sLr.: of the three lines di:cctl;' above ). Ny.-nber of an _F^als s::Zied histologically %%'::ca were net ...- cluded in t_he e:_'e:::':e nu:: bers, i.e. which had diec established : apillor:a befcre termination of exoerir::ecL: dif:erence between :..e :ic_res without and wit: para ::_s=; iL L:e s2.^.:E a All an'-:-als, ....__c__ _.. :.i?e _abie, :hat died bef:re :cr=?za- tion v.,it: c::: establis :ed _ arilioma, had been ~.ynder treac-:.e_: for at least 9C 'x'cc:;s. T r ~..rr. v~r CTR CQh(TRRCT6 0053366 `' 11225107 C~a~' N~`"°~ 0'~~ °~~ ~'

=ablc }L r~,es::lts cf 1=istopa•`.clogic S::=ies in Group F No. o°- A nimalst Where Skin (~ Eistopathological Observatio::s Histolo Wae Studicd and gy ~ - Where Gross ti'isual Ex- + ~ ( Ca r- l:ype Hy- . In- tilast f amination Had Established I~ ~•* Ca _~ao and/ - ~1-T ke ra - per- l flam- Cell :7or- + r' Ot::e: 1" RQ! NL P ~ RP C~,e i I or Pap ce: i tosa p a- sia ma tion~ Ac- __ cLIM. ( r.ia i i I I i 11(0) i I 1( f ~ i I I 0) ! I T ~ 1 T I T I ( \ ~ ' I1\1/ I I I I { ~ I ~ - ~ T ~ T T T I T 1 ~ T I 3(3) (0), I / T I 3(3) i 1 I - T - i I a)~ J)' i ~ _ i o~(3) ~ 8`''8 j I i I - I I ( 01 I - C j 2 I 2 .. 1 4 j i C ,_; c( )I I. I 2 2 : I 5 6 5 ~ ( Cl 1~, C 0 0 - 1 1 1 1 j C ~O a )I I 1 I 1 1 ` 2 6 6 _ 0 I 3_ (Z 4) 1 1 - 2 8; 23 31 23 23 1 ~ I 2 I 2 3 I i ;'I 1 1 8 i I I i I CTR Cah{TRRCTS 40536%' 11225108 i r" CTR MN 042393

Le;•=: ds :o Tab1e :-_ tNumbers in parentheses refer to anir:a'_s survi.•ing at te- m.inatio:: of experisnent. * P= existing papiLo:na; RP = regressed papilloma; C~ = questionable papillor: a; RQ = regressed caesti::.- able papilloma; NL = no visible lesions. Derraal or epidermal; seuamous ceL carcinorr.a, unless sta:ed otherwise. S :)er-:=_1 c: en_der:^el. ~ Dcr..._.. ~ D=-....a_ a::c eo der-:a1. .j ::•_ rs of a-i--=.s (dead or a_iv e at ter-:i.natica; u:::;,•_: gressi.: v=sible panillo-•ras (estaais::e_), but vr_t^ :.._-_ =ove^ carc?no: :a (i:::lsdi=g cues:io::alolc :a:-c- , nor:a) or papil:o:•:a (sec s•,::- c: the t:.ree 1ir-es di:e.t-2;: above). a=:-..__.s st-.idiec :_-•ologica!': no: au.:.bers, i. e. which l.a'_ d:ed .::_. o::: e<_:z'D-:isied caoi:io-a before terr..i-a^or. o: exoe=i-=.e-.:: _`iz re> n__ o=_-_.__._. :. - :..- --..... ab_-t. ~) _._• a-:irn _-., i^.._,.=ec -.n th:c _a:-_. t::a. _?ec bcfc:_ ._....--._ estab:isne•_ papiLlor.:a, : ac 'cccrr ur.dcr trcc:..._r._ ic= a: least : n.•eeks. CTR CCHTRRCT5 005368 11225109 CTR HN 04~~~~~

a /I ':able XI: Results of l:istopataologi:. S =-dies in i;ntweated Control Mice' No. of Animals Wbere Skin Histology Was Studied and Where Gross Visual Exa--ni- nation Had Shown 10. Lesions Totz_' -Nos. Si. Ca and/ or PaD 0 .E-iistopathological Obser.ztions Hype r y- - Mast - per- 1-T flam. Cell kera- er pla - ma P.c- tosis sia tiona ~ cur-.:. C' T t c + + i T Othe- s'raved until after sacri..ce. ` Je-Ta'_ ~- epide:: ial. = Je-^ a1. C and epider: a:. CTR CCNTRRCTS 005:969 11225110 C" T R M N 0 42°t 9~~

a Tab:e ~^ / 1 Nur.:be: s o: A nimals with Papillomas and/or Carcinomas of the Skin by A.1: :vfeans of Observation * No. of Ani--naist I Total No. No. of t without Grossly i of Animals I Croup P-n izr.._1s witb Grossly ~isi~le Pa: s Visible E stab- lished Paps$ wit:. 1-Iistolog't- cally Proven Carcinorr:a cr Papillo:na ~ wit: Ca I Effective ar.d!or No. of F Pao by All :.r.i:::als' ~_2-."/s c_ _ OSservat'•o i j (i) (iij (iii) (i , ' (••) ! A i i 5 l0 c: I e 0 I 0 12- c c e „-:~ 1 No. of Animals Studied ol- ly His- ogy and \ot In- cl::cec ir. the Effec; tive No. ' i Nc. cf An:-::als ac-it : Ca Ad;ustec: anc, or Ef:e-ti•:e ' Pac in ~~ No. " of A•?- 1L•S:=C: E:fec ve (ti-1 ~ ( .' , i 1 7 ; 131 12= s ! Z.. _ -; 3 _ res from co:u-r.-. I':. Jesc or ali•:e at terr•ina:ion. _.=•6-essed or existi;.g. - :..-icluding ar..:::als wit: q-.;estionable (vis:ble) :api'?omas (Q), v,-it: recresse'_ c,uestionable papilLo-=as (RQ) and wiL`:oLt v:sible sl:?n les:ons Tables VI to XI (second last line). ~ (iv) = (ii) + (iii). § From col_rnn (vii) of Tal:le IV. 5 F rom Tables VI to XI, s..za of the three figures on last line. G (•-ii) _ (v) + (v-)• e (viii) _ (iv) (1OO)/(vil). cTC2 CoNTRacTS 0o53%'a 11225111 C T R M N 0 4 12", 3 9- G-

i a51e >u'•: -Nuzr.be` c-' A+irnals •x:t ; Sk?r. Car cinor nas and Malignancy of Lesions ~ - ~~ No. of !~i:'ials with H:stopat+hological'_y Proven Skin Carcinon:a ir. Eac: Group per \o. o: ;{ Mice Studied Histologicallyt Group ; Observatio:.s on Cross Visual Inspection De i ; Tota_ No. of hace .,-;:. s g- natio Carcirso: :a n . azillc: 2 uest:o-- F:egresse I able ?apiLz':::a 4 'I 9aDi-,lO.^..2 Regressed G*,:estion- able ~ PaDillOr na oo Lesions I Per To~I No. of tifice Sb.:: iez by ril6izlOsy PexcTo;.;:'. of `:icef v::. roveL Ca and/or Pa= (i) A 2 i,14 1 (57. 2) i 3i° (33.3) ~ 1i3 (:3) I G/1 i 2,'4 E j i - Gi13- I C/13_ i C/1.' L - - (== ' /y= E - 0%'_ : /43 (?.C) ~1%- (:3) F 1%: Ci' 0/32 ( 1~-' (1.9 .%- i:- L. 2L C`- ' 2 ;'5 51Z1 (?3.6) ; 1/- (3:) ~ 0, _1 f I Z.'3-"' (C. 3) 1 " ~31.50 -.: .. . ' t ?erc:-: i-. oare::::.---ses. 'h ?\o- regressed at tS.:e of de=_th or sacri-`ice. Co:r.pletely re;ressed. ;: rom Tables VI t::rough )Q, sums of f:E::res in last seve n lines of third co'_ur.•.a under : eading of "Histopathological Observations." CTR caNTRaCTS 0053~' 11225112 CTR C~IN ~~~~397

/ C Table };'.' Gross a•__opsp F-:-._ings at Ter::~aat'-== of Experirnezt ( Autoo sy Finding s (No. of Anir-:a:: ) Total otal No. VoN o? i I ~ r G-oup o. Ar:imal. ~..als • Spleen Liver I"iidneys Ovaries L~te~ss ` De'sio- o n V: :ich %c ithout M I ~ ~ ~ -ycro- i ~ ~ na:ion i A. top...; r bnor::zal ° ~ ~ _ : C2~SST ~ i I «'as Done~ :-top sy x*t ~ ~ o 0 ~ c I = indigs ^. m o I v i C m v = _ : (i) ! (iil I (iii) (i~j (v)'(:-i) (vi~~~(:i:i)i (~ (a) I (Xi) (~i) - ----~- ; _ o ~ - - i ~ - j ~ i 1 ~ - - ~ 12= fl ~ (40 .0} 16! 3 2 1 _ '. ! f 2 2 ~ ' . i - I ~_ C I -- ('6. 7) I i - I I :'_ 3c (o~.-) ~; 32 (3C. c) I I 2 C i - i . j . 5 _~ !2 S: 2) ~571 :C: t e end of 104 veeis of treatr ent. isceot s:c:z lesior.s i: the t:eate~ area. T Percent of total numbers of -:ice autopsied ir. parentheses. a) One adrenal t•, mor. b) One tu::•ior beneath skin of treated area. c) Lung congestion. cl One tumor at back of neck. e) One enlarged bladder. ?) Number o_° miscellaneous tv:aors. CTR Ca~4TF~RCTS OQ53 ~ 2 11225113 ~''~~~ C`~R ~~N +~4~''~~

'1'alrb• :: VI Distrihution of '1'unlors 1•'uwrld ut. Alltup.y Arc(rnling to '1•ype of Tunior, Site and Group of Mice ------__ - - -- ---- _ __.-- - ----------- --- * Hlstopathulopic 1•'indinl :; (Nos. of Alllrt)als, with Group Designation in Parentheses .Anatorn- h1.(li};n:ud 1'luuurs Nonmalignant Tumors Other l,esi(Ins ical Site or Group Dc eigna- Squa- 1_y111- T(rtal G ranu - Total N(i . mous 1(eliculunl No. of of Non- tion Cell Carci- noma Adenocarci- noma C:r•lI S:r rcoma I,Irrr- r~n~a Filrru_ 9a 1'l'(l1I lil hlali} - nanl lutrruls I lcrnan I;iorna Adenoma losn Cell Turn o r malig- nant llrmora Inilanl_ tnation t;y:;t Spleen I(lZ) I(C) l. 1(A) 1 ~ l.ive r 2 ( il, 1)1 ) L 2(I1, F. ) Kidneys 1(C) Z(A, I)) 1(ll) q Adrenals 1(IS) 1- Ovaries 1((;) t 1 1(C) 1(l.i) 7. I(It) Uterus I (A) 1 1(F) 1 lntustines 1*~~ 3(t:, 1). ~(1) Lung Subcu ta- ~ 4(A,'-B,I, F) 11'(>;) 1 neous .f(1~t:1JJ i --- Group A 1V 2 j t ~ Group li l 3 t 5 1 1 C) Group C 1 t 1 I ~ Group D 1 ~. T 1 1 1 or Group E 10 + 1** 2 1 7D Group F 1 I 1 1 ~ Group 0* 1 1 Total No. por Turnor Ln 1 & I (1 t i 1 1 5 * Group 0 Is group of unlrcltr•.l. Im:rhavcrl crrn(rol tni('c. 0 t Animal also had an invasiv(• srlu:llnrrtiv r-.•I1 r•;trcirnvna in ll,• trcatr•d aru:t rlf the :rkin. $ Animal also 1ta(1 a hislrrl-Ki(:lllv Irrrrvon 1ral>illonla in lhr trcnlrrl .Ir(•a ol thc .9kin. 0 uli/;llt inflatn- ~~ *k Anitnal wlth a 1•rnnnly vinild.. '1.rlrill~rnra Ili.ll Irn.l rr•/;rr'.. .(•rl, .rn.1 I~.r.l nr• n..•r~• 11i.tn rrlilrl Irylrerpl:tsla, ~l ~ mat.ion an(1 tuast cell ar (rnnulaliun <ru hi:+lul(ll!y. ~ cJ Aninlll (lied prior tu tr•rru n. li.rr~. 7 C~Sf, t N 3_t-

MORTALITY OF MICE IN GROUPS A, B AND D ;0 2C 3C 40 50 60 70 80 90 100 r:,,V BEF o4~' :,==K s : ig•.: re 1 Grc~; .. ZOC l..:ce rece:viag 160 :::g precondecsed ccadensate in acetoae per week. Group B 200 hlice receiving 0.32 ml 5°'0 1anolin in acetcze per week. Group D 112 Mice receivir.g 160 mg instant smoke conde:.- sate per week. CTR CONTf~RCTS QQ5~%'~ 11225115 C TR MN 4~.42 4,00

MORTALITY OF MICE IN GROUPS C, E AND F 100 ~ ~ 0 ? e0~- , 1"%_GR0UP C i^- I 6_ 7: 8C 9^ o~ iG NUr.i__-. ;,- , __r.5 Figu'e Z C:rc•,:a C _._ :.1i_e rece: ir._ C.:'_ acetone Yer •x•ee'r.. con- Grcup ZE 112 ce receiv:-Lg 16~ mg instant 6moke densate and C. 32 ml 5r/0 1anolia in acetone pe: week. Group F 112 l.iice receiving 160 mg icstant smoke con- densate and 0. 32 ml acetone per week. CTR CGNTRRCTS 4453%'5 11225116 w ~.- CTR NN 042-410-13 .

AVERAGE BODY WEIGHTS OF MICE GROUPS 8 AND C ti F- x w 3 r 0 GROU?S A, D, E AND F oIG- cc L ~ cc < < 10 20 3:) AC 5C 60 70 80 9J 100 I;: VJMBcc r= \'=c{c _ igur_ 3 Groups A D, E ar.c F Experir:enta'_ gro_ = of ZCO ....__ (Group A), or 112 mice (Groups D, E and F), receiving 160 rr:z smoke condensate per week (precondensed smoke condensate in Group A, instant smoke condensate in Groups D, E and F); an:- mals in Group E also received 0.32 ml 5% lanolin in acetone per week; those in Group F also received0.32 ml acetone per K'eek. Groups B and C Control groups of 200 mice re- ceivi.ag 0. 32 m1 5°,'0 lanolin in acetone per week (Group B), or 0. 32 ml acetone per week (Group C). s CTR CaHTRRCTSi 0053776 11225117 CTR HN 042-402

RATE OF PAPILLO•41A AP°EARG,NCE i i ~ 99.9 w J C 1 B.. 9C :_- i _~ ?C 40 50 6. ~ Figure 4 Group A 200 Mice receiving 16C mg precondensed smoke condensate in acetone per week. Group D 112 Mice receiving 160 mg instant smoke conden- sate per week. Group F 112 Mice receiving 160 mg instant smoke conde: - sate and 0. 32 ml of acetone per week. cTR coKTRAcTS 11225118 CTI ! HN 0424,.,.R 3