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al \ ~p ASSOCIATES~ICAL 5221 RIVER ROAD, BETHESOA, MARYIAND 20816 ~,~hi__p, CTR COHTRRCTS 029167 11248917 E:XFi1BIT NO.-L S.N 0 CTR ~~~ ~RES

FINAL REPORT THE COGNCIL c'OR TOBACCO RESEARCH-U.S.A., :YC. CONTRaC';: C':R-0030 "SKORE I:1HALaTIOY STODIES IY !SICE" !SICRCBIOLOGICAL aSSOCIaTES 5221 RIVER ROAD 3LTItES0J1, MARYLAND 20816 PROJECT D[RECTORS: DR. CaROL J. HENRY OR. RICHARD E. t00RI FEBROARY 28, 1984 CTF2 CONTRACTS 029169 11248919 CTR HN 04-3956

7tnal Rapor- Contract: CTR-0030 ADDITICNAL CCYTQI3CTOQS TO ':!?SS^o ?ROJSCTS FROM SlICR0820LCCICAL aSSOC:a:'SS :VCLCDS :''iS eCLLCWI:7G: ti s. M. D e n i s e Av e r y Dr. 3harat 3hooshan Dr. Leonard 8illup. Dr. Rodger 0. Curren Mr. David R. Danaie Or. Marshall Dinovitz Ms. Sue Goanell Dr. William C. Hall Dr. [in-Rai Hwang Dr. Ren iC. Kanaqalinqam Mr. Rob Ryanko Mr. aqustin Lopez Dr. Ronald A. Lubet Ms. Cindy E. McKinney Mr. H. Doyle !!ull inax Mr. Raymond Hims Mr. Thomas Rude Dr. Carrie Whi"-t:e Special t4anks to Ms. Janet C. Stinnett and Mrs. ?atricia' HarSin Eor taei: help in completinq this report. CTR caNTRacTS 029170 11248920 CTR HN 04,39. E-55 7

linal Rapor: contsac:: C:'2-0030 TABLE OF CCNTEYTS A38RrVIaTIONS ............................ ...... 4ACE NO. i SCM!'.ARY ........................................... 1 I. I:1TR000CTION ..................................... 10 II. FACILITIES AND EQOIPMENT .......................... 12 A. Description of General Operations ............. 13 3. Description of Facilities ..................... 14 C. Description of Equip:aent ..................... 14 III. C:'R-100. (1-100) CO-CARCINOGENESIS AND CHRONIC INHALATION OF 2A1 CIGARETTE SMOKE ................. 42 A. Evaluation of SEi II Smoke Exposure Machine and Its Animal Containment Qnits .............. 43 8. Determination of Expected Mortality Rates With the SE:S II Smoke Exposure Machir.e ........ 44 C. Effects of Exposure to 2A1 Cigarette Smoke On MCa-Treated and Ontreated 9C3F1/Cum Hice.... 45, D. 3iological Activity of Chemical Carcinogen Induced Lung Cancers .... ....................... 52 IV. CTR-101a. CHRONIC EXPOSDRE OF SC3F1/CII,`1MICS TO 2R1 CIGARETTE SMOiCL ........................... L01 A. Introduction/Objectives ....................... 3. Data Collection/*ianage.aent .................... C. Daily Observations ............................ D. Cocumentation of Smoke Generation ............. E. Cocumentation of Inhalation of Smoke by 101 103 103 10S BC3F1/Cum Mice ............................... 1.06 F. Oocumentation of8ody Weights ................. 107 C. Survival ...................................... 108 H. Microscopic Evaluation and Analysis ........... 108 I. Effect of 2R1 Cigarette Smoke on Mice Pretreated with 3ap ........................... 114 CTR coh{Tf2acT5 029171 11248921 CTR HN 043915-6

*lnal ReQor: Con::aet: CTR-0030 TABLE 0F CONT=:ITS (con:ld) ?AGE `70. V. CTR-1013: EXPOSGRS OF 3C3F1/CC`i MIC3 TO 2Z1 and 3A1 CIGaRETTE S::ORE .................. 203 a. :ntroduction .................................. 203 3. Smoke Exposure Reqimen, '"oxiciny and e{lstopatholoqy ........................... 204 C. Dosiaecry .................................... 205 0. Shor:-Te:a Marke:s to Monitor tse Effects of Smoke ............................. 206 S. DisPOSition of AQiaall ........................ 207 ?. Summary of Results ............................ 207 VI. COROLL.IRY ST?DIES: ?HaR.KACOICINETICS 0F IvFiALE7 :!ATERIaLS .................................. 271 A. Vicotine, 3aP, and DTC ....................... 271 3. Catschol in Ciqarette Smoke ................... 272 C. Cateclol As a Chemical Aerosol ................ 273 0. TPA as a C::emical aerosol ..................... 276 VtI. REPERENCES ...................................... 291 VIII. ??9 LICaTIOVS ............... ...... 296 CTf2 CONTRRCTS 029172 11248922 C TR NN 04.%39..ffl=%_5q

CTR coHTRacTS 029173 11248923 CTR HN 043960

?inal Regort Con:rac:: C':R-0030 ?.BBR.'. VI.1TIONS ABBREVIATIOYS AAC aCN AH AHH A:JCv ASC AO 36 CCM, 36N 3 A P 3AP-7,3-DIOL C0Ft3 C.V. D2J,D2N DNA DP!i DTC, 14C-DTC Ha HB HE?A H0 HYP I? IT YIC 4-D1Q0 0DC ALVEOLaR ADEVOCaRCIVCNA aLVrOLaR COMPRESSING NODULES • ALVEOLaR HYPERPLASIA ARYL HYDROCaR90N HYDROXYLaSE ALVEOLAR :10N-COHPRESSI:IG YODULES ADENOSQUa.K00S CARCINOMA CyCLaSSIFIED .1DEJIOCARCINOMA C579L/6 C".M, C57BL/6N INBRED wOCSZ 9E;IZ0(A) PYRE;lE 7,8-DIHYDRO-7,8-DIFiYDROXY- BE:]ZO (A) ?YRENE CaRBOXYH_MOGLOB IJJ COEFFICIE:JT OF VARIATION DBa/2J AND DBA/2Y, INBRED MOOSe: DEOXYRI30NOCLEIC ACID DISI.4TEGRATIOyS PER MINUTE DOTRIACONTANE, CARBON-I4-DOTR:ACONTANE HElSACCLUTIaATING ANTIBODY HE.`SOGLOB IN HIGH EPFICIEHCY ?ARTICULATE AIR FIL:ERS HYDROXYCREA HYDROXYPROLINE I:lTRAPERITONEAL, INTRAPERI':0`7EaLLY IvTRATRACHEAL, I!ITRATRaC;{EALLY LITER LaBELLI:IG INDEX MICROBIOLOGICaL aSSCCIATES 3-METHYLCaOLAN'rHRE;IE ;iILG:GRA.K (10-s grams) METHYL METHANE SULFONATE !1ANOGRA.'t (10-9 grams) NICOTINE 4-NITR0Q0INOGI:lE-N-1-OXIDE ORNIT'{INE DECARBOXYLaSE i CTR coNTRACT5 029174 11248924 L.J' )) 1 l~/ 7 7 0413C.36 1

?Saal Repor: Cont:act: CTR-0030 138Rl.'VIATZCNS ABBREVIATIOYS (cont'd) ?A-`Ll PIGMENTED ALV:,'OLAR MACRO?HAGE ACCQHULATION PDC POORL'i DI?FERENTIATED CARCINOMA PEC PLAQOE FORMING CELL PSI POQNDS PER SQQARE INCH PRESSURE RCS RETICOLOM CE;LL SARCOMA RDS REPLICATIVE DNA SYNTHESIS SCC SQUAM00S CELL CARCINOMA SCE SISTER CHROMATID EXCHANGE SD STANDARD DEVIATION SE:! II3, C SMOKE EXPOSORE MACHINE SN SQQA.KOUS :SETAPLAS IA SN SQOA.lSOOS NEOPLASN SRSC SHEEP RED SL00D CELL 3H-TDR TRITIATED THY*lIDINE TPA 12-0-TETRADECONYLPHORSOL-13-ACETA:E TPM TOTaL PARTICOLATE MATTER ODS ONSCHEDOLED DNA SYNTHESIS OR "DYA REPA:R" OG MICROGRAM (10-6 qrams) GM MICRON (10-6 aeters) ti cTR coHTRacTS 029175 11248925 CTR NN 0439.wu-2

CTR COHTE2ACTS 029176 11248926 CTR HN 043963

8ina1 7epo:c Con::ac=: C':R-0030 S:. Kl4AAY Su'uF-k ZY . ZI... I. *VTROCCCT.CY Tsis :s the Final Report for Calt:act 0030 entitled, "Cigarette Snoka Znhalation Studies in yice." This report descrizes: 1) the facilities and equipaent, 2) the :esults f:cm three major smoke inhalation studies, and 3) the :eaul:s from corollary studies that supported tae three major programs. The five chapters that -make up the Final Report are sus.zarized here. II. ?AC:L:T:?S AND SQO*.?y*NT The cigarette smoke generation and animal holding equir:ent were designed to provide high exposure to fresh aainscrean cigarette saoke on a daily basis for a major portion of t::e 11=et:me of the animals. ':::e =acilities provided the following: o Tao Smoke Exposure machir.es (SEM LI 3 and C), one stiam exposure machine, three animal contai::ment units, twenty-four exposure modules, and 400 animal restraint trays. o Capacity to expose 480 mice simultaneously, and up to 2000 mice per day, to cigarette smoke under conditions where greater than 90% of the smoke partfculates were deposited in the respiratory tract of the :nices o Doc~.Laentation of the quantity of smoke presented to the mice during each exposure session. o Safety systems t::at assured exposure of -tice to saoke only under preset exposure conditions. o Generation of cigarette smoke under conditions controlled for cigarette type, smoke aerosol concentration and smoke partic:e size. o Capacity to 'louse up to 10,000 mice at a tiae under conditions that insured a clean and controlled environment. 1 CTR CONTRRCTS 029177 11248927 CTR HN 043964

p:^ai iepo:_ Contract: C':Z-0030 SQ?N.A eZY III. CTR-100 (I-1c01. C0-C?IRCI\CG2:7S5:5 A:iD C'ROVIC He1L.dT.,,V C: :a CiGARiT':IS SM0X-7 The first c:salat:on study _':i:ed :ent_cky Reference 2a1 cigarette ssoke ~(tiiqh ;ar-lov nicoti e) a: a dose of "1 aq/tota: particulate =atter (':P`4) per day per co;:se lu::q. The experiment =ad _ou: rajor objectives: 1) to dete:m:-e the icpact of _;e ciqaret:e sroke ;ene:ation equi ^ent and delivery system on t~e test aai3als (3C3:1/Csa female mice), 21 to determine t~e toxicological ef:ects* of daily exposure to 2a1 whole cigarette smoke in 3C3?1/Cus -nice over a 12-15 aont:: period, 3) to characterize the susceptibility of 3C3F1/=- and C1H/anf C::a mice to l::ng cancer after t:ea=ent wLth inovn chemical carcinogens, and 4) to dete:mine the effects of _aily exposure to vho?a ciqaret=e smoke on 3-methylcsolant!:tene (MCa)-ind;:ced l::aq tuno:s. A total of 3476 mice were put on test :o: this study. Mice ve:a observed during and i=ediately after daily smoie and sham treatment. ?or the smoke expcsed animals, :rhile a certain aaouat of adaptation occurred, t`:e aninals never accepted the =oka exposure without some aqitatio.^n or struqqlinq in the holder. They also occasionally demonstrated some shallow breathing or gasping during exposure. Iamediately after exposu:e, ssice in the smoke exposed groups were :ethargic, ataxic, and hypothermic. The sham exposed animals also demonstrated agitation and struggling while restrained in the holders, but generally appeared normal after exposu:e. Cacboxyhemoglobin (COHb) levels in the smoke exposed =i:e averaged between 40 and 50% after each exposure session. ".`.e results ::crs the 2A1 smoke, sham, and shelf control groups uil: be presented f?:st, followed by the resu:ts ::oc the !!CA-t:=a:ed group. The i=pact of the exposures and :elated -anipulatio.^.s :esu::ed in "S0t of the animals dying in the smoke or sham exposed groups by 36 weeks on test. :Z addition, neck abrasions were cbserved lo both smoke and sham exposed mice. Two equipment modifications were recommended: temperature coapensated flow taermistors to monitor smoke/air :lov. _h:ouqa the modules and polyca:5oaate inserts to cushion the neck edge of the restraint. :t:e theraisto:s were designed, developed, and utilized. ?olycar5onate neck inserts were designed but their atilization was not approved. Data from all 6ham exposed controls shoved that the spontaneous incidence of malignant lunq :=o:s in CTR-100 (all alveolar adenocarcinomas) was 0.5%. Smoke exposed animals were observed to have no malignant lun,T changes during the study. The most frequeat lesion observed in the smoke exposed g:oups was pigmented alveolar macrophage acc,=ulation (PA!!A). After 20 weeks of exposure, the incidence of PA:d.a was 100=. Two other r. 2 CTR CONTRRCTS 029178 11248928 CTR MN 04,39EGS

?iaal Repor: Contract: C-R-0030 SG' W a Y lesions were associated 'dith smoke exposu:e, squa=ous -e:aplasia of the t:acaea and :zi-:tis of the nase. T:eatzent of 3C3?!/Cua aice with MCA zes::d ted in -0'2 3il:=esent :ocbiaa:i:ns o• `sistooat`:oloqic diagnoses in the lunq. alveoloqenic ?esic,s we:e classified i.^.to 4 cate:::ies: alveola: ,ype:p!asia (ii)r al•reolar non- co:np:essi-q'^..odu!es (AYCY), slveo:a: coop:essi-q :9u:es (aC;t), and alveola:adenoca::incaa ;A aC). Sq;:amous lesi ^s were classi::ed _.:0 3-_.eaocies: squa.mous =etapiasia (3M), squaaous ceoplasa iSYI. a^d squanous cell carcinoma (;CC). The other carcinoTas four.3 ..^!__ed poorly di==e:entiated ca:c.-oaas (PDC) and a va::ety z f aixed csrc:::ocas. The ca::i::ccas metastasized and even:•:ally caused the death of the ar.iaal. The proq:essive nat::re of tnese :esions was documented and a scheme depictinq the evols:ian o: these =ali;.^.ar.cies was posts!a-ed (see ?iqu:e 29 and Sec::oz %nci!larl s:ud:es with other inown carcinogens and o:"er strains of zice suggested: !! C3H/anf C:La and 3C3: !/:-za sice were idenc.cal in :hei: s::scepti5ility to ~lCa-induced __c:q _a^ce:s; 2) a dose :esponse to MCa .-duced lung t;:~o:s was tse:ved; and 3) the carcinogenic activ::y for lung tissue was =ete:=i::ed to be yrea:est :o: MCA, =^lloved by 7,8-dihyd: -7,8-dihyd:oxy 5enzo (a) py:e::e (3a?-', 3-diol) , both o: uhic:: •:a:e -uca more active than :,enzo(a)py:ene (3aP). Daily exposa:e :3 2A1 cigarette smoke did :ot alter the types of MCa-iaduced p•::3onary lesions observed -__oa:ed to sham expose•d mice. The:e we:e slight variations in the incidence and 3ist:ibution of the lesioc:s as a function of 3_:a:ica of sao:ce exposure. analysis of individual lung lesions s:"owed that the `:Ca-shaa exposed ;:ouP ::ad a 1iqher inci3ence and/o: sho: te: :a:eacy _°oc all ca1:?::a.^.: 1u.^.g t=ors than the ':C1-szc)cs exposed g:ou?. 3noie exposed _ice developed a slightly ..i;::e: .^c:denca of wC:1, AC:1, and/o: Sythan the sham exposed,aice. analysis of the develoc.^.ent of either these latter lesions o: =aliqnant :esions was also pe::,z:=ed. Daily ex?osu:e to 2a1 =iqa:ette smoke did not alte: ~e incidence and/o: dist::~:ution of t:.ese mCa-i::duced lesions wnen compared to the saaa exp:sed ccn::ols. iY. ':R-101/1. CRR7y:C ZX?OSCeZZ CP 3C3:!/CC?S `!:CE °0 2R1 A2?T"E S1:OiC3 This second :csa:3:ion study was the fi:s: fes;qc:ed to use .lonq-tera or "lifeti:e" chronic smoKe exposu:e. Future studies .ere to address suc.'h variables as specie sensitivity, smoke exposure regimen, smoke dose, cigarette type, and d•s:ation of exposure. However, after initiation of this second study, it was decided not to pecfoa any other Lonq-te:a smoke inhalation studies. Thus, the main objective of this second inhalation study was to deter_ine :::e potential biological ac:ivity of daily 3 CTR CONTRRCTS 029179 11248929 CTR HN 0439-66-

?:.^•al Report :3nLrsc=: C-Z-:O3G SVnVf7•!ZY exposure to Kentt:cky Re!e;"ce :a1 :ga:e:-4 smoke .`•::;.`, _3: - •.l'1 ..:cotinel under one soeci::- se: a: exMe: -en:a1 ---d: .. s.a secondart objec:ive was _;ed_:e:mina::on of t..e poss:b:_ ,e::ec: 2: d a i : y e x P o s u : e : j s : a < e 3 ~ :en:o(a)pyrene-ind,.ced "ne .1•=be- 0: :z:ce an :esc .__ .-? s_cie s-an aad siel? qroups -as::3'a^d 439. :esrec:.:e:y. 3a? :as _ :en int:at:acaea:lf :o an :.`.er ;:aup of :^•:ce. --e ---be: ~: oice cut on test :ar .-e 3a? - saoke, 3a? - s-a, a-d Ha? - s:.el: ;:oups was 320, 257, a:d :30, :es;.ec: vely. 3a? :as ;• :ea =h;ee ::nes at b!veekly .-sr~als and smoke/s;:a3 ex::s;::e ~:as initiated one :+eek after .;e :as: 3aP t:eatxent. Smokz ex:cs:::e concinsed on a daily basis :zt 1:0 weeks. The :.-se ~f :R1 :;arec:e smoke was dete:=:-ed :o be 0.2 =q '°?!i -er day =e: =:•_se ^q The results :ro3 _"e 3o<e, sham and shel: v:1 be p:esented :::s:, :oL:.;wed by the :essl:s .-:?. .-a 3a?-t:ea:ed groups. The major cl!nlcal si;ns ia ^e smolce exposed, s-a: ex?:sad, -^d sae1= =ontrol mice :+e:a as ::1:ovs. 1) The -'asc::s ::r o:e~1 =eath of t:^e 3ice vere ei-.`.er =^di:.or.s that randa-:.y -2- nai-aLs P:cm the study S!.e. smoke cr sham expos:::e--ela:ed, ho:der-:elated, or doc=ez:ed ai' or smoke flow :....:e.2s1 , or '_evelopiaq diseases that litely :ed to the death o:.ne aa::al. 2) Rate of weight gain vas sl:ver in the sham and s.:<e ;:..-;:s -c=_va:ed to the shelt :^:::::-s. 3) ~aily COt•tb LeveLs sve:s;ed :7.2%, 1.4% and 1.81`:r :::e smoke, shaa and :espec=ively. - Among the shelf ocn::sls, 50% o: the -mice =fed Z: s-:ca -eo?las;fc diseases as -e=a: -.oietic cancers ;-33t) a-d j b:osa::o3as ("13:), -dhi:e 40% 3:ed of non-neo?1as::: :asi--rs s::ca as congestion/pne•:-oa:a neph:itis a-3 a :nriety of incidental :esions ("23%) . :hes.e sace ::pes o: :esiozs were observed :n :'.^.e smoke and saaz axpose3 ..i:e. ~atailed analyses of '::e `:1:owin? :esions vere -,ade in r_o~ce and sna3 exposed animals: cancers, 2) zon-le:?:as:ic :espi:atory tract lesiols, 3) other ::on-neoplastic :es::-s. 4) all maliqnancies, 5) head and neck :!brosarco=as, an3 5) ::ecatoaoietic t=ors. all lung cancers obse:ved in the 3C3F1/C::3 -.=e =ere alveolar adenocarcinccas ~aAC). ~to squa:ous call car:::::=aa o: other pulmonary carcis:mas were cbserved !1 the s=o<e, s,am. a; s:nelt control groups. A total of 19 AAC were observed 2u: Z= a total of 978 animals at r:sk in :=e smoke exposed .;:ou=, wni:e 7 ;aC were observed out of 531 s-a.z exposed animals at ::s:c. :a:a analysis (see Section :7. i.) ia :our different ways i-d'._3:ed that no differences vere »se:ved between the smoke .. snaa exposed groups at p< 0.35. 7.:e :acidence of l+AC was ;:ea:er i1 the saoke exposed qroups to the sham exposed ;:o.:ps CTR CONTRRCTS 029180 11248930 ~ CTR N 0439-6:~P'?`

?:na 1 Repo: : Cont:3c:. _-R-J03J 5:.F.-.AtY (p-0.17) and tie Latency of expression •:as dec•eased :n :=e sn o<e exposed g:c::?s ccepa:ed :o t::e sna= ec=ased ;:oups Occsrraace o: avCY or aC!1 vas 38/973 :c: sr:oia exposed ;:oups and 21/551 for _he shaa exiosed ;:o::;.s ;;.•J.5= (:acidence): p0.33 tl3tencY)!. T::e occ_::ence of ei:"e: ,aC o: \C4 ;(:elded -e =a:;est 3i::e:e::ce be:veen the s:o~ce a^d s,s.3 erposed ;roups. :'he _°.nai inc:dence ot XAC or ACN :as :9. ?'3 :: . .ne s~oxe ex • c?a:ed posed c.." :a 23/53i ia ` _^e s:naz ax:asad y:oup These ~a _z:ide.^•ces were ^ot de:ent (pv0.59). -== _;e :=ocs~ •:pea. d =ace ;::.:k:y in .-e s~c;ce exposed ,:o••p p=0.:?) -. A total of five ::on-neoplas:i: :espi:ato:y _:ac: :esions were analy:ed: ?At*.A, congestion, ati:is -edia, snd oti:is exte:::a. The overall incidence :: ?iIKa was :5t dzi-a is considerably love: than that obse:ve3 .- the previous =i;a:ec:e smoke study. The nuxber of ?XYAs inc:eased with exposure, hu: only :eac:led 35t durinq the :ast 4 months o: ezposu:=. Congestion was a:at`er common '-esian in Soca the smoke and s=am exposed ani=als. The occir:ence of conqestian was greater in :-e sham ezcosed ani=als compared to the scc. Ke exrosed •_.ce !c0.02). The :i:ia1 incidence of rhinitis was noc different ~e:veen the s;noRe and sas.-a exposed gcoups (p%0.97), but the lesion oc_u::ed slig::;:y =ore :apidly in the smoke exrosed =ice (?-.25) . The latency pe:i.d :o: otitis media was sno::ec in saoRe exposed =ice p< 0.01) and according to one aaalysis, the final incidence o; t::is :esios was also higher in s-moke exposed ani=sis (p-0.05) . The final .-.cidecce of otitis extecna was Zot different between the smoke and s:a3 exposed groups (?0.iS). Hovever, :he:e :re:e i:.dicacioas :::at this :esion occ::•:ed with a shorter late.^.cy in the s3oke exposed q:oup compa:ed to :ie sham exposed ;:o~; (?'0.10). The ot-er r:on-neoplastic lesion chse:ved was nepk::? :s, a•^n in::a=atc:y :esion of the kidneys. '=ere were no d:::a:ences becveen the smoke exposed and sham exposed ;:oups. A comparison of the occa::eace of all aali;nancies demonst:ated a 27% incidence for the smoke exposed and a 29% :::ci,r .er.ce :o: :`e sha_~ exposed animals. The .. ._idence and/o: :atency was not different between :he two ;:oups. One oartic,:lar type of t_eaor, head and neck :i5resaccoca, was obsesved with a higher frequency :Z smoke exposed =ice ccmpa:ed to the sham exposed mice. "=ese t:LZo:s were aggressive and metastasized to tiss::es far removed :'-e .primary site. A total of 29 head and neck :ibcosa:c:=as •Je:_ observed in the smoke exposed aice. c:apa:ed with 8 in t:^e sha3 exposed q:oups. This difference is hiq:z:y significant (p< J.05). 7zposure to smoRe was associated with both a higher inci3e^ce and shorter latency of head and neck :ibrosa:comas. The zec::anism cy which these :--=o:s developed is unclear. S ' CTR CONTRRCTS 029181 11248931 CTR MH 043966

*:za1 Re?or: Contza_-: C'3-0030 Scli`,•AdY Hematopoie:ia = nphosa:c:-as. ce:' sarcozas, and =ysp oc; ::c c: : yapao i3 :euke:a ias . The :o t3: iacider.ce of :.`.ese _was 131. :d '-he s-o:<e exposed and :91 :~e shaa exposed an._als. ~s:ng `ouc d:_`e:e::_ ,e:,od s : a^nlysis, -he .-c:de^te .;: _-ese =ocs :as :ea:e:, and =~e =3=e.^.^y ; er.-:d was si:..: _e: :1 .'e s:1am ex--Csed ._Z~a:2v t3 e S73oRe eX?zsed aZ:ma/5 Acalfsis a: _~e 3a?-t:ea:ed ;:ours :as 'a z::j :-;ed as desc::bed above. :he ..,.:dence of PAMA in tae Ba? - smo<e exposed ;:oup was 513. For _::e aaimals axposed :o :R1 s-o<e without 3a? .:ea:=ent, :^e _.._i:e::cs of PAxA -was less than .:t. ._ would seea :.`.a: t:ea:=en: wi::z 3a? aodi:°ed the -ac:op-age :esponse :esul:ing in h:g':e: .::cidence of these p:,:.en:ed ce1:s in ::e _:ng. Dai'_y expos:::e :0 231 _iga:st:e saoke !•-.-::g _:ze __ce 3a?-i :3_ce3 L_-q .r.ors ,•.eveloced did ::ot al:e: .-e .•.c:_'ence o: :a:e.^.cl in _,e 3a? • scoke ^,:oups c0mpa:ed :o _::e Ba? - sc:a= ex;osed q:oups. V. C:R-1113. EX?:SGR3 ^? 3C3F1/C:?t `S*_CE '0 2R1 5 !0KE During 1979-1980, aa exposu:e reqiaen was :eveio?ed that :esul:ed in low toxicity for botl 2R1 and 3a1 _i;a_et:e saoke. :hese expcsu:e coc=:::oss allowed the se'.^.eduli::;, o: a:onq :erm study whe:eby the S:ological effects of smoke '::= bot:1 _i;are::e types could be s'_mu1:a.-.eously evaluated. Suci a:ang :e:3 study zcpioying ::zis saoke exposu:e :egizen was p:oposrS, a?proved, and .ai::ated d• -ic:g the :330 contract year. :a Ju'-y :930. -oweve:, ._ vas deci"ed that _...s s:,:dy saould not :e and that saoke ex?osu:e ua!er -hese newly defined -d-iors •:oul3 be _:~ited to the lea;::: of iae durir.q whici aai_a_s in C:R-L0LA :+e:e to be exrosed to smoke. T`:e exreri=en:s coad•:e:ed in the ..:R-10:3 were desi;zed to evaluate =er:aiz sio: -te:z _:xi=ological endpoiz:s ;at may be :elevan: to !e:e=:•^.:^g the po:e^:fal Siologi:al a:tivity of whole ,.igare::e s-oke. ;he smoke exposu:e :egi-en :ot in this s:u:y did not include :est periods between' successive ciga:et:es, •:sed an exposu:e pe:iod of 15 seconds :er minute, and contin_ous exposu:e for :7 •':uns" in 140 micu:es. This :egiaen :esul=ed i1 -30 •.:g :'?!t deposited per day ;er =ouse Ls-q, an averaqe C:e?b of 13.9%, and 3reater than 95% i:%cf=ence of ?A.'iA by 15 weeis of daily smoke exposure. The following assays ~ere selected for :hei: ?ocen:ial =o predict or monitor L:ag-te:M e::ec a of _:qa:e::a s;:o:<e: 1) inhibition of pulcona:y :NA :epai: in vit-oi 2) s:icjla:ion of pul=onary DvA synthesis in vit:ol 3) iaduct::n of pulaona:y a:yl 5 CTR COh{TRACTS 0229182 11248932 Cf R PIN 041"3969

?inal Repor: Con:ract: --TR-0030 Sa;'..--IAAY hydrocarbon i:ydroxylase (aHH) activity; 4) induction of orni:hine decar5oxylase (o0C) activity; 5) aug:aentation of the DNA 3amaqinq effects of particular lung carcinogens; 5) induction of sistar chramatid exchange (SCS) In bone marrow; 7) alteration in certain physical -aaracteristics such as Lung weiqht (wet and dry), body weight, and bioc::emical c::aracteristics such as DNA, protein and iydroxyprol:ne LeveLs in lunq, 8) alteration of i3mune c^iPecence, and 9) alteration in rate of atre:ia (i.e. oocyte =oxici ty) . Osing the discontinuous exposure reqi:ien as defined in CTQ-L00, inhibition of DNA repair capacity (e.q. unscheduled D`tA synthesis (ODS)J occurred within 13 weeks of •daily exposure to 2a1 or 3a1 cigarette smoke, but was unaffected by exposure to 2Q1 cigarette smoke for even up to 52 weeks. Osinq the continuous reqimen as defined for CTR-1013, no DNA repair inhibition :+as observed for either 3A1 or 2R1 cigarette smoke even after 52 weeks of daily exposure. Thus, the inhibition of DNA repair (QDS) by cigarette smoke would seem to be dependent upon cigarette tyYe (i.e. 2A1, 3a1 >> 221) and/or the exposure reqimen (i.e. 30 seconds smoke per minute >> IS-20 seconds smoke =ec 3inu=e). Replicative DNA synthesis (ROS) in vitro was stimslatad 2 to 4-fold after 9 weeks of exposure to 2A1 or 3a1 cigarette smoke, but not after exposure to 2R1 cigarette smoke using :::e hiqh dose, conventional exposure regimen. ROS was stimulated -2-fold in mice exposed to either 2R1 or 3A1 cigarette smoke ssing the continuous exposure regimen. ONA :eplication as measured by tritiated th 1-3 id:1e incorporation into lung cells in vivo (Labellinq Index (LI)), was increased 3-to 6-fold in lung and tracheal tissues :com 2R1 and 3A1 ciyaret=e smoke exposed mice. No increase in Gt was observed in bladder, colon, liver, kidney, or spleen. 7-vidence was presented to suggest that daily exposure to whole smoke (ei ther 2R1 or 3A1), while stimulating normal DNA replication, may induce unsc::eduled DYA synthesis or repair synthesis as well. Daily exposure to 2R1 or 3al cigarette smoke failed to augment the L: induced by the potent lung carcinogen, 3aP-7,3-dio1. Thus, both cigarette smoke and certain chemical carcinogens (e.g. 3aP-7,g-diol) were capable of stimulating DNA synthesis in pul=or.ary tissue, but these effects did not seem to be additive, synergistic, or antagonistic. AHH was induced in the lung after exposure to 2R1 or 3AL ciqarette smoke using any exposure cegiaen investigated. Pulmonary AHH activity was stimulated 4-to 5-fold after 3, 6, or 9 months exposure. Renal AHH was induced after daily exposure for 9 months. aepatic AHH was uneffected at all time intervals. 7 C.TR CONTRRCTS 0291e3 11248933 CTR MN 041397`0

Fi-al Repor_ Consraet: C:Q-0030 SIIMLKAR? Pulmonary 00C activity, a potential marker 'or promo-ian, was induced -2-fold within 3 hours after daily exposure to 2R1 0: 3A1 szoke for 3 or 5 months. zxposu:e to 2R1 or 3A1 cigarette smoke :ailed to cause single strand breaks in the DYA of pulmonary cells. Smoxe axposu:e also :ailed to influence the level of 0:1a damage caused by 3a? or 3a?-7,a-diol. Sister chromatid exchanqe (SCE) was induced in bone marrow cells of mice after exposure to either 2R1 or 3a1 smoke. The iad uction was -2-fold and was first observed after 1 week smoke exposure. Continued exposure for 4, 12, or 46 weeks showed no further increase ta the number of SCEs. Increases iz SC=s persisted after cessation of smoke exposure for at Least : week, s+het::er the mice were exposed initially for 1 week or 46 weeRs (p < 0.05). °xposure to either 2R1 or 3a1 cigarette smoke using the continuous regiWen resulted in an increase in the cellalari:y of pulaonary tissue. Daily exposure from 1 week to 52 weeks caused a time-dependent increase in lung wet weight, lung dry weigit, lung CvA and protein, and lung hydroxy?.roline. This i,crease in tissue 3ass was likely the result of increased cellular proliferation. This proliferation was probably a:inal manifestation of the increased DaA synthesis rates observed ia these ani=als. Daily exposure to 2R1 and 3A1 cigarette smoke 'lsinq di:=arent exposure regimens failed to alter the immune sta:--,s of 3C3P1/Cua mice. I=mune competence was determined by .:e:-:e plaque assay. Veith.r acute nor chronic exposure caused i.:.munosuppression in either HC3P1/Cum or 3aLa/c C.:.,n mice. Rates of atresia (i.e. natural loss of• oocytes from the ovart) were also uneffected by chronic exposure to 2R1 or 3A1 cigaret:e smoke. While it has been suggested in the literature =::at there is a relationship between cigarette saokinq and early onsat of menopause (i.e. when the ovaries are depleted of oocltes) , oocyte toxicity was not observed in eC3F1/C.:.3 aice after 52 weeks of daily smoke exposure. VL. C.^.ROLL.IRY STDDIES: ?HAe'2lSOCORIVETICS CF IVHAGEJ The deposition, distribution, and clearance were deterrined :or nicotine, 3a?, dotriacontane (DTC) and catechol in cigarette smoke. Data showed considerable differences among these four classes of chemicals. yicotine. 9ap and catechol showed rapid zedistribution from the lung to other tissues and clearance froa t;e body. The half-li:e for internal :etention (tl/2) was ess a CTR CONTRRCTS 029184 11248934 C TR HN 04 3 9 ~ I

Pinal Report Ccn::ac:: CTR-0030 SIITSl4ARY than 1s minutes for nicotine, Less than 1hour for 3a?, and less than 20 minutes for catechol. OTC si:oared little :edistribstion from the Lunq, with a tl/2 of greater than 24 hou:s. The deposition, distribution and clearance of catec::ol as sinqle csemical aerosol was shown to be identical :o catec::ol in cigarette smoke -- i.e. the pharaacokinetics of :atec^ol are not altered by :ae presence of the other chemicals ie cigarette smoRe. 3reater than 901 of the izhaled cateqhol, eit::er in cigarette smoke or as an aerosol, was found as a beta-ql-.:curonidase- or aryl sulfatase-sensitive conjuqate in the urine within two hours of exposure. The deposition, distribution, and clearance was also detec:iined :or 12-0-tetradeconylphorbol-13-acetate (TPA) as a single chemical aerosol. The pharaacokinetics of aerosolized ;'?A was investigated as a potential promoter in a two-stage (initiation and promotion) carcinogenesis model systea sreci:ic for l:nq tissue. Results from two expariments showed that over 631 of the initial TPA deposition was found in the -espi:atocy t:act. TPA was rapidly cleared from the lunq via the blood, •aith increasing acc=ulation in the liver, stomach, and intesti::es ::p to 8 hours after exposure. By 48 hours, over 70% of the inhaled TPA was found in the feces and 20% in the urine. aerosolized '?A induced a specific biochemical event in the lungs, that is, induction of 0DC activity, within 3 hours after inhalation. TPA thus, exerted its effect i=ediately and was then removed f:om the lung. The pharzacokinetics data suggested that aerosolized TPA may have effects in the qastrointestinal tract, in addition to the respiratory tract. These data supported the usa of T?A in a tvo-staqe model for Lunq carcinoqenesis. 9 CTR CONTf2RCT5 029185 11248935 CI R 1 I 7 I 0439.72

CTR CONTRRCTS 029166 11248936 CTR HN 043973

CTR CONTRRCTS Q1g 180 11248936 CTR MN 043974

Tinal Repo:: Con:ract• C:'2-a030 IYTRODCCTION r. IVT:t00CCT:0V/03J?C'":V?S Chronic smoke :ziaia:fon studies were designed to deterai::e if potential biological effects occur in inbred strains o: mice 3•sr:^g 12ng tera exposure to whole cigarette smoke. :n humans, data to date suggest that cigarette smoke possesses weak biol:gical activity. 3ecause of this weak biological rotential and because of the conditions under which humans are ex;.osed to cigarette smoke, it is a necessary requirement that any smoke exposure study use: 1) conditions whereby the smoke deposition and distribution parallel that of human smokers, 2) duration of exposure which parallels that of a human smoker, 3) an animaL model which is capable of developing those types of biological lesions associated with cigarette smoking In humans, and 4) a number of animals sufficient to arrive at a signi:icaat conclusion. Several approaches were taken at !!icrobiological assaciates (Y-N) to develop a aurine model =or lung carcinogenesis along with techniques and procedures for quantitative smoke exposure s:udies which take into account the above requirements. Essentially, three major smoke inhalation studies were performed. The first of these (CTR-100) utilized the Rentucky Reference 2A1 cigarettes characterized by high tar/low nicotine content; the second (CTZ-101A) used the Kentucky Reference 2RL cigarettes characterized by high tar/high nicotine content; and in the third experiment (CTR-1013), 2R1 cigarette smoke was used to expose one group of animals, and a second group was exposed to 3A1 cigarette smoke which, like 2a1, is characterized by high tar/low iicoti::e. The scope of the first two studies was the assessment of :::e carcinogenic potential of cigarette smoke either alone, ~r synergistically when a known chemical carcinogen was administered pcior to long-term smoke exposure. The third study provided an::7als =or the evaluation of short-term assays that had :::e Po:ential to be early indicators of possible toxicologic consequences of exposure to 2R1 or 3a1 cigaret:e smoke. Corollary studies describe the deposition and distribut'ion of szoke constitutents and selected aerosolized ciemicals after "zose-only" exposure. Over 10,000 mice were exposed to smoke from over 300,000 _igarettes at the inhalation facility. The studies required the 53M II 3 and C smoking machines, a sham exposure machine, 3 ariimal contai=ent units with 24 exposure modules, 3 optical sensors with B strip chart recorders, and 400 animal restrain: trays. Smoke exposure related manipulations included ir.dividual ani~al identification (by ear tag), individual.vaccination against Sendai virus, individual loading a.^.d unloading of an:mals °or daily smoke exposure, monthly weighing of each animal, and a;.pcoxi::ately 5-10 data entry points for each animal within any 10 CTR COh4TRACTS 029187 11248937 C TR MH 04~.."~ 9 f'1E5 .

*inal Reporc Ccn::ac:, ::'R-0030 iYT3cOCC'::ON given experiment. additfonal daily ef:ort in technical sup;,o:- sorvices included autoclaving :ood and beddi::q, washing cages and -dater Sot:les, and disman:eli.:q and cleaninq smoka exposu:e equi:ment each day (2i anical =antain=ent nodules, 400 aniaal -es::ain:s, _~e smoie ezposed su::aces c: the SFM 11 3 or C, _ie :lcw the:=is:c:s, and :=e optical sensors). :'inally, another '_evel of a::::: in professianal support services was :squi:ed 'or ::ec:opsy, ;is:ology, pat;oloqy, data evaluation, re-avaluation, and ::nal analyses of each experiaent. '"`se Level of effcr: :equi:ed to ;,erform these studies amounted to approximateiy 11,000 aniial :elated manipulations each day. T`:e following report desc:ibes the experimentaL '_indings in five areas: facilities and equipment necessary :or "nose-on:y" exposure of zice to cigarette smo<e (Section II), coca:cinoqenesis and chronic inhalation of 2A1 cigarette smo<e (Section LIr), chronic exposure of 3C3F1/Cum mice to 2R1 cigarette smoke (Section iV), exposure of 3C3£1/Cua aice to 2R1 and 3A1 ciqarette smoke (Section V), the pharaacokinetics a: innaled materials (Sect:on VI), and the references (Section '!II). A list of publications f:o:n !4ic:obiological Associates supported by The Council :or ':obacco Research may be found in Section vtt2. 11 ICTR CCHTRRCTS 0291e8 11248938 CTR MN 043-976

.CTR CONTRRCTS 029189 11248939 CTR MN 043977

?inal Report Conc:ae=: :':R-0030 iACLL'.:':-s & ZcQ:?'tEVT II. ^aC:LIT*YS aYD :QD:p!!3:J'" ?OR "•;OSS-OYGY 0 V. ~J . ~....t . ...... J•~ The ^.iteria %.sed in desi.;ni-g the facilities and eTa°;-en: vere based on :^ose factors that `aave been reported• to 'oe :mpor:ant in cigarette smoke-associated ,c:aan diseases. ;'hese :actors i:.c-":de high exposure to f:esh mai.zst:eam cigarette sao:ce on a daily basis for a major portion of _;e *lifetizte o: :::e i::dividual (1,2). !ven under these conditions, however, :~e resultant smoke-associated diseases are observed in only a sma:l ::action o: exposed individuals. ':hus, the :ollo.ri,g set of condi:ions were followed in order to most closely approxisa_e the human situation: o Ose of an exposure system ahere the lung is the -ajo: _ar^,et, o Use of a smoke generation system capable o; pcovidi^g large quantities of _°:esh cigarette smoRe, o Cse of :arge numbers of animals so as to detect _`:e . biological activity of substsnces with weak Pocential, 0 2rovide :or aaily exposures over a-:ajor por:ion of t`:e lifetime of the animal, o 'tonitor and -docu.•tent the ytitantity of smoke presented to the ani=al during each exposure session, o Provide safety systems In order to assure exzosu:e zc ani=als to smoke only under preset exposure cor.di::ozs, and o Generate cigarette smoke under conditions where °acto:s are controlled, such as: cigarette type, smoke a2roso: concentration, and smoke particle size. This section will provide a generalized description of t;e operations of the Smoke Inhalation ?acility, a generalized ~escription of the facilities, and a detailed desc:iption of :he cigarecte smoke generation equipment and the animal exposure sysrem. 12 • C.TR CCHTRRCTS 029190 11248940 laa' T 1'`6 V I 1 7 0 4 or.i arf 7 6

Y:-a1 Repor: Con:=aC-. CTZ-0030 . rAC::.2T:ES & EQQ2?KE:JT A. :esc:iption of ::e Ceneral ^.perations . ~., The squ:~3ent and procedu:es ~escrioed ::ereia :+ere desiqned for standardized, doc::men:ed izhalation exposure of :a:;e numbers of mice to cigarette smoke. Mice were chosen as :=e a1i7a1 -ode1 for these studies because: 1) ecor•omy fn animal ic:sband:y operations were possible; 2) large a::3be:s of ;enetically diverse inbred strains :rere available; 3) stsaias of =ice were available in which susceptibility to Lang cancer following treatment with chemical ca:cinogens was ;enetically regulated (3,4); and 4) animal colonies :+ere available that were well defined in terms of types of biological adventitious agents ao ^ally present. It is especially important that :::e -acuse colonies be free of `wcoplasma pulrzo^is and two :es?i:a:ary virusas, Sendai virus and pneu.monia vir::s of mice (5,0), ali mice used in these studies were vaccinated agairst Se^dai (see Ref. 3-6) for discussion). Smoke was generated on the Smoke Sxposure -Maciiae, ter=ed SS?S :2, a lar?e capacity dynamic smoke exposure sys:ea (7,3). The S'.`! II uses reverse s3okinq (positive puff) and a;i::aatical:y loads, ligyts, puffs and distributes the smo)ce f:om up to 33 cigarettes to the animal containment system In a flowing st:eaa for "aose-only" exposure of the animals. Smoke and air flow aonitoring devices :rere provided to docuaent exposure levels'a-d also to provide safety for accidental over-ex?.osure.or for mac.^•iae -alfu.^.ction. Smoke levels :rere quantitated :;roug•~ the use of an optical sensor interfaced with a strip-chart :eco:der (9). .3L pu:f-by-puff profile of the s3oke total par:._s:a:z zat:ar ("?~i) delivered to the animals was docu.•:ented :or eaca exposure. Curinq S years of use, the SEa II proved to be a :ema::<ably ::ouble f:ee precision inst:sment, utilized 5 to 8 ;ours :er day, 5 days per week. A unique feature of this inhalation system was that the azi=a:s were exposed to the saoke aerosol only via the ^asal ari:ice for "nose-only" exposure. Croups of five aaiaals •;ere ::eid in a"stock-like" holder usinq a combilation of a r.eci slot and restraining spring. The r.ose of each animal passel :;:oug1 a fez:al :::bber dam diapnragm. forming a seal which p:even:ed exposure of the bod y to the snoke aerosol. The animal :oztai:.ment system was efficient and easily used. Mice can be : apidly loaded and unloaded from the trays and the :rays ran ':e easily placed on the :aodule for the exposures. in such a:a,r.er, 430 aice can be exposed in a"^oaa-only" fashion to smoke .cder =1:e same conditions from a single SE:4 II. 13 CTR CCIHTRRCTS 029191 11248941 CTR HN 043S47'.3

?inal Repo: :oncsact. C:'R-0030 PaC2L:T.-S & sCOI?!iXNT 3. :esc:iption o: ?acili:ies all 3zo<e exposu:es at XA were .a::ied oa: i-1 a dedicated facility, with rocros arranged in 3 gene:al areas: smo<a ;ar.er3tion a:eas, inimal exposure and :1o1d :.^,g 3:ei3r and nai::•e.^.anc e.ance areas. The exposure Labora_ory vas pera:ad as a bar:ier facility. Access was :estric:ed to aut::or::ed :acson^el and :olicies were in force to prevent admission of 'isease agents that might comprcaise the ::ealth status of the anicals. A 11 personnel entering aniaal-containment roons were suitably dressed, and all incoming materials were either sterilized ic carefully cleaned. The facility was equirped with sta;.Ca:d laboratorl support services, including a ron-reci:cila:i.^.q, fresh-fil=ered air supply, :dEFa and charcoal :iltered exha,:st systems, an oiless air supply, and a standby elec::ical generator. waintenance areas provided cleaning cnpabili=ies :zr anixal related equirWent and for the smoke inhalation equi;_en•. C. Cescription of ?qui;m.ent 1. Smoke Ceneration The Sr.`i LI was designed and sanufactured by ?:ocess and r.stv~.2ents, Corp., 3rooklyn, M.Y., under contract =o "he Council for :'obacco 3esearc)S-u.S.A., Inc. (Fig.l) .-he 3:4 an automati^ smoki::q machir:e which generates a continuo_s st:ea_ of sao<e and delivers it either whole or dilited :o: bioassay. Reverse smoking was ::sed in this design beca::se _ deli•:ers the smoke aerosol without a vacuum generating device in :;e smoke lines. Most reverse smokir.g sys:ems •.:se a movable pu:= cha:ber =::at seals over tl:e cigarette and forces air _irough the cigarette generating the puff. Subsequent designs !S:a .::) lave teen developed using normal -moking (Process and :^s:::-en:s, Corp., 3rooklyn, :).Y.). In the SE:S :I, the ent-:e smoking -ec::anism was locatad under a sealed clear plastic der..e 'he dome was pressurized and puff air was pushed through the -iqa:et:e by positioning t`:e butt of the cigarette in f:ont 21 a sli"er block, where the smoke was diluted and sent to :::e smoke delivery system (Fig.3). The dome sealed against the base plate of t::e cnachine and is hinged at the back so that it can be ocened =or access to the c=ponents inside. The entire smoking sequence was automati: af:er loading a hopper with cigarettes, sealing the doae, and activating the control system. Cigarettes were taken °::m :::e hopper by a pne,=a:ic loader and injected into cigarette '.^.alders fixe3 in a:otating d:::' (?ig.2). The d:Wz rotated stepvise and :ar:ied :;e block that seals against the inner surface of :,e 14• C.TR COh{TRRCTS 029192 11248942 C TR HN 0439.'60

_r:nal 2eporz Cons:ac: CTR-0030 ?aC.n:TI=S y :CO:?w?:1'" drum, removing the butt of the ciqare:te from the dome ataospae:e ar.d positioning it :n the smo;ce delive:y system at nea::y atmospheric pressure (see ?!g.3). Ccme pressure then =:.rced air :::rough :::e cigare::e generating the puf:. ?uf: vol;:me was set ay adj::stir•g the dcze pressure. ~t the puF:ing position, the liqhter, ahigi .a_sr.si:l in:ta:ed :a.=p 'ocused at the :ip of the cigarette, was automatically actuated during the Liqhtinq step•. This lamp •-•.ay be manually operated to light or relight single cigarettes. The d:-= rotated stepvisa by Ceneva move^aent, ma<ic:g one complete -evolution per minute. As thirty cigarettes were puf:ed sequentially, each step was of two seconds' duration, gene:atinq a t•ro second puff and puffing each cigarette once ;er ainute. After a preset number of puffs were taken, an autoraatic eject system vas actuated. The ejector, pneumatically operated and consisting of spring-loaded metal :ir.ge:s, moved =oraard, grasped the cigarette butt, extracted the butt :::m ,;e holder and dropped it into a butt receptacle (chute). To prevent smoldering of butts, which could add smoke to subsequently smoked cigarettes, a st:eam of CO was released into the butt chute during the ejection cycle io extinguish the fire cones. :he machine could be operated in either a single cycle or a recycle mode. In the single cycle mode, only one batch of 30 cigarettes were smoked. At tse end of the smoki.^.q cycle, each cigarette was sequentially ejected and the machine was au:omatically turzed off. In the recycle aode, instead ~: stopping a:=er one run, cigarettes were automatically loaded c::o the dr::rz :or a second run as cigarettes of the first run were being ejected. In this manner, the machine will continuously prcdc:ce smoke :or as lor.g as desired, or• until all of :;e cigarettes in the hopper were used. The air flow scheme for the SEM :: ^as one blower system, which used :ocm air. Air was provided :or done p:essu:e dilution air, and purge or breathing air (see Fig.3). The a:r passed through a hursiditication system and was then split between a dome pressure regulator and the purge and dilution :Lo:+ metecs. Dil•ation and purge air were regulated by need:e valve ad;•,:stzents. The dome pressure regulator was adjustable and ect•aally maintained a differential pressure ac:oss t::e cigarettes, as measured between the dome atmosphere and the slider block. Air frca the pressure regulator then entered _::e same area and was defused through an air filter (see Fig.3). 'lery little of the dome air was consumed by puff generation. The bulk of air moved smoothly around the edges of the t:ood a::d efficiently carried the side stream smoke out of the smoking area through the vent in the top of the dome (see :iq.3). ?::f: ai_ 15 CTR CONTRRCTS 029193 . 11248943 CTR HN 043981

Final Report Cont:aec: C°Z-0030 TAC2L:-'I's3 G £C022KlJiT passed through the cigarettes and the smoke aerosol entered the slider block. Dilution air was added to the aerosol in the slider block. The diluted smoke passed to the smoke distr:but:on valve that directed the smoxe to one of four animal expos.::a '.:ni ts or ani=al exposurs units or to the vent. The distribution system was proq:a:zmable to establish exposures of various duration vitain the one-minute smoking clcle. If an ani=al ex-zosure channel was not receiving smoke, it :eceived b:eat:::ng air. IZ the smoke distribution valve (?ig. 4), an inner cone was :otated in increments by a solenoid-driven stepping soto:. Smoke entered this cone at A and exited at 3 tirougl one of 4 smoke delivery tubes (C in Fig. 4). Four smoke outlet =::bes were connected to the four animal exposure units, but smoie .1as delivered to only one exposure unit at a time. Air entered :-e upper cha:aber (Z in Fig. 4) and three exposure units :eceived breathing ai:. The distribution valve may be positioned so that the exits from the delivery tube (0 in Fig.4 ) were aligned with _;e smoke Vent tube (F in rig. 4). Exit 8 will then he sealed and all the smoke will be vented. The distribution valve was proqra=mable through a drum prograsmer. Any desired exposure sequence using all four smoke outlets and the venc may te progra=ed. operating experience with the SE:t II has sioan :iat the smoke generated was quite similar to smoke prodsced by laboratory analytical smoking machines in partic:e size (13),'gas ccaposition, and in nicotine and TPM concenc:ations (3). 2. Smoke Xonitoring/Doc,-:entation/Safetf syste:n The purpose of the Smoke and ?Lov':ionitori-g Sa_°ety Svscem was to deliver the s3oke and ru:ge air from the SE`i :I, to auxiliary breathing air to the test ani-,a1 -cd•:1=s, to p:otecc :::e animals from possible operator error or ^a1=•,:.^.c:ior.s Of eq::ir:ent, and to provide documentation of exposure levels. The inst:=entation was also designed to protect animals from :verexposu:e to abnormally high doses of smoke particulates and carbon =onoxifle, as well as to protect them from suffocation due to an inadequate volume of breathing air. Accurate measurement and recordings of the exposure doses of :'P:i were also been provided. The output from the distribution valve of the 5=4 *.: consisted of four separate smoke channels. A description of ho:+ :hese channels were routed and utilized will be given. tn the exposure system, each of these four channels was fed through a monitoring and safety network into the animal exposure :ocm where each channel was split into two parallel streams, one to the 16 C.TR CONTRRCTS 029194 11248944 CTR ~~~ ~4~39082

rinal Report Zonc:ac: ^TI-0030 :ACSL:T:ZS~: !QGi?!!E:1T upper and one to the lower sections of the animal contain=en: modules. As can be seen in Figure 3, the sodules could be operated in tandem. :ach module ::eld 30 nice :1 i•s -.:ppe: section and 30 mice in its lower sect:on. :Sice aera loaded onto each side of a module. One smoke c:ar.nel p:ovi..ed smoke or air :or two wodules containing 120 mice, and the four channels of a 5=:4 II p:ovided expossre for 430 -:ice. The hinged modules, to he desc:ibed in detail in the following section, cqntained the aice ia a nose-only con:iquration, sucz that the a1izals had to breathe the air or smoke in the module c::annel when :;e module was closed. It was imperative therefore, that a s3oke/ai: mixtu:e or fresh air alone be forced through tye closed .:.odule at all times. The latter was provided by an auxiliasy air supply independent of the Sc'.r! II (see e ig. 3). The final safety devices in the smoke delivery system aere t::e flow sensors, were designed to automatically open the modules to room air, if necessary (see :ollowing section and Fig, 6 and 7). Piqure 9 is a photograph of the smoke -:onitoring recorder bank console. Contained in this console were ei3zt aonitorinq and recording units su:ticient to monitor the smoke generation and delivery from two SL4 IIs. This console was located in the SEM II Room, but projected (inside a metal housing) into the animal exposu:e room. The recorders and console drawers could be opened into the SIr`S Room for service. Pigu:e 9 is a view of one of the t:ro auxiliary, air safety system racks with associated components for four channels. A module rack in the animal exposure room can be seen through the window. The major canponents in the Ssoke and Flow Xon::ari-g and Safety System can be briefly described as :ollows: a. a smoke particulate sensor in each channel to continuously measure the smo<e concentration (?ig. 10 and 11), b. an =pli:ier-integrator net1or< to condi:ion and evel transform the sensor response into a high l electrical signal linear with instantaneous smoke concentration and also to provide a signal proportional to the iltegral of sao'<e concentration, c. a three pen strip chart recorder for eacl channel to record the instantaneous con- centration of the smoke or particulate as :rell as the TPM integrated with respect to tice (?ig. 12 - the third record ren was provided as a spare) , 17 ' C.TR CQNTRRCTS 029195 11248945 _L' 1 A. _' I • - , 0 4 3`/111/ 8 3

rinal Repa: _an:ract: CTs-0030 : AC:..:::L$ & :QQ:pX:.N^a'. d. an ala:3 (on each caanZel) that act.ated i: ;=e instantar.eous smoke concent:ation exceeded a preset lavel, .6r e. a ti4e sequenced alara :o actuate :he •,sa sa:ety valve i: d~ration of szoke ia ~. :e saoke/ai: cycle exceeded a preset _imit, f. an auxiliary air :low safety valve ac:•:ated by the smoke alarms and by cessacion of smoke or purge air flow (see eig. 9). g. a flow sensor located at the outlet of the exposure systess on the exit of the two st:ea.ms of each channel (see :igs. 6 and 7); (When smoke oc air flow fell below a preset value the !:o:+ sensor caused the flow safety valve :o automatically switch to auxiliary air, h. a tiae delay circuit to actuate a quick :elease aecaanism that opened the exposure modules to room air if auxiliary air did not flow through the system within 8 seconds after :he :low safety valve had switched to auxiliary air, and i. an air pressure switch in the auxiliary air liae to cause the exposure modules to oren :ocm air if auxiliary air pressu:a fell below a pre=et level. 3. aniaal Containment System The ?rimary components of the nose-only aninal con:ainaent systea are presented in rigu=es• 13 to 20. A !esc:i;tion of the important aspects of this system follows. a. Mouse Trays The mouse trays were used to restrain a y::::p of five mice by confining their necks within slots just aide enouq:n :o :-old the neck but narrower than for the head or body o= =ae aaimal. Mice were held in these stock-Iike holders usir.g a :o:zbination of the neck slot, restraining spring, head clip, and =::in rest (Fig. 13). Each tray had 5 slots, 11/32 inches :+.ide _* :o'afortably hold average size inbred aice. The desi;Z will accomodate a reasonable variation in neck size. Special ::sys •aith adjustable slots :+ere available. In Figure Ii is shown a tray loaded with naice :eady for '_oading onto an exposure module. As each nouse .+as la CTR CaHTE2RCTS 029196 11248946 HN 043964 CTR N

E'inal Report Contzac_. ^TR-0030 FdCi:.iT: zS G 3QQI?!'S:N': loaded onFo the t:ay, the neck sprinq was Iowered onto :;e :cp c f the r:eck and sacured by _ie reck spring catcl. a::ead c1:p attached to each spring :=obilized :::e head. The chin rest at the bottzm of each neck slot acted as an additional :est:sint and inssred that the nose of the ani mals was aligned :+ith :-e =cae-saa:ed opening on the exposure module. A :emovable s=ainless s=ee1 screen plat:ora fitted within the :ecessed bot::m of =•`•e t=3y to support the mice. Sxc:e:nent f•on the mice :assed :irough the screen and was contained within :l:e tray rxcess (see =:g. i3) . The tray supports, shown in F iqure 13, jount_d on the 4odule ar.d the mouse trays fit on the tray suppo:-s. Raised• back edges (tray stops) on the ends of the a~s of :;:e tray supports held the tray in place. b. Exposure yodules The exposure modules accomnodated 50 nice and were designed so that only the nose of each mouse pro:r-:ded :nto the smoke/air channel. Figure 15 is a photograph of an asse^,bled module in the opened ;.osition with one mouse tray installed. Smoke entered the module through the two smoke tubes (Fig. 15), f:oved through the smoke passageway or channel (upper and 1o•+er) and exited through the smoke tubes on the opposi:e end. The mice to be exposed were rest:ained in :^e stock-like holders that fit on the opposing sides of the mcd::' e in two tiers of 15 mice per tier per side for a total of 50 -,i:e. ':`e =iers aligned with the passageways and as the mice in t:ays were loaded onto the module, the noses of the animas s prot:::ded through a dental rubber dam diaphragm, foraing a seal that prevented exposure of the body to the smoke aerosol (see Fig. :5 =or a schematic of the holding system). Fiqu:e :7 is a ::a.^.sve:se, cross-sectional drawing of a module and Figu:e i3 shows an end-on view of a module mounted on an exposure :ack. 3r.e enti:e side of the module was hinged at the bott:m alor:q i ts length so that it could be swung open to expose the passageways or channels to room air. In this manner, the mice breathed room ai: whenever the module was open. The 0-ring seal prevented :ea<age of air or smoka from the module when it was =losed. Z:ect:=aglets held the modules closed. The magnet keeper, ~ounted on the hinged side of t';e module and the magnet, mounted on the exposure cack, may be seen in Figure 18. The magnets were pouered by a direct current source and released autocnati-al:f causing the modules to open i: the auxiliary air line pressure dropped below 5 psi, or if one of the two flow sensors in a _aannel detected a preset, low-flow :ate of either smoke or ai:. 19 CTR CONTRACTS 029197 11248947 CTR HN 0439,85

*inal 2epo: ' Concrac- -':R-0030 :,AC-?LaT:ZJ a ZQQi2uVr c. =xposure Rack :"he 2xposu:e ;ack -eld all o.' :::e coapor.eacs --a: ~ake ::p t"e ani:al contai r.en: syst::a. :t consisted o: a basa vit^ 3ver_:=a1 supports to viici are at:ached 4 horizontal c:oss suYpor:s of _i::e:ent levels. Zach level or =sanne1 ;eld ::+o 1odu:=s sZr a total of eight. ?igu:e 19 is a v;aw o: a saci vit; ~ods._s -ou..tsd and camponents labeled. ?ig:::e 20 shows a:ack assambled a.^.d loaded with aniaals durfng an act;:al exposu:e. 20 CTR CCNTRRCTS 02919e 11248948 Cr TR HN 043966

Ti.^.al R.po: Cznesac:: ^TR-0030 ?aCI:.:T:_S~r -rGQI?D!:-vT FIGGRES 1-20 21 CTf2 CONTRRCTS 029199 11248949 C TR MN 043967

FIGURE 1. The StM Serlef 11 22 CTR CoNTRACTS 029200 11248950 ~TR HN 043968

in1nI r1a,1u uwI 1un 1 i1CUNf 2. Ihe swjklny mechanlsm Is huused under a se.led plastlc dome. lhe dome Is hinged and Is slKwns here ! 1.. flr uoen uusltlon_ / I

OIINL-UWG 1G-14/a~// N t ftaiOME TER 00 l J P ~ ~ 4 ~ fHjME ~ PItESSUItE .,q C) REGuLATOft 0 ~ ~ M3. ?U II[UMIDIF1Elt ,~ C") f "'i fiLOWER l,' 1 U1 4 0 f U11 N AIR INfAKE t"'o 0 N N U ~ - SMOKE ROfOMETEH U U FIGUNL J. Fluw Jii.ji.un uf llle SEH 11 eMN.Ac Jelivery oystcm. ISTftIDUTION U VALVE L PuNGE Atft ANIMAL CONIAINMENT/ I VENT r-j i ExPOSUHE UNIT A r VEN1 -VENf -VENf

^RNL-:'+vG 73-2CI8R ~vaLV: CRIV_ --- H afT ~ SMCK;. CUTLET N9E F P '.:p C-E aI R INL.: T 7,L'9E 0 ~lOTT: SHOWN 'NIT4 iCP CCV'cR c"IFr FIGU0.E 4. The snoke discribucion valve. 25 cTR coNTRacTS 029203 11248953 G 7"R MN 04-3991

ua/Mtt 'r. S_/ ''l'tu'.1 au'Kr ~w aawa - -:_J ttM I/WM --'----- 1 Oal/hwlww.w.t Y.aWt ur tt 0 t ul~. I -~~ •u.r LNr AYI ttOrYlllr t/u+a~/llt MIVt ' - ~rl _ l _1 fw.r I~ /o.a.Il1. f.xtwuo qtvtatww/l r~t w t tr~ a.. t a.w.t awt _ j--------- . C LY* -,- ~--~- • 1-1-1---u_={- I tt WIti1-.w. .Y //li ANIMAt. EI(i'OSUNt: I/OUM uwwtc ------- ~ {'_`~-~ 4H.rwl. Y I / I 1 . FIGUNE S• Plplny Jiryr:YY of une uf Ihe fuur chuunel exyusure syatcr,a. Unit is sIluwn lu uutwal u//eniny /lwde with StM xmUke/.aIr yuiny to r/udule.

v FICURE 6. A typlcd) awdule fluw sensor ready fur use. t

W ~ 3:) C7 ~ Ln ~ ~=~ N ~1 ~ '10 O 0IfNL-OWG 110-1494211 l COMPENSATING TFiERMiSTOR (Tcl FENWAL GA45M2 AIR FLOW ~ . -- FLOW THERMISTOR (Tf) FENWAL G(332M2 -----' N0.1- COMMON (E]K) NO.2-COMPENSATOR (R) N0.3-FLOW Tt1ERMISfOR (Itl USE WITfi 12/t LONG 3 WIRE CABLE, TC-WriiTE, Tt - REO, COMMON- EiLACK Figure ). A lutdMJy dfJM/Uq uf thc (luw aenaut 114"winq IuteruJl uun,1truuiun. 4 -- -- 2 %l in. --

i I ---- ~~ - fLOW SEN5U11 "In - UIOIfAL INTEGRAT011 ~~ MLTE/I L'UN(ItOllE11 , • •. -P W - '! •• I f- -. FLUW ~; ALAIIM AOJUSf • ry .1 _ / _ 0 ` SONALEfiT I!P)r ~ `~AN AwUST~ ` ~" INT tGf1AT01( l fESET UUTTON ~ ,lAnl SwITCII• ' . ..~ ~~ THII INl)ICATUII~ 1 • ~• ' " 2EH0 ADJUST r"w "'w*Pvrw' 1 - ---- i N b m O/INL PIIUIU-~1611MpA . INIL[iliAlUR' • HOLD SWITCII ~ I ~--- 7~i I t""s+n~~ r-tr1i~11, IIGUIIL 8. Overall view uf amul,e Rwniturlny tonwln. k

Figure 9. One of the t.•o aux11(ary air safety system racks. 30 CTR COHTRRCTS 029208 11248958 CTR MN 043996

PIIOTOTRANSISTOR LIGIiT EMITTING DIODE (LED) / 0 FIGUHE 10. Ihe liylit eiwit(lny JiuJn (LED)-plwtutfaual5cof uolt. ~ C> I ~`'~ 40

FISURE 11. The smcke-iensor consists of the LEO-onotocransistar conbinati*n -outed f1usA alcng ;~le inside .+all of a rigid plastic tu*a OrouSn ~ni:h smoKe ;asses. 32 CTR COHTRRCTS 029210 11248960 CTR HN 043996

0 =.-. . 0.00.4 C~ T-g~ c_ ~.lF r = - - JI . .. t%t ;9 ..d. . ~. ' ~ y -- ...- . -- =!:~2= '2. ?uff-by-puff arofiles and total integrated values of sr+oke Darticuiate r.atter from all 4 channels from the Si:?l 11 8. Ten puffs taKen Interri;;ently using 30 seconds s.-+oke - 30 seconCs air are s`own. 33 C-rR CONTRRCTS 029211 11248961 C TR HN 0439991W

u P NtCK SP/lINO CATC.1/ I" NECK SPHING I/EAU CLIP \ . . IIGUNC 13. Muuac tiry -,IwMlny Supyurl. IIEMUVAULE SCREEN PLATFOIiM uuNl ri w 1 u a,az wUA ~ ""~AUJUSIMLNI ~ IIOLES /IHAY ~SUI'PUI/T AIIM ~~ AUJU:: f MEN f SLU f I .

35 CTR COHTRRCTS 029213 11248963 C TR MN ~''. 44001

SAFETY CIIAIN I 1 I ~ uttNL rINrIu Ibctl WJA l.Al CII I/ULE fUl/ SUI'1'UNT" CLIP STUU fIGU1lE 15. An uPen module esirnjly wilh onc tray IustallnJ. SMOKE PASSAGEWAY (UPPE H) Qfi1NG SEAL

ORNL-UWG tlO-tf997 0 K TYP 1 u 11 1 HEAI] RESTRAINT rItAU rtts lrtAlrv i-1 1 11 11 `.,-NOSE SEAL W v (RUBBER) fIGUHE 16. Thr nuse only eKposure technlyue usioy the abalule anJ mouse trry. L-0 En

i •n0il,3n/lq wn, ~ou/alu~ fiu~r,nqc ajnFwm P jn unll7ac ccn/~ /I 1N~'ll l 71V:1: ON i1VNnnNV:U1x1./ 1/7•111 'M-yi IIVi.I nva nrlNN/ N~nNnM wrM Y7N11•N•M AVQ N1OwnN 7/ VNrMNV aAip,l ~17MI1- ~•'/~ 71v M ?Nn•l l179NW sR119-110 .04•1NN/1 9NNln1 xLLL/3 V7 !N1/1Nlw)1'1 N7.Iilt/ llvM 00,4 ' S 1V 7S !fNl// O M7d11>t IlNnrrt-~~ i 71vMrn1NVlAlOd 117MI1-M•~'~ 71 V Id »InNn - Nlnn/m nnm rvn/l •11e fN NVn N 111nnN l IvNnawvllinA w-mn11= w-w1 71V Id nvn Awvwrn/vlc

i I I w .o

i 1 A O FIGUKE 19. A rack asseml.ly wi th modules in pl.ce. J Ii

41 CTR CONTRRCTS 029219 11248969 CTR MN 044007'

CTR CONTRRCTS 029220 11248970 CTR HN 044006

gi.^.aI Report Con:rac=: C^R-0030 C':R-100 ::I. C^R-100 fI-100) CCCaRCIVOGE4Est5 AND CHRON:C a:JHALd:!.^,`1 C•: 2a1 ^.:~;d.:t7T"'Z S`!0{' In June, 1977, _-e F'_rst large scale cigarette in::alation ;:•:dy aas izi:ia:ed. ':'yis study was designed to assess ~.~=cq•cal .sk of !cw nicori^e-nig`: tar cigarette smoke .n a mcdel ani,al s.~s:em. :he exper:cen: :,ad several objec:ives: o cetermine expected mortality rates associated with _ e use of these facilities and equipment. o Determi.^.e _::e effects of exposure to whole 2a1 ciqa:et:e smoke in 3C3F1/C::m mice for a 12-15 month axpos;:;a ceriod. o Charac_erize the suscepti5ility of 3C32'1/CLm s1d C3H/an= C=n mice to lunq cancer after treatment with '<newi c::e^:ical carcinoqens. o Determi::e the effects of whole 2aI cigarette -.m.oke on 3-met:nylciolanthrene (MCA)-induced carcinomas. :he smoke exposure conditions and the distribution o: experi,ental animals in C'R-100 (1-100) are outlined below: o Reference Ciqarette: 2a1 - 0.5 aq nicotine, 40 mg tar. o Standardized -:xpo- :en puffs per cigarette; sure Ccnditions: 30 sec =oke exposure Zer minute; two sessions oE S cigarettes per day (10 sin rest between saccess i•: e ciqarettes); exposure for 5 days per week. o Smoke Concentration: 10%. 0 :'?"i '.eposi:ion:. Approximately 1 -q day rer mouse. per o Smoke Ex?osure Groups: o MCA and Smoke -sxpo- Smoke Zxposure Groups: 765 3C3°1/C~:.m mice :or smoke (325), sham (220), and she1=-control (220) groups. Additional 2,160 mice assigned to smoke (750), sham (720), and shelf- control (690) groups. 42 CTR COHTRACTS 029221 11248971 CTR NN 044009

*inal 3epor- =ont=ae=• C^R-0030 c:R-L00 0 c!Ca-t:sa=ent and MCA (250 ug) was given Smoke 3xposu:e: iZtratracaeally in 0.02 m1 gelati-s-saline a total ~f 9 times at bi-weeilt inte: vals over an 13 week period. Smoke exposure was initia:ed 3 days a:ter first xCa t:eat.zent. o Chemical carcinogen- Additional 551 C3H/anf C:a treated Groups: mice treated iatra- tracaeally with MCA, 3aP, and 3aP-7,9-diol. A. Evaluation of SrM I:3 Exposure Machine and Its Animal Containment C;ni:s °::e previous section (Section II.) presented a detai::d discussion of the smoke generatinq sys:acn and the ar.i-ai containment units. Some of the studies that led to ::;e _inai configu:ation of these systems were initiated in C':5-130. ?ou: areas were studied in this experiment: thermistors, :`:e :Lov safety system, exposure modules, and animal restraints. Tl:ersistors were used to =onitor the floa of air/smoke at the end of the exposure modules. Studies carried out in C':R-100 showed t::at temperature-compensated tiernistors were needed because as the exposure modules warmed dari-q ^or=a1 working hours, the relationship between ai:/smoie :1ov and a=o::n- of cu::ent :equired to heat the probe to constant temoera:•::e was not Linear. :1us, a pair of thermistors was developed with one eabedded in the polycarbonate module housing block and one prot:sding into the air/smoke channel. Flow was monitored by :::e dif:erence in carrent required to heat the thermistor :n the air czannel conpared to the embedded taeraisto:. The ::se of :ar;e and more visible direct reading gauges with defined "trip coints" marked on the gauges were the final changes. ~ T!:e exposure modules and ani=al holding t:nys were a::ered so as to minimize leakage of air/smoke during axpossre. :: •.as decided that larger diameter "0"-cings were needed :n :::e exposure modules and stainless steel inserts were needed :1 :=e -odule where the noses of the animals pcot:sded. These c::an?es insured that greater than 4 li:ers/min of srooke or si: w ed through each channel in the exposure modules. Six dif:erent animal holding t:ays were eval_ated over a two x,onth exposure period. The holder that was ac:yl:_, ad;ustable, constructed with a teflon neck edge and regular `;o: ;: id was determined to be the best. Additional -ioditica-:.,ns :i 43 CTR CONTRRCTS 429222 11248972 CTR f fN 044010

*inal Report Cone:ace: CTR-0030 CTR-100 the ;.eck edge (i.e. t`e use of polycarbonate 'inse:ts) were also suqqestad, but no decision was made sntil C:'Q-1Jla was on test (see Section IV :or details). ':he :!ov safety systes was preset to allow at least 1.3 L/-Iin of flow at all ::aes. A toxicity study tlnde: CTI .t-1~0 :o:+pa:sd animal survival with flow rates of 1.0, 0.5, 0.25, and 0.17 :./.,a ini Flow was measured with a :otaaete: and pneuxotsc:^oq:aph. Mice died only at air :low :eis than 0.5 L/min for 20 -tin. The "alarz point" was set at 1.0 :or all :utire stsdies. 3. "~etermination of *xpected Mortality Rates with the S:a I:9 Smoke Exposure Machize The animals in all nine groups of CTR-100 and t;e cir:Lmstances of death are presented in Tables I and 2. In ts:s groups of animals not exposed to smoke or sham, q:eate: than 301 of t::e animals ^ad complete nec:opsies and either died i:I tie caq., were sacri:ic.d because moribund, or were sac:i::ced at the terainaticn of the study. For those animals exposed to smoRe (i.e. Groups 7, 9 and 9) and those sham exposed (i.e Groups 4, 5, and 6), a total of 55% and 401 of the animals, respectively, died of exposu:e-related problems. Thus, over the :40 week observation period, approximately 50% of the animals exposed twice per day to either smoke or air (t.eI snam), died o: exposu:e related factors. Approximately one-hal: o: the animals died by :+eek 30 to week 36 for either the smoke or siam exposed groups (see Table 2), with approxizately one-hal0 of c::ese aini-als dying o: exposure :elated Eactocs. The most co.--:on cause of death seemed to be a combination of agitation, head movement and 3ocumented improper air/smoke :lov,t::at :esulted in suffocation of the animals. The circumstances su::oundinq the death of the animals are very important when attempting to anae lze the :esul tant histopathology associated with these ani-als. These analyses will be discussed later. 44 i CTF2 CONTRRCTS 029223 11248973 C TR HN 044011

:'inal Report Con::ac:: CTR-0030 C:'R-.00 C• Effects of Sxposure to 2a1 Cigarette Szoke oa ;sCA-treaced and :'nt:aated 3C3?1/C;:m Mice 1. Analysis of Weights of Mice ':::e mean mouse weights at 4 wee< intervals for Groups 1, 4, and 7 are shown in Figure 21. 3y 100 weeks, the mean weight of uninoculated shelf controls was about 42 graas, (Group 1) the sham exposed about 27 grams (Group 4) and the smoke exposed mice about 23 grams (Group 7). The difference betaeen the untreated ar.d sham exposed groups was apparent between 5-10 weeks on test and this difference is significant p< 0.05. Rate of weight gain in the sham and smoke exposed groups was also different (?.< 0.05). mean weights for .the groups of animals that were treated-with :!Ca are given in Figure 22. These MCa treated groups reflected the effects of smoke and sham exposure, in that the smoke exposed groups weighed the least, followed by the sham exposed group, followed by the shelf control group. Rate of weight gain a3onq all three groups was different at p< 0.05. Analysis of variance was performed to determine the lavel of siqnificance. 2. Analysis of Carboxyhemoglobin Levels To document that smoke exposure resulted in deposition of gas phase constitsents (and by inference, also smoke particulates) in the respiratory tract of mice, the p ercen^ carboxyhemoglobin (COHb) in blood (obtained from the retro-or5i=a1 sinus) of these smoke exposed mice was determined at :onthly intervals (Figures 23 and 24). The percent C.Hb was correlated with the amount of smoke ta which :ie mice were exposed, and during the standard exposure regimen of 5 cigaret:es, averaged between 40 and 50:. (The 5 cigarettes were given twice per day). Thus, it seemed that the amount of smoke ~!eposition was dependent on level of smoke exposure and that the mice did not alter this level of deposition after being exposed on a daily basis for at least 25 months. Treat:ent aith MCA had no effect on the levels of smoke-associated increases in percent --CHb (Figure 24). 3. Analysis of Nicociae Levels in Pooled Urine Samples !licotile is the prizcipal alkaloid present :n cigarette smoke. It is associated with the par:icslate pnase of smoke and can be found with the total particulate :natter (TP-4) 45 CTF2 CCNTRACTS 029224 11248974 CTR MN 044012'

!inal Repor: . Contract: CTZ-0030 C:'R-100 collected on a Cambridge filter pad. Dosimetry studies in ouz laboratory have shown nicotine to be rapidly cleared f::a :he upper respiratory tract and absorbed .ui.»the• blood (see Section VI). It had a half-life of '1.0 hour hnd. was excreted in :he u:ine eit::e: unchanged as nicotine, or as its major netaboli:e, cotinine. If nicoti::e levels in urine could be cor:elated with total dose of cigarette smoke, then a nondestric:ive pracedure would :e available fo: monitoring the level of smoke exposure within _;e mouse. In order to explore this possibility, urine samples f:ca each experiaental group were collected :or analysis. Orine from -20 animals was pooled to yield about 1-2 mi11i1iter of sample. Urine was collected by gently stroki::q ("ticklinq") the mouse's ventral region. It was filtered through a 0.45 W-a 3illipore filter before storage at -70°C. Ur ine samples were removed from the freezer about 2 hours betore anallsis. ;`:e following procedures were used: a. Sample Preparation 1. 3asify 1.0 al sample of urine with 1 ml of SN NaOH. 2. Extract of 5.0 al of diethylet::e: containing an internal standard (fenfuramine, 1.0 mg/vl). 3. Vortex the dilution for 15 seconds, centrifuqe at 1500 rrcn for 10 minutes. 4. ?'ransfer the upper layer, about 4.5 ml, into a conical centrifuge tube, add 100 ul of 24 HC1. 5. Vortex for 15 seconds and evapora=e the ether layer under a stream of nitrogen. 6. 3asify ehe aqueous phase with 300 ul of 54 NaOH; add 50 ul of =et::ylene chloride. 7. Vortex for 15 seconds, cent:if'.:ge at 1500 rra for 10 minutes. 8. Inject 2 ul of the organic phase (lower layer) into the gas chrcmatograph for analysis. 46 CTR CaNTRACTS 029225 11248975 CTR HN 044013

nnal Repo:: Cans:3c:. CTZ-0030 CTZ-1v0 b. Gas C::romatographic Conditions Inst:=ent: Perkin ':mer 39203 ~etec:or: ?lame Ionization Detector Colc:mn: 7% Car:owax 20 a/3t six ring ?P1/2t KOH on 90/100 Cascarame Q; 6 ft x 2.:m I.O.; glass Te:nperature: Injectoc 22S°C Detector 2S0°C Oven: Initial- 1S0°C for 2 minutes Heating :ate- 160°C/min. ?inal - La0°C for 4 minu:es c. Quantitation Samples for a standard curve were pcepared by adding 1.0, 2.0, 3.0, 4.0, and 5.0 ug nicotine (in Lo0 u.l ethanol) to 1 ma1 deionized s+atec and extracting these sa-sples in a manner similar to the urine samples. The standard curve was prepared by plotting peak height ratios of nicotine and intermal standard (fenfuramine) versus nicotine concentrations.. ?eac height ratios of unknown samples to the ilternal standar3 •:ere calculated and the concentration of nicotine in the :n'<-aw: sam?le was determined from the standard curve. Table 3 shows the results obtained for samples collected from ?xperiwent CTQ-L00 during Hay, 1973. :ata :;.dica:ed that nicotine was deteceed in the urine of animals :ol:owi-g exposure to smoke from Low nicotine cigarettes ;2A1). Gar;e day-:o-day variations may be attributed to: 1) poor sensitivity of the detector, and 2) variation in the time be:•aeen smoke exposure and, and 3) urine collection procedure. 4. Analysis of Histopathology 71 summary of the numbers of 9C3F1 mice surviv:-g'at 4 -weei intervals is presented in Table 2. The time :or 501 survival was 90 weeks for shelf controls, -70 weeks !:: 3el-saline treated, -32 weeks for sham exposed, -36 weeRs :3r saoke exposed, and -32 weeks for smoke +:!C!i exposed ani:als. :he single major cause of death (-501) for animals either sha..5 or smo<e exposed vas death on the module during smoke expossre 47 CTR CONTRRCTS 029226 11248970" C TR MN 044014

?inal Aepor • Cont:act. CTZ-0030 CTR-100 (Table L) . For this reason, the histopathology data on t::ese mice have been analyzed by incidence of various lesions observed per given 4 veek per:od of stidy. For :hese analyses, all anizals aere includedd regardless of the reasons for death. The numbers o: histopat::ology samples diagnosed for eaca of the groups of aice ars shown iz Table 4. A discussion of _ ese :esul=s folloas: a. pulmonary Histopathology of 3C3?1 Mice Exposed to 2a1 Cigarett2 Smoke The incidence of pulmonary histopat::olsgy iz smoice exposed ccmpared to sham exposed mice is presented in the first four colu--ins of Table 5. The two sham exposed control groups (Colu:..ns I and 3) were observed to have similar types of lesions. The only lesion observed in the sham-exposed group with any frequency was congestion. Lov levels of pigmented alveolar macrophage accum;:lation (?AMA) (2/400) were observed. The only spontaneous t;-=ors observed were two alveolar adenoca:cincrsas. Therefore, for this observation period, a total spontaneous incidence of 2/400 (0.5%) malignant lung tumors was observed. Smoke exposed animals developed no malignant changes over this exposure period (see Coluns 2 and 4). The most ::equent lesiol observed in the two smoke exposed groups was pigr<ented alveola: macrophage accuaulation (246/579). Two other lesions Vere associated with smoke exposure: squamous netaplasia (Sa) of the trachea and :hinitis of the nose. The incidence of PaHA, SM and rhinitis as a func_ion of :ime of exposure to whole cigarette smoke is presented in Table 6. The observation period for the smoke, sham, and shel: control groups extended up to 140 weeks. 2AMA was first observed a::er 3-16 weeks of suoke exposure and every mouse that died after 32 weeks on test was observed to have ttiis lung :esio^. Syua=ous metaplasia of the trachea was first observed at 64 :+ee'<s a° smoke exposure and for the period of t:3e 64-114 :ree<,s, the total izcidence of this lesion was 14/56 or 271. Ho such lesion was observed in shelf or sham exposed animals. Rhinitis was first obse:ved at 36-44 weeks of smoke exposure. For the :eriod af 35-140 aeeks, the total incidence of this lesion was 15/135 jr 12%: Only one mouse expressed this lesion in the s:.elf or sha-a exposed animals. 48 ' CTR CONTRACTS 029227 11248977 C f R HN 04401E5

?! nal Repor: Cont:act: CTR-0030 CTR-100 b. ?ulmonary )ii stopat::olcgy of Smoke Sxposu:e plis T:eatsent aitz MCA . . •. A si:m.-sa:y of the total incidence of selected pulaonary lesions for the :!Ca-sham exposed group (269 mice) and for t^e MCa-smo:<e-exposed group (406 mice) is given in Co1=ns 5 and 5, :espectively, of Tab1e 5. T:ea_zent with MCa had 1it:le effect on the incidence of smoke induced ?AMa's. Tye total incidence was lower because 451 of the :!Ca-treated ani:als d:ed aith lung cancers before the time when a high level of ?AMA's were expected to occur (e.g. 32 weeks). Greater than 90% of all animals that died after 48 weeks of smoke exposure :+ered cbse:ved to have ?1Me1's (see Table 7). The same types of MCA-i.^.duced pulmona:y lesions were observed in sham or smoke exposed mice (see Table 5). There were slight variations in the incidence and distribution of the '_esions as a function of time of smoke exposure. ;1e dist:ibution of the pulmonary lesions as a fsnction of time of smoke exposure are presented in Tables 3-14 :or alveolar hyperplasia (AH), alveolar noncocnpressing nodules (aNCa), alveolar compressing nodules (aCH), alveola: adenocarcinoma (AAC) , adenosquamous carcinoma (ASC) , squaaous setaplasia (Sri), squamous neoplasm (SN) , and squamous cell carcinoza (SCC), respectively. A description of these lesions is presented in pages 54-55. The observation period for the :!CA-treated animals extended up to 88 weeks. T!:e Hantel-Haenszel statistic was used to determine significant differences between groups. 1) Incidence of AH (Table 8) Total incidence of this lesion over the 33 week :,bservation time was 52/269 (199) and 104/406 (25%) =or the s;= and smoke exposed tiice, respectively. The time at which 501 a: _::e lesions occurred was 29 weeks for sham and 36 :+ee:<s !0r smoke exposed animals. The distribution of _'.^.e incidence of the A)t lesion at 4 week intervals suggested that smoke exposed > sham at pa 0.072. :'yus, smoke exposure had a slight enhancing effect :,n incidence and/or latency of MCA-induced pulmonary AH. 2) Incidence of ANC?t (Table 9) Total incidence of ANC:1 over the 33 :+ee< observation time was 27/269 (10%) and 55/406 (141) for the siaa and smoke exposed mice, respectively. The time at which 50% of :::e :esions occurred was 48 weeks for the sham and 36 weeks °or 49 CTf2 CCNTRRCTS 029226 11248978 CTR HN 04,4016

?inil Repor= Contrac=. --:R-0030 CTR-100 the smoke exposed groups of aice. The distribution of the incidence of t::e aNC:7 lesion at 4 week intervals suggested =;a: smoke exposed > sham at p= 0.37. Thus, smoke exposure ::ad no effect on the incidence and/or Latency of :!Ca-ind;:ced aYCV. 3) Incidence of AC:1 (Table 10) Total incidence of this Lesion over the 33 week observation time was 31/269 (12t) and 65/406 (15i) for the sham ar.d smoke exposed aice, respectively. The time ar which 50% of .::e lesions occsrred was 48 weeks for both the sham and smoie exposed ani3als. The distribution of the incidence of the AC:7 lesion at 4 week intervals suggested that smoke exposed > sham at ~= 0.19. Thus, smoke had only a slight enhancing effect on the iacidence and/or Latency of wCa-induced AC:1. 4) Incidence of XAC (Tab1e 11) Total incidence of this lesion over the 33 week observation period was 46/259 (17%) and 53/406 (13t) for the sham and smoke exposed groups, respectively. The ::me at which 501 of the lesions were observed was 48 aeeks for the sham and 52 weeks for the smoke exposed mice. The distributlon. of tie incidence of XAC at 4 week intervals suggested that sham > smoke at p= 0.015. Thus, smoke exposure depressed the incidence and/or latency of MCA-induced aAC. 5) Incidence of SM (Table 12) SM was the first squamous-tyce lesion of pul.-..ona:y tissue observed following treatzent with c::e:ni:al ca:cincgens. The total incidence of this Lesion 3aring the 33 :+ee< observation period was 92/269 (35%) and 111/406 (27%) for the sham and smoke exposed groups of aniaals, respectively. The t: _e at which 50% of the lesions occurred was 20 weeks for the sham and 24 weeks for smoke exposed animals. The distribution of the incidence of the SM lesion at 4 week intervals s::ggested that s::a3 > s:moke at p= 0.28, therefore, smoke exposure ::ad no effect 3 n _::e incidence of MCA-ind.uced SM. 6) Incidence of SV (Table 13) SN has been defined to be a well contained Lesion c::aracterized by pleonorphic cells, keratin production, ....t :+nere no evidence of growth through a basement membrane ias 50 CTR CONTRRCTS 029229 11248979 CT~' V-11f,1 0440174

?inal Report Contract: CTR-0030 CTS-L00 occurred. Total incidence of' this Lesion for the 38 :ree~c observation period was 28/269 (101) and 48/406 (121) for tl:e s:,a.-t and smoke exposed groups of animals, respectively. The time at which 50% of the lesions occurred was 29 weeks for sham and 32 :+eeks for the sioka exposed groups. The distribution of the incidence of the SN lesion at 4 week intervals suggested tha: smoke > sham at p^ 0.24. Thus, smoke exposure had no effect on the incidence and/or latency of :!CA-induced SN. 7) Incidence of SCC (Table 14) The total tncidence of this mali.;nant lung cancer was 101/259 (381) and 128/406 (32%) :or sham and s3oke exposed animals, respectively. The time at •rhicz 50t of these lesions are obse:ved was 36 weeks for both groups of mice. :he distribution of this malignant tumor at 4 week inte:vals suggested that sham > smoke at p= 0.0024. Thus, ,xposu:e to cigarette smoke suppressed the incidence of latency o; :•.Ca-induced SCC. 8) Analysis of Latency of Lur.g Lesions and Analysis of Combination of Lung Lesions A complete description of t! e time when these Lesions were first observed and the time at which 50V of the treated animals were observed with tlese lesions is given in Tab1e 15. Data were consistent with the hypothesis that =or alveologenic lesions, the prog:ession was likely to be AH --> a:JC:t --> aCN --> AAC and for squamous lesions the p:ogression may be S:S --> SJt --> SCC. Also, the latency of SCC was -::ch shorter than the AAC (-36 weeks vs. -50 weeks) and probably :eflec:ed the rapid growth, retastasis and event::al death caused by SCC. The biological properties of these t•::aors will. be 3iscsssed later. The incidence at 4:+eek inte:vals of all -,alignant :esions observed in sham and smoke exposed mice is presented in Table 16. Analysis of these data showed that when all malignant t:r.sors were included, that the sham controls had higher tumor incidences than the smoke exposed ani:sals (p 0.0012) . The higher incidence of malignant :•:ng =-=ors in the saam controls compared to smoke exposed animals probably :eflected the method by which the animals were taken off _est. Smoke exposed animals had a slightly higher incidence of a.NCY, ACN, or SN coctpared to sham control animals (p•0.10) (Table 51 CTE2 CONTRRCTS 029230 11248980 ~TR HN 04,4018

Final Report Con=rac:. CTR-0030 CTR-100 17) . Thus, for certain lung lesions, (i.e. ANCN, aCN, and Sa) smoke > sham, and for other '_esions (i.e, AAC, SCC, ASC and ?CC) sham > smoke. Analysis of t::e incidence of all lung lesions (see °3ble 13) in mice as a fznc:ion of tixe after MCA t:eatment demonstra:ed that subsequent exposure to 2A1 ciqaret:e saoke did not alter the incidence and/or distribution of t::ese lesions :+ien _-.npared to the sham controls (p0.21) . This lack of difference bet:+een smoke exposed and sham controls was not a function of cczser•iative statistical analysis. Data in 'Table 11 and 14 demonstrated statistically significant differences (p <0.05) between smoke exposed and sham populations where t::e-total incidences of lung tumors were 171 vs. 13% and 331 vs. 311, res:ectively. Thus, if -40 lesions were observed over a given ti-e interval (e.g.-2 years) for the smoke exposed and sham animals, very small differences (less than 10%) could be detected between these groups. D. 9iological Activity of Ct:emical Carcinogen-induced Lung Cancers , One of the controls of the CTR-100 study aas the g;oup receiving only ;tCa. This group was designed with two different protocols: one scheduled protocol included random sac:ilice of animals at predetermined time intervals, and the other scheduled protocol included allowing the animals to die naturally and observing the reasons for the death. These two studies require di~t:erent methods of data analysis. The serial sacrifice schedule was designed to provide data regarding the incidence •o; pacticular lung lesions at specific time points a:ter MCA ::eatWent. The method of data analysis for this protocol :equi;es the assumption that the lesions were probably not the cause of death and incidence analysis using the :Santel-Haenszel test statistic is employed (see Table 19). This is the same analysis as was rer=o:med for the smoke exposed and sham animals discussed in the p:evious section. For the study when the animals were allo:+ed to die, an actuarial method of data analysis is used. A sum=ary of these factors required for calculation of t,=or probabili:ies may be found in Table 20. 1. Response of 3C3F1/Cucs and C3H/Anf Hice Probability analysis Was used for certain aspects o: =TR-100 (3C3F/Cum treated with MCA), CTR-L01 (C3H/an! Cs= treated with ;1CA), and CTR-al (benzo (a) pyrene (3a?) and 7,a-dihyro-7,8-dihydroxybenzo(a)pyrene (3aP-7,8-diol) given to C3H mice). Cetails as to the materials and methods used in these stu,dies are given in References 11 and 12. A comparison of :::e survival cirves for C3H and 3C3P1 mice treated 9 ti.-Ies a: 52 CTR CCNTRRCTS 029231 11248981 CTR I-IN 0-4-40 ISI

rinal Repor: Conc:act: C'TR-0030 CTR-10o biweekly •iztervals with MCA is presented in „guze 23. ':ye :i3e for 501 survival following MCA treat=.ent was ~identical :or boca strains - 33 ~.+eeks. Control C3H mice expressed a hig;~er spontaneous death rate than the 3C3P1 mice (-70t vs. -90t survival) for this 68 week period of observation (?:y;:.e 25). The ?robabilility of an animal dying of a pulmonary tsmor as a °-znction of _i--e after MCA treatment is shown in ?i;ure 26. Tu:aor ?robabili:y was identical for the two strains with a:inal c::7ulative probability of -0.9 for dying of lung cancer by 68 aeeks after tzeatment with 2250 ug :iCA. By 88 wee:cs after MCA *_reatment, the treated animals reached a c~ulative probability of 0.99, whereas the probability of a control animal dying of lung t.:mor was less than 0.006. Qse of this method of analysis in C3H/an: mice treated with either 3 (750 ug), 6 (1500 ug), or 9 (2250 uq) doses of MCA is presented in 8igure 27. The probability of an aniaal dying of a lung tumor at any given time interval is :ela:ed to the dose of LCA. Analysis of these data showed that 9 doses of MCA resulted in a higher :ung tumor probability than animals treated with 3 doses (p=0.078) . By only 29 weeks after cseatcent, the probability of lung cancer was higher (p (0.05) in the ani mals treated 9 times compared with those treated 3 tises. By 33 weeks after treatment, animals treated with MCA 9 times were higher in t=or probability than those animals treated' S times (P <0.05). A comparison of the carcirogenic activity of MCa, BaP, and 3aP-7,8-diol is presented in Figure 23A. Oata vere analtzed over a 72 week observation period post t'^emical treatment. At approximately equal doses, MCA was ;ore carcinogenic than 3ap and 3aP-7,8-diol by 24 weeks post trea;-en: (?<0.05). 3a8-7,8-diol induced a higher probability of death by 1ung cancer compared to 3aP by 60 weeks after chemical treatment (?< 0.05). During this 72 week observation period, th e 3a?-=reated animals developed no higher probability of 1.:nq cancer than the gel-saline treated control animals. By 34 :+eeks, :zo:+ever, the 3aP-treated animals were observed to have -nore Lsng cancer-related deaths than the control animals (data not s'.own). Most (-85i) of the lung t:=ors were AAC :ather than SCC in =`:ese 3aP o: 3aP-7,8-diol-t:eated mice. BC3F1/Cum and C3H/Anf C•= mice that died following MCA treatment had a variety of pulmonary carci::o3as. Over 95% of lunq t::mors observed were SCC, AAC, POC and ASC. Table 21 presents a comparison of the dis:ribution 0! culmonary carcinomas as a function of time post MCA treatment o: 3C3°1/Cum :nice. Three animals that died before the end :0f the ::eataent period were found to have SCC. The :irst SCC was observed at 12 weeks after initiation of MCA treatment, after only five intratracheal MCA instillations. Of the 224 ani=als that died during 14-weeks on test, 190 (95!) were obse:ved :3 have SCC alone or in combination with other malignant r-=ors. Zur:^g the same time interval, only 49 (221) of the animals were 53 .CTf2 CCNTRACT5 029232 11248982 {..I' I y I ! / IN V' -1I I 020 .

F:r:al 2epo r : Contsac:: C:Q-0030 CTR-100 observed to have any evidence of AAC. However, of tl:e 74 ani.rals dying after S0 weeks on test, 67 (91t) developed AAC alone or in ccabination with other t'=ors, and 27•(361) developed SCC. Analysis of the biologic behavior of the :!Ca-ind--ced SCC and AAC in 234 3C3F1/C::m aice showed that of the 172 SCC's 'abse:ved, 90 (521) were extensively invasive and/or ^etas:asized to ot^er Major organs, especially the aear:, kidney, and bror.c:zial Iy:nph .^.odes. The sost p:edominant rou'te of =etastasis was by dfrect invasion of the pulmonary vein with extension to the left atri= of the heart. A total of 62 animals developed AAC alone or with malignancy other than 3CC, while 70 had both SCC and AAC. of ;::e 62 animals with AAC, 29 (471) showed extensive evidence o= invasion and or metastasis to various organs. AAC most often invaded the pleura, zediastinu:a and thoracic wall. The distribution of lung cancers in C3H/Anf C- =i=e as a:unction of dose of :'.CA is shown in Figure 238. The anicals that died of t~arsors early after MCA t:eataent (less ::zaa 40 :+eeks) :+are usually found with SCC (overall mean of 85f for all of these groups, Figure 238). Animals that died of tvmors late after MCA treatment (greater than 50 weeks) were usually found with AaC (overall aean of 931 for all three groups, Figure 298)*. Different doses of MCA failed to alter this response. For all three ;!CA treated groups, the mean expression time for 5CC :+as.33 + 4 weeks, and for AAC it was S7 + 6 weeks. As the dose of MCA was increased, the number of anima-Is dying with evidence of 5CC over the entire observation peciod was 19/72 (261), 20/62 ;321), and 61/104 (591) for the 3x (750 ug) , 6x (1500 ug) , and 9x (2250 ug) MCA treatment groups, respectively. Conversely, as the dose of XC.A increased, the n=ber of animals dying with AAC decreased (46/72 (63t), 37/62 (60%), 55/104 (53%) for 3x, 6x, and 9x, respectively. The postulated progression of c::eaicallj-ind---ced 1::r.g :esior.s is presented in Figure 29. A brief description ~: ::ese lesions is presented below. Alveolar Hvoerolasia (AH) was defined as t:^e ~cc•:::ence of proli erat:on or alveolar inilg cells which were asual:f adjacent to terminal bronchioles in the peripheral portiocs of t:e lung. The normally flattened alveolar liniaq ce11s are low cubiodal with somewhat basophilic cytoplass and :aund hyperc::roaatic nuclei. Mitoses were rare. These rtasses of cells were 1 xm in diameter, often multiple, and not observable grossly. Alveolo enic 4on-Comoressin Vodules (ANCN) were defined as rourd ed massas of ayperp ast:c a veo ogenic cells, app:ozimately 2=m to 3 a.m in diameter, which did compress t::e 54 C.TR CCNTRACTS 029233 11248983 C 7 R NN ~".4,40~' 1

*inal Aeport Contsact: CTR-0030 C:R-100 surrounding parenchys:a. These nodules were barely visible grossly. Mitoses were observed cccasionally. alveolocenic Co3v:essins Vcdules (ACN) were deFined as zorpholog;:ally simtlar :o alveologenic zon-comp.essil sod ules. However, there was compression of the surroundi;.q lanq ?arenc::yma. Nodules located in the most peripheral portions o: :^e 1inq :snded to invade t^e pleura. Alveologenic Adenocarcinomas (AAC) were defined as large :,amors aorpnologically similar to alveologenic :ompcessi,g nodules with usally one-fourth to one-half of a Lung lobe involved. Cells were often cubiodal or colu.nnar, arranged in a papillary or acinar pattern. vuclear PLeosorphism and i,vasion of the pleura was present in some cases. PulWonarv Sauaaous Metaolasia (SM) was defined as :rell-different:ated squamous c• s:rnica appeared to arise f:oa alveolar lining cells distal to the terminal bronchioles. a-=all 3asses of squa3ous cells, usually non-keratinized, often filled one or multiple alveolar l::mena. Sauamous Veoolasms (SN) were defined as well circ~sc:ibed squa3ous lesions wnicn were often keratinized, generally larger than squamous aetaplasia, but there was no invasion of blood vessels or metastasis. Sgua=ous Cell Carcinomas (SCC) were defined as t=ors composed of well-differentiated squamous cells, with characteristic "keratin pearls." Mitotic figures and nuclear hyperchrczatism were often noted. These tumors most ~Dften resulted in vascular invasion of the pulmonary veins and/or =etas:asis to the left atri=. Adenoscuamous Carcinomas (aSC)' were defined as tumors arising in t.~.e teratna airways aitj formation of both acini and keratin pearls. These t•Wnors were thought to be "collision" tumors which possibly represented a mixture'of developing squamous cell carcinoma and alveolar ader.ocarcizema. The serial sacrifice experiment of C':R-100 was designed to provide data reqardinq the incidence of these various :.ing lesions at specific time points after !!CA-treatment. The criteria for the statistical evaluation of incidences of lung lesions are provided ia Table 20. The observed incidences are presented in Table 23 for lung lesions induced in 3C3F1/Cu.3 temale mice after intratracheal inoculation of :SCA, after 9 gelatin-saline inoculations or as shelf controls. A total of 193 mice were sacrificed in 13 intervals over 60 weeks. A large number of lung lesions were observed. The uninocula:ed and gelatin-saline control -nice demonstrated almost no pathologic alterations in lung tissue. In the MCA t:eated groups, after 24 55 C•TR COHTRaCTS 029234 11248984 i.rr TR Hf'i 0440.t- 2-

'inal 2epos: Contrac:: ..TR-0030 CTR-LO0 aeeks, the incidence of noraal lungs was zero. As the time after tsea=ent increased, the number of mice available for sacrifice decreased :+ith. the result that by 36 weeks, :ess than 501 of the mica scheduled :or sacri:ice were alive. The tumor incidences at these :iWes indicated t;at 441 of the mice sacrificed were diagnosed to ^ave (SCC) and 111 had AAC. Thus, it seems likely that many of ;;e animals scheduled to be taken off test during :;ese :imes were dyir.g of lung cancer and thus, vere unavailable for _his incidence study. Based on these observations, the serial sacrifice data have been evaluated in teras of the progression of lesions only up to 40 weeks, since the incidences of lesions observed In the remaining available animals :ay not be representative of the incidences in the total populaton. Data from Table 23 are summarized in Table 24 for alveologenic lesions. AH was the first lesion obser•ted (L2 wee<s) and the incidence persisted up to 40 weeks. A:1CY fi:st appeared at 12 weeks along with aCN, however, the latter was cbserved at a much lower incidence. alveologenic ca:cinomas aeoeared first at 24 weeks. Yon-alveolar lesions which inclu-de SCC. S`7, etc. occurred throughout the study. Data from Table 23 are plotted as percent incidance for each lesion in ?iqure 30. These data indicated that the aH lesion remained at a level of 40-60% incidence, that ANCa occurred before aC4 and increased to 40% incidence, that aCN followed A:JC:1 and increased rathe'r rapidly to 70% incidence. AAC was the last lesion to appear and gradually increased in incidence to about 351. T•he non-alveologenic lesions remained between 10 and 30% incidence. These incidence data seemed to support the morphologic observation of the progressiveness of the alv.ologenic lesions. A similar analysis of the distribution of squa:nous lesions :s presented in Table 25. SM was one of the earliest lesions observed and reached the highest incidence at 16 veeks, 2:reeks prior to the last MCA inoculation. The incidence then decreased to zero at 40 weeks. SN and SCC seemed to occur together with Lower incidence of SN. These data did not suggest a progressive behavior for the squamous :esions. SCC had the zig::esr incidence of any carcinoma during this 40 week period and =`:e inctdence appeared to be declining at 40 weeks. :n cont:ast. :'able 21 showed that AAC was increasing at this time. These data would support the concept that SCC grew rapidly and appeared ear:y, while AAC were slower growing and did not appear until after the incidence of SCC was declining. The numbers of lung lesions per animal as a function of sacrifice ti9e are presented in Table 26. Statistical analysis of these data showed that there was a significant (p< 0.05) time factor in the distribution of these pulmonary lesions- Z: seemed that as the time after t:eatment increased, the nusber -if animals with normal pulmonary histology/pathology decreased. that the n,=ber of animals with 1 or 2 lesions per lung generally 56 CTR CONTRRCTS 029235 11248985 C~' ~'' PI~~ ~" 4~~ 02 .~

?iaal Repor: Can:ract. :':Z-0030 CTR-100 increased until 27-28 weeks on test and then decreased, and the number of animals with 3 or 4 lesions per lunq gener3111 increased aita t:rte. Thus, it seemed that not only was t::ere a progression of :acyologic caar.ge .+ithin the lu:.gs of these iice, but also a nu=ber of :aese lesions were simultaneously proqressing within the lungs of the same animals. Over 65 unique types of leaions and combinations of lesions were found ia the 193 mice evaluated (Tab1e 27). It is readily apparent that the lung of the inbred mouse is capable of developing many aetaplastic and neoplastic changes in response to carcinogen treatment. 2. Transplantation Sxperiment A major effort in evaluating the so:pnologic progression of lesions (see Figure 29) was the verification of the classification of lesions by transplantation. The sources of the tissue were the visible lung lesions at autopsy of t^e -%ice scheduled :or serial sacrifice. Procedures for selec:i^g and transplanting tissue were as follows. If selected for passage, the pulmonary tissue was excised and transferred to a retri dish on ice. T5e lung masses were described and docu3ented photographically. Discrete masses greater than 3 x.9 were chosen for passage. One half of the sass was removed leaving •the remainder intact in the Lobe for microscopic histopataological diagnosis. The mass was then minced with sterile scissors. Dependinq on the size of the mass, one to two 3illi'_iters of Hanks' 3alanced Salt Solution (HBSS) with 40% fetal bovine ser a were added after mincing. Clumps of cells were broken ::p by slowly passing the material through various gauge needles until it :+ould pass through a 23 gauge needle, Yewbor::s were i:%oculated intrascapularly with 0.1 nl/aouse. ':ice were cbse:ved for six months after inoculation of tumor :issue before being considered negative. If tuaors develcped. :=e :::mors were removed and divided to provide samples for subseqc:ent passage and histology. Tumor tissue was minced and :aocs:ated into other newborn animals, as described above. A su-ary of the data obtained and the n~-:ber o: passages are in Table 28. A total of 84 tumors ::om 68 nice was transplanted into over 1500 newborn 9C3'1/Cua mice. Three :esions transplanted: AC:J, AAC, and SCC. These three appeared to transplant at the first passage (P1) in only about 50% of the cases. For example, of the 20 SCC transplanted, only 10 :or=ed t::=ors at P1, 9 of t::ese 10 grew to P2, 6 of these grew :o P3, and 4 of these grew to P4. AC:1 was successfully t:ansplanted sugqesting that this morphologic lesion contained within it "e capacity to progress to frank carcinoma. 57 CTR CaNTRACTS 429236 11248986 C TR MN 044~",.~'4

riaal Repor_ concrnc:: c:R-o0a0 c:R-10o a sa:nple =•.:.3or genealogy is presented for an SC-- in ^able 29. Of the 5 aice into whica the original _s=or was passed, only qne developed an SCC (vuriber 0095 -?1) . °hat t•r,;.or was passed ^:o 7=ice in P2, 4 of which developed (n,.=bers 01i1, 0142, 0144, 0:45 -?2) . =aca of these t=ors was :':en passed :n:o addi=ional newborn ani=a2s. With t::e ezception c.^.:=e mice which develoced aiveologenic -_.-~ors and one va h develoced a poorly 31!:erenri3:ed c3rcinoria, al?, of =~ese _:3ors :+hica s::bseq::en:ll developed af=e: passage ~:ere diagrosed as SCC. 58 ' CTR CONTRACTS 029237 11248987 C T R N N 0 4 4 02. =5'

Fiaa1 Repor: CoAt:aC:. C:'5-0030 C;~-100 +.. TaBLZS L-29 59 CTR CONTRACTS 029238 11248988 6..r TR l ) ) ) 044026

rin.l Rcyuct Concr.ct: CIN-UUJO ItOU OuruSLJ1UN UC ANIMAIS M U M• L A 0 E A N 1 M A E S E.ruwNL--EkAU~ T[Sr Or- GNWf EtrObuNL INCAIMENI INIIIAC / Of NICF ACC.UINIAL UEAIMS REIAIEO OEA1M1 IN CAl! MUkIbUW! KIllCO COU A60UAilUM (ICUMIMAI) SACNIEICC NO M(CUtN'St UI NwK Nune 220 ] - 2) S1 12 )5 (U) 41 02 Nune Gel-S.11n. 220 111 - )1 32 2 104 (2) li 0) Nune MCA 690 ) - 1/S 210 0 255 (0) /l u/ Sw Nwie 2lU 02 91 22 l0 0 U ([) 17 OS SA.. Gel-S.Ilne 220 OU 111 16 ) 3 O (4) 10 116 SA.r MCA 200 61 122 48 3B 0 0 (0) 11 0/ 2A1 Srul.e Mwse JLS . S) W, 2S Itl 30 U (1) 12 0tl 2A1 SwAe Gcl-S.11ne 310 10 11.1. 21 17 0 U (]) 14 09 2111 Swu/,e I1LA 430 91 21L• 62 33 U 0 (0) 24

SZOVP~',~ N14 NIO 0668bZ11 OtZ6Z0 61ObN1N0O d1z) 19 a ^ N N N__ C C ~y r QQ r ~. e y. V a. a V w N N N r_ oS Naa e s Naa $ V rV CS~~•NQ.~oPNa a o0'Naaoe Ns ^= r ~p ti _N N N ~ MO~.~o000~NNwa+Y V~$OODC ¢raiOr55 ~ .,,~. a..PVOOV.ep ep r-- NNw_==ieam ~~~ 0~ 00_vw P- V~C V awwaI PVC N N w a V V P O. O P e Nw H~ 0~ P e~ C .o G a v Q ~ o~rw+PVeVOwo~.s$:~woae~CPNO r NNN~r y•y.y~ POvv00 rr~0 rC .._ N ~ ^ -NwMONa vNwva p PD e P.00•.r wr• V V•.~C CwvC N~ ~ C NNNNV w w.aV OP\V~ `rC ~'•/N.r Vp .O.': O wwP V~C+PONNPC w~ 10 wv10v•... Pw _ V Pw~f ~.aPr C ~NNNaN~ V D~wP.O V V-wP_ V warNV ~O C N N N N. r rNNVaPO.L C.•~ V V...e ..' N ~ NNw~'a a~.~OP\O.OVDawa Pa O wP PO~O_ ~O V_ ~O~ V NNNw • ~OO_ wP ~ N~~~ ~' _ wa P Pr C OO COOOOV.r NPNSO+ Va V~.GN ~P ~O ~.+ C ~ Orrrrrlra VO~O_ 1.0NO Nrw1i Pa PwC P~

=tra1 Reoort C:.ntrac:: C-MR-C030 TA3LE 3 VICOTIYE L:'/:LS I•'1 'J2IV: :OLL:CTC] r:A-CH3C3F1 "IC'c EX77Sc7 TO 2A1 C:OZZE-II: S:tOCE.a ~~icotine ) ^ate of C:llec:ion ,e.e Smoke • ^e sali^e :.^cca • 4CA S/1/73 -c 1.0 1.5 5/3/73 -c 1.5 2.2 5/5/78 1.7 2.8 4.7 5/3/73 2.0 1.9 2.7 5/9/73 1.4 2.7 3.2 5/10/78 3.2 2.4 3.5 5/11/78 4.4 1.7 3.2. 5/15/73 3.8 2.7 5.3 5/16/7P 2.1 2.5 2.7 5/17/73 2.5 2.3 3.3 5/18/79 2.6 2.3 3.0 5/22/73 2.2 3.5 17.0 5/24/73 3.0 2.6 1.8 5/25/73 2.7 3.3 • 4.2 5/30/73 1.2 1.4 1.5 'sUrine samples from 20 mice per group were collected at 1-3 hours ;cst s-oke expos:,re. Smoke exoosure conditions were as descri:ed far C7 i-1C0 s•udy (see ::xt). ,fice had been exoosed to 2A1 sr..ake for •.11 ronths ce~:re ~ ::!ese irine samples were collected. DVicotine levels in sham control mice +vould not 5e detected. cNo samples collected. 62 CTR CONTRRCTS 029241 11248991 C T R N N 0 4' 4' 0 022 9

1111+1 Mrlw11 TAIII1 4 Cunltat.t: CIN UUJU NtttM[NS O[ 111SIOPAlIC1lOGY SAf9'llS OIA(:NUSIu Gruuy / 01~ 02'- -U1 --U/ ---0:~ - IM. - 01 W CLe.llal IWne L•Sa MiAL None G-S ICA Noao G-S [. usure Mune IWne Mune Sha0 Shar Shar S.ulcc S.ole _~---'----------'-'-----° -- 09 MU S.++le Intrtgal Mcrls 4 0 2 l 20 1tl 20 2/ 24 11 B 0 0 1 19 12 12 16 30 241 12 10 le 14 V )z 11 32 1] l) 16 10 10 l1 ll Il 20 14 iG 22 20 •] 0 S 1 7 22 16 11 20 21 20 Z0 1/7 4 6 23 12 20 47 28 11 9 60 1 1 1tl 12 16 21 32 10 9 71 1 11 2/ 9 12 46 36 0 3 AS 12 9 25 14 21 42 40 1 4 67 2 4 11 13 19 27 44 0 4 42 0 4 12 1 11 241 48 1 6 46 4 1 16 11 0 16 62 1 1 lA e 4 10 11 21 1? $6 0 6 21 2 4 6 5 6 6 60 0 6 22 3 0 1 le Is 12 64 / 0 6 1 e 7 21 14 12 66 1 1 3 1 12 2 11 9 9 12 S / N 1 1 I 9 I 1 )6 1 0 O 9 2 4 1 4 3 160 0 0 2 6 2 0 3 6 2 84 6 4 1 4 3 1 7 4 0 utl S 1 0 tl 1 2 0 O 0 92 6 2 0 ] ~ 0 I . 0 0 96 17 6 1 1 4 0 1 0 O 100 5 /1 . • 0 1 1 0 O 104 6 10 0 2 0 0 0 1 100 10 / S 4 0 1 0 • 112 S 1 2 S 0 0 0 116 9 9 1 0 0 0 0 170 6 3 3 0 I 1 3 124 6 1 2 1 • 0 0 12t1 3 1 1 2 1 2 I12 3 1 0 0 0 0 IlL 4 4 1 3 1 2 1/0 !- -1 - 1 -- ~ ~- - 101A1 167 1/9 64) 201 110 Z69 ltll Jul 106 . U 4.11! yrl.tln-sallnc sulullun, 1.1. 9. 0.02 a1 pet Iowaulallun L. 7•.O ~f HCA'In 0.21 9tlatl.•s.lInt tulutlun; 1•1. 9 0.02 .1 pr. Iau ulatluo c. ?AI C1y..cttc s.u1a; 10 c19arettc/Jry J. In1.r.aI n..d,cr I.dlulas tl.o un tes( In !.ma /lae yerlu0.; I.c. 4 teyccsenls rr.ls 0 1; tl; 5-ll atc. 1udlt.les y.ouy uff lcst

F1uJI Rcpurt Cuntract: CIR-UOJU SUtN1A1tY 01' SEI ECTEO PUI.MONARY iIISTOPATIIOLOGY FROM 8C3F1/CUN M1CE TNEATLU WITH MCA AND/OR CIIRONIC EXPOSUItE TU 2AI CIGARET7E SMOKEa a JA 0 J-4 0 i N~ W 5-4 o. P CheWlcal: Ulaynosls Exposure: no visible lesions ply.alv.oac.accum. pneumonia 9ronchlolltis congestion alveolar.hyper. alv.non.comp.nodule alv.comp.nodule adenocarc/nooa adenosquamous carcinoma squaawus metaplasla squamous neoplasa squamous cell carcinoma TAULE 5 None Shaa Yune Smoke Gel-saline Sha Gel-saline Smoke !1CA1i Sham MCA Smoke 113/205 82/2b1 115/195 97/293 27/269 35/4Uu 1/205 167/281 1/195 179/290 0/269 112/406 - 6/281 1/195 10/298 1/'lb9 - - - - 1/298 18/:69 21/406 70/205 34/281 65/195 24/298 9/269 20/406 - 4/281 - - 52/269 100/406 1/205 3/281 1/195 1/298 26/269 54/406 1/205 1/281 - 1/293 31/269 65(406 1/205 - 1/195 - 47/269 53/406 - - - - 1/269 4/406 - 2/281 1/195 2/298 92/269 112/406 - - - - 28/269 48/406 - - - - 101/269 127/406 aMlce were treated with MCA 9 tlwes biweekly at 250 ug/0.02 ml gelatln-sal/ne, or yelatln-saltne alone. Conditions for smoke exposure consisted of 10% smoke, 30 seconds smoke alternattng with 30 seconds air tur a period of 10 minutes. Ten such exposures were given each day In two sessions. Shao exposure was the same but without smoke. All necropsied mice are Included tn this suaruery. bAnilQJls with /YetJStJtIC twuors, IyiuphusarcumJS, sarcuwas FS nut hcre.

Final Reoor: Contract: C;R-C030 TABLE 5 INCICE`/CE !.F RE3?I;^'TCRY '2r1CT LESIOYS i'i 3C3F1/C,:4 HIC: #S A FJNCTI':N CF T:4E ..FT;Z =X?CSCiE T J 2A1 C::v:r~-i i: Su0!CL3 •4eeks On Test Lung Pa"A Trachea Sy 4ose Rhlnitis 4 0/27 0/27 0/27 3 1/16 0/16 0/15 12 0/32 0/32 J/32 16 . 1/14 0/14 J/14 20 6/16 0/16 0/15 24 2/12 0/12 0/12 23 7/12 0/12 0/12 32 9/9 0/9 0/9 36 14/14 0/14 1/14 .0 13/13 0/13 0/13 44 7/7 0/7 1/7 •i8 13/13 0/13 0/13 :2 13/13 0/13 1/13 56 5/5 0/5 0/5 so 18/18 0/18 0/18 64 21/21 6/21 5/21 68 10/10 5/10 3/10 72 9/9 2/9 2/9 76 3/3 0/3 2/3 ao 3/3 0/3 0/3 84 7/7 1/7 0/7 88 0/0 0/0 0/0 92 1/1 0/1 0/1 96 1/1 0/1 1/1 1C, 0 1/1 0/1 0/1 1C3 0/0 0/0 0/0 0/1 0/1 0/1 112 0/0 0/0 0/0 '15 0/0 0/0 0/0 i20 1/1 0/1 0/1 124 0/0 0/0 0/0 '23 0/0 0/0 0/0 :32 0/0 0/0 0/0 136 0/0 0/0 0/0 140 0/1 0/1 0/1 166/230 14/29Q 1. 6/2?0 Incidence is defined as the number of -nice with a scecific lesion divided by the total number of rice which died and were necroosied durir•g eacm time ericd. Lesions included are: ;ignented alveolar -acro:hage ac:::cule- tion, squa-cus .r.etaolasia of the trac^ea, and rhinitis of the nase. 65 CTR CONT9ACTS 029244 11248994 . CT~.'' HN 044,032

Final 2eoort Contract: M-0030 TABLE 7 I?iCICEVCE OF ?I.uE`1T_0 ALVEOLAR !"ACRCPyAuc ACCU4ULAiIOY (?;,YA) I4 'iIC"c AS A FL"1CTiCN OF TIME AFTER MCA T<cAT~E4T A.-N0 EX?OS.nE T 0 2A1 CIu:+RETi c SMOK: AND SMy EX?OSI:RE 3-3 aee(s -.n T est' MCA + Sham 4CA + :,-oke 4 0/20 0/31 a 0/12 0/23 12 0/31 2/17 16 0/20 5/22 20 0/22 2/20 24 0/23 a/47 23 0/18 2/27 32 0/24 8/;6 36 0/2S 3/41 40 0/13 3/27 44 0/12 9/24 48 0/16 11/15 52 0/10 3/12 56 0/6 6/6 50 0/3 12112 54 0/2 11/12 58 0/2 9/9 72 0/1 3/3 75 0/4 3/3 30 0/0 2/2 34 0/1 0/0 ?3 0/2 Totals 0/269 113/406 alncidence is defined as the nuaCer of mice with 7A!t4 divic!ed by t~e tota1 number of aice +hich died durinq eicn time period and were necropsied. b'iCA trearnent and s-+oke exposure as :escribed in Table 5. eeks a fter fi rs t.:CA treatment. 56 CTR CONTRRCTS 029245 11248995 CTR HN 044+~". 33

Final ;e^.or: Contract: CTR-0030 TABLc 8 I?iCIOE'lCE OF ?Ul?iCNARY ALVEOLAR HYPERPUSIA (AH) Itl 3C3F1/CU!1 MICE XS A FUNCTICN OF T;ME aF'iER MCA TR?ATuEYT AYO EX?OSURE TO 2.a1 CIGiRE7, E_ SMOKE At10 SHA4 :X?OSURE.a'b ";eeis :n -iSt' yCA • SFamd 4CA + Smoke d ; 0/20 0/31 3 1/12 5/28 12 13/31 3/17 16 2/20 3/22 20 1/22 1/20 24 5/23 8/47 29 4/18 5/27 32 1/24 15/46 36 3/25 14/41 40 1/13 3/27 44 3/12 9/24 43 9/16 9/16 52 2/10 5/12 56 4/6 5/6 50 1/3 6/12 54 0/2 5/12 58 0/2 5/9 72 1/1 0/3 76 0/4 0/3 30 0/0 2/2 34 1/1 0/0 39 0/2 Totals 52/269 1C4/4C6 3:ncidence is defined as the number of mice with AM divided by the !Jtal ^.~~Cer of mice which died during each time period and were necropsied. b''Ca treaLment and smoke exposure as described in Table 5. `teeks after first `iCA treit.-nent. 'Inclder.ce of AM In both groups .as determined using t`e Mantel-4aenszel s:a;isti:; snoke *>snan, 2 • 0.072. 67 CTR coNTE2acTS 029246 11248996 CTR ' 11N 04.4.034,

Final Reaort Contract: CTZ-C030 TA8LE 9 INCICE'1CE OF AL'/EOLOGEYIC 4CN-CCMP3:S5ING 400ULES (.SVCV) iN 3C3F1/CCy NlCE AS A FuNCTI04 OF TiuE AFTER NCa TR_ATZ!E'(T XVD :XPOSURE TO 2A1 CIi.tRETT_ SuCKE A.i0 SriAH ::(?CSJR'c.l'b ~ ,?eis :n T•s•' uCA * Swan ~ '~ ~ A ~A + SroF~ ~ 0/20 0/31. 8 0/12 0/28 12 2/31 1/17 16 2/20 0/22 20 0/22 1/20 24 1/24 2/41 23 0/13 2/27 32 1/24 7/46 36 2/2S 6/41 40 2/13 3/27 44 1/12 5/24 48 3/16 3/16 52 4/10 2/12 56 4/6 2/6 50 1/3 3/12 54 0/2 6/12 53 1/2 i/9 72 0/1 1/3 76 0/1 2/3 30 0/0 1/2 34 1/1 0/0 33 1/2 0/0 Totals 27/259 55/406• tlncidenca Is defined as the number of mice with ANCN divided by the total n.-oer of .nice which died during each tfire period and were necropsied. ~`!CA treata+ent ancd snoke exposure as described In Tabte S. "4eeFs after first HCA treat.r.ent. 3incidence of ANCM for both groups •.as deternined using the "antel-Naens:al ;:a;4s:'=; sroKe > shnm, p • 0.37. 58 ' CTR CQMTf2RCT5 029247 11248997 CTR NN 0440ti~'Z55

Final Repor- Contrac::. C?R-0030 TABLE 10 INCIOE.'{C'c OF ALY:OLCucVIC C~'MMSSiVG yCCUL'cS (A-C4) IN 3C3F1/C-M MICc AS A FL'NCT:CV OF T:!!2 •ar t=R `!CA .iREAT,`•!5'VT ANO cz?OSuR: 70 2A1 C:;v.R.Ti: 3i".CICr .a4D SPAM EX?OSURE a.b de•is •n Tsst` uCA + S~am uC A + SToke S 0/20 0/31 8 0/12 1/28 12 0/31 0/17 16 0/20 0/22 20 0/22 0/20 24 1/23 3/47 23 1/18 2/27 32 1/24 3/46 35 3/2S 6/41 40 2/13 5/27 44 4/12 7/24 48 5/16 4/16 52 5/10 6/12 56 0/5 4/6 50 0/3 8/12 54 1/2 5/12 58 0/2 4/9 72 0/2 2/3 76 4/4 3/] 30 0/0 1/2 34 1/1 0/0 38 0/2 0/0 Totals 3I/259 55/4C6 ncidence is defined as the nunber of rice with AC`1 divided by the .ota1 n,c,ber o-f nice which died during each time pertod and were necropsied. '`!CA trea Cent and smake exposure as described in Table 5, `~eeks after first MCA tresc,.ent. I Inciz:nce of AC4 for both groups aas determined using the "antel--aenszel statis:':: smoke) shaa, p a 0.19. 69 CTR CaHTRRCTS 029248 11248998 CTR NN 044,03"y.'r

Ptnai Reoorc TABIe :1 Contract: CTR-0030 INCI:EHCE OF PUL'!CNARY AI,'/EOLOGE'IIC a0E40CAxCI'iCF'A (AAC) IV 3C3F1/CU4 MICE AS A Fr'4CTICV OF TI!:E AFT'e= "Ca TREAT!!E.4T'UJ EXPOSURE TO 2A1 Ci;.1RETT'c S!!CxE ANo SHAM ExPOSUZE.3'b ae!ks on 'estc __`!Ca + Snam d "CA + Snoked 4 0/20 0/31 8 0/12 0/28 12 0/31 0/17 16 0/20 0/22 20 0/22 0/20 24 0/23 2/47 28 0/18 1/27 32 2/24 4/46 36 4/2S 0/41 40 2/13 3/27 44 7/12 2/24 48 8/16 8/16 52 8/10 6/12 56 3/6 4/6 60 2/3 4/12 64 2/2 8/12 E.d 2/2 2/9 12 1/1 3/3 76 3/4 3/3 30 0/0 2/2 34 0/1 0/0 38 1/2 0/0 Totals 46/259 53/4C5 j;ncidence is defined as the*number of rice with AAC divided by the total numoer -of mit• which died during each tire period and were necroasied. byCA treat.rent and smoke exposure as described 1n Table S. c~eeks after first MCA treat.r,ent. d:nctdence of 4rSC .`or both groups was determined using tye "ant:1-~IaensZel s:a:istic; Oam>s-+oke, p a 0.015. * 70 cTR coHTRacTS 029249 11248999 CTR VIN 044w403-7

Final Recort Consrac:: C7R-0030 TABIE 12 INCIDE4CE OF PULMOHARY SOUANOUS VEOPLASH (SN1 IN SC3Fl/Ct;,M MICE AS A FU`ICTIOH OF TIME AFTER yCA TR:ATME'iT APIO EXPOSURE TO 2A1 CIGARETTE SMOKE AND SHAH EXPOSURE.3'b '.'a•<s on T astc yCA + Sham d '-ICA + Snoke I 4 0/20 0/31 8 0/12 '6/28 12 2/31 0/17 16 2/20 1/22 20 2/22 3/20 24 4/23 8/47 28 2/18 5/27 32 3/24 10/46 36 6/25 6/41 40 1/13 4/27 44 2/12 2/24 48 1/16 3/16 52 1/10 2/12 56 1/6 0/6 60 0/3 1/12 54 0/2 0/12 58 0/2 0/9 72 0/1 1/3 75 1/4 1 /3 30 0/2 0/2 34 0/1 0/0 38 0/2 0/0 Totals 28/269 48/406 itncidence is defined as the number of rice with S`1 divided by the total nunoer ef --nice which died during each time period and were necropsied. y'!G1 treatment and smoke exposure as described in Table S. cWeeks after first treat.rent with 4CA. d'ncidence of SN for both groups was determined using the Mantel-Raenszel statistic; sr+oke > sham, p, • 0.24. 71 C.TR CCNTRRCTS 029250 11249000 CTR NN 4~',.,4403-B

Final qe^.ort Contrac:: C"R-0030 'aBLE 13 IHCICE`ICE OF PULMOHARY SCUAMOUS CELL CARCIVC"A (SCC) IV 3C3F11C:;!i yICE AS A F,iVCTICH OF TI!'.E AFT;Z !ICA T3:.iT4:YT AV0 E:(POSURE 70 2A1 CIiARE7Tc S!!OKE CR SH.L4 ea?OSEO.a'y Weaks in 7estc "CA + Sham d '4rA * Sroked 4 0/20 0/31, a 0/12 0/28 12 0/31 0/17 16 1/20 1/22 20 3/22 4/20 24 8/23 11/47 28 10/18 17/27 32 20/24 20/46 36 17/25 21/41 40 11/13 16/27 44 9/12 15/24 48 12/16 7/16 52 6/10 5/12 56 3/6 3/6 60 1/3 3/12 54 0/2 2/12 5!, 1 /2 1 /9 72 0/1 0/3 75 0/4 0/3 30 0/0 0/2 34 0/1 0/0 38 0/2 0/0 7otais 101/259 12E/406 ilncidence is defined as the nc,.:.ber of rice with SCC divided Sy the total nurter -of mice which died during each :1.-e period and were necropsied. b`!CA treatient and smoke exposure as described In Table S. c'.1eeks after first :reatment with yCA. d:nciCence of SCC for both ;roups was :eter:eined using the "antel-~Jaenszei s:atis:t;; shar) smoke, p a 0.c024. 72 CTR CCHTRRCTS 029251 11249001 CI I S I / I / 0440,239

Final Report Contract: CTR-0030 TABLE 14 I!ICIDENC'c 7F ?UL'"CNARY SVt;rL`10U5 `!ETA?USia (SM) IV 3C3F1/C;:M MICE AS A P:4CTICN OF TI1!E AF'i:R MCA TRE,1T`!E4T ANO E:(?OSUR: TO 2R1 C:.i.+AcT• _ SMOICc :+VO Sri:.M :x?OSJRE 3'b :ieeks :n est 'aCA + Shamd '!CA ~ :Toked 4 4/20 3/31 3 3/22 5/2C 12 19/31 3/17 16 14/20 6/22 20 18/22 14/20 24 12/23 30/47 28 5/18 10/27 32 7/24 12/46 35 4/25 5/41 40 1/13 5/27 44 2/12 3/24 48 1/16 2/16 52 1/10 4/12 56 0/6 1/6 50 0/3 2/12 64 0/2 1/12 68 0/2 2/9 72 0/2 0/3 76 1/4 1/3 30 0/0 1/2 34 0/1 0/0 38 0/2 0/0 Totals 93/259 123/4C6 }Incidence is defined as the number of mice with SH divided by the total~^:r..oer of mice which died during each time period and were necropsted. bHCA treatnent and smake exposure as described in Tab1e• S• c'deeks after first treat.'nent with MCA. dlnciCence of Stl for both groups was determined using the yantel-Haens:el stittstic; sham *.-smoke, p • 0.28. 73 .CTR CONTRRCTS 029252 11249002 C T!-. i" )N 044040

t inal Itepurt Contract: Cllt-UU3U TABLE 15 CUMPARISON UF IIME UF OCCURRENCE OF PULMONARY LESIONS IN BC3F1/Ct1M MICE AFTER 1REATMENT WITII 3-METIIYLCIIOLANTIIRENE AND EXPOSURE TO 2A1 CIGARETTE Sf10KE OR SIIAM EXPOSED. Time first observed (weeks) PAMAa Sfta SNa SCCa AIIa ARCNa ACNa AACa St14,b -d 4 11 18 8 9 21 31 • Smokec 10 4 14 15 6 9 6 21 14 A Time at which 501 Incidence occurred (weeks) 0 Sham `I 20 28 36 28 48 48 48 Smoke 48 24 32 36 36 36 48 52 aPlymented alveolar macrophage accunulatlon, squamous carclnoma, alveolar hyperplasla. b A total of 269 anlmals were necrupsled. cA total of 401 anlmals were necropsled. metaplasla, squamous neoplasm, squamous cell JNot observed in this yroup.

Ftnai Aeoor! Contract: CTR-0030 TAgL: 15 I4CI0E:YCE OF PUL'4CMARY Tr'MORS IY 3C3F1/C:,'`i MICE AS A r'JNCTiv"~V OF TIME AFTER MCA TREAi'"E4T :.VO ".XP:SUR"c TO 2,11 CICv:R?i: SMOKE CR Si+1„4 cX?OSEJ ao Weeks on Testc MCA + Shamd MCA r Srroked 4 0/20 0/31 g 0/12 0/28 12 0/31 0/12 16 0/20 3/22 20 3/22 4/20 24 8/23 13/47 28 10/18 13/27 32 20/24 23/46 36 18/25 21/41 40 11/13 18/27 44 12/12 15/24 s8 15/16 11/16 52 9/10 8/12 56 5/6 6/6 60 2/3 7/12 64 2/2 11/12 68 2/2 2/9 72 1/1 3/3 76 4/4 3/3 80 0/0 2/2 84 0/1 0/0 aa 1/2 0/0 Totals 125/25? 169/4C6 a 5 _c d Incidence is defined as the n=,ber of mice with a lung tunor divided Dy the nu;nber of the total number of mice which died during each ti-e period and •mere necropsied. Lung t:mors Included .rere: SCC, :,AC. ASC, :'aC and poorly differentiated carcinonas. WCA was given 9 ti,,es biweekly at 250 ug/0/02 ml gel-saline. Condi.ions for smoke exposure consisted of 1C% smoke, 30 seconds smoke alternating with 30 seconds air for a period of 10 minutes. Ten such ex;,osures 4ere given each day in two sessions. Sham exposure was the sa:ne but without s-moke. Weeks after first treatnent with MCA. Statistical analysis of sham versus smcke-exposed groups suggests that sham >smoke, p • 0.0012. 75 CTR CONTRRCTS 029254 11249004 CTR VIN 04404e"12.

Ftnai Raaort Contract: CTR-0030 TABLE 17 I'tCICcYCc OF ?UL".CthRY ANCY, ACM ANO/CR SU Ili 3C3=?/CL'M F:i'AL'c •'!;C" AS A 7i.iVCTION 0F TI"E ~IFT:R TRE~1Tf'E!IT alTr{ 'CA A;;0 CjRONIC SHAM zX?0SUR3 CR hCA AtiD CHRC.IIC ---- 2Af C IiARE T'i : SMOKE eXPOSJRE a'A 'aeeksc MCA & Sham d MCA & Smcke d 4 0/20 0/31 a 0/12 1/28 12 3/31 1/11 16 4/20 1/22 20 2/23 4/20 24 5/24 12/47 28 3/18 8/27 32 5/24 1-3/46 36 8/25 15/41 40 4/13 10/27 44 4/12 9/24 48 7/16 13/16 52 7/10 7/12 56 4/6 4/6 60 1/3 8/12 64 1/2 8/12 68 1/2 7/9 72 0!1 2/3 76 4/4 3/3 80 0/0 2/2 84 1/1 0/0 as 1/2 0/0 Totals 57/259 1:5/4C6 a b c d Incidence is defined as the number of mice with a lesion divided by the total number of mice that died during e3ch time ;eriod and were necropsied. MCA treatment and smoke exposure as described in Table 5. Weeks after first MCA treitment. Statistical analysis su95ests that sctoke 3, sham, p0.10. 76 CTR COHTRRCTS 029255 11249005 CTR HN 044043

Final Reoort Contract: CTR-0030. TAOLE 1B :4CIGENC_ OF PULMONARY ANCH, ACN, Stl, A.1C, SCC, ASC, POC, AND OiriER PRIMARY UJ!'$= !'ALIG?1ANCIES Itt BC3F1/CUt1 FE'"ALE :1ICE AS A FUNCTION OF TIME AFTER TREATMENT 6tITM MCA AND C4RCNIC SNAM EXPOSURE OR MCA Ai10 CHRONIC 2A1 Si•10KE EXPOSURE a,b 'aeeksc MCA & Sham d MCA & Srsake d 4 0/20 0/31 8 0/12 1/28 12 3/31 1/17 16 4/20 4/22 20 5/22 7/20 24 12/23 22/47 28 11/18 24/27 32 21/24 36/46 36 23/25 29/41 40 12/13 23/27 44 12/12 20/24 48 16/16 16/16 52 10/10 10/12 56 6/6 6/6 60 3/3 12/12 64 2/2 12/12 68 2/2 7/9 72 1/1 3/3 76 4/4 3/3 80 0/0 2/2 94 1/1 0/0 88 1/2 Totals 151/269 239/406 a;ncidence is defined as the number of mice with a iesion divided by the total number of mice which died during eacn ti.re :eriod and were necropsied. b..MCA treatment and smoke exposure as described in Table 5. Weeks after first MCA treatment. ' d Statistical analysis suggests that smoke > sham, p• 0.21. 77 CTR CCNTRRCT5 029256 11249006 CTR NN 04-4044

F1na1 ieoort Contract: C:,R-0030 TABLE 19 STATISTICAL EVALUATION OF LUNG 'U`tOR DATA incidence Data A. c"numerate all animals as to the number and type of lesions. 3. Construct table listing each lesion or lesions, including non- :scored for each time interval. One animal fits into only one category and is counted only once. C. If possible. list those lesions found in animals that died during :his same time interval, but were not scheduled to be taken off at this time. 0. C:,nplete incidence table by adding all the data for a given row or column. E. Construct and expected-value table that is just like the observed table. (Surn of a given row x sum of a given column civided :y sum of all rows and columns.) F. :crapute the variance of data at each time interval by the follcwing for-nula: (E animals with lesions x L animals without lesions z' E animal~ qr~uo A x L animals group 8] .(total numoer animals) x total number minus 1 3. C.~aloute X2 analysis ("antel-Haenszel Test) by: X2 '( :xi - E estimates xi -.5)Z : L variance ri 78 .CTR CaNTRRCTS o2gz5"r 11249007 CTR I/N 0+404S

Final ReDort Contrac:: CTR-"v030 TABLE 29 -STATISTICAL EVALUATION OF LUNG TLMR CATA Tunor Probabil i ty A. Assure :.hat when tumor is found.in a dying animal that this tu.-or was a factor in this death. B. Catermi r.e the number of animal s that di ed and the number of ani-al s with a lung lesion for a given time Interval. (e.g. weekly, b!aea,cly, nonLhly). C. Construc: table with all observed tu.^nors and time intervals. 0. Ceter..nine matching expected values for a given value by multiplying the total of that row X total of that column, divided by total of rows and colwnns. E. Cete rsire rest of expected values by subtraction from column to:al . or row tatal. F. Ceteraine variance of data by: vi =1: each rowi X E each columnt 2 (total sum of all rows and columns) (total - 1) G. Cetermir,e each expected variance by subtracting this variance f-:.m sc:;a of each row and column. H. Sum each row, column, for tumors, non-turnors, variances, etc. I. Either evaluata by: (!EX X , obs exo E Variances or 79 , £ Xabs - £ X exo £ Variances CTR coHTRacTs 029258 11249008 CTR MV# 044046

Final Report Contract: CTR-0030 iAaLE 21 OISTRI3UTICN OF LUNG CANCERS I:f BC3F1/CUM MICE a NUMBER OF ANIMALSb ~iE:YS ,, Y0. ON TEST TJTAL' TUMORS SCC BOTH SCC+ ACC+ OT'ri'eR AAC SCC+AAC OTHER1 OTNERe TU40RSf 0-169 - - - - - - - - 17-19 2 2 0 0 0 0 0 0 20-29 30 8 66 1 1 2 0 2 30-39 142 6 87 12 33h 1 2 1 40-49 76 0 29 9 31i 2 2 3 50-59 5-4 1 4 26 191 0 4 0 60-69 13 0 0 8 2 0 1 2 70-79 4 0 0 3 1 0 0 0 80-89 2 0 0 1 1 0 0 0 90-99 1 0 0 1 0 0 0 0 b c d e f 9 h i j• Mice were treated with 9 lntratrachael doses of 250 ug MCA In 0.02 ml GS at - biweekly intervals for a total dose of 2250 ug MCA. The diagnoses sor 374 animals were individually arranged in 7 categories. The numoer of animals that died or were killed when moribund is given f:r eacn time interval. Includes 1 anicwl with SCC and lymphosarcoma (21 weeks), 3 with SCC and ?OC (28, 43, and 48 weeks), and 1 with SCC and POC (33 weeks). Includes 1with AAC and carcinoma of bronchogenic origin (37 weeks), 1 aith AAC and AU (45 weeks), 4 with AAC and POC (30, 46, 58 and 60 weeks), and 3 animals with AAC and ASC (51, 52, and 56 weeks). Includes 5 animals with ASC (2 at 27 weeks, 2 at 40 weeks, 1 at 66 weeKs), 1wtth sarcoma (37 weeks) and 1 with AU and POC (45 weeks), and 1 with POC ;52 weeks). Animals (25 rice) that died during the treatment period are not included In :-ese analyses. T!:ree animals that died during this period had SCC (12, 15, and 15 weers) :ncludes 1 anical with SCC, AAC and POC (34 weeks) and 1•aith SCC, ~.~.C, and fibrosarcocu (25 weeks). Includes 1 animal with SCC, AAC, and AU (43 weeks) and 1with SCC, :AC, and POC (48 weeks). ' Includes I aniral with SCC, Ar1C, and AU (53 weeks) and 1 with SCC, :AC, and sarcoma (57 weeks). (From: Reference 13) 80 CTR COHTRACTS 029259 11249009 CTR IwIN 044047"`

Final Recort Contract: CTR-0030 TABLE 22 3IOLOGIC 3 u{AVIOR OF SQUAMOUS CELL CARCINOMA (SCC) VS. ADENOCARCINOFIA (AC) Primary Lung SCC ?rirarv Lung AC Biologic Other Tissues No. of vo. of Behavior Involved Oiaanoses Diaonoses Invasion Vascular 45 4 Pleural 5 21 Rib Cage 11 9 Chest Wall 4 2 yediastinum 0 7 Metastasis Heart 25 3 TfLymu s 0 3 Trachea 1 0 Mediastinum 4 4 Rib Cage 4 1 Chest Wall 1 1 Bronchial Lymph Node 9 9 Cervical Lymph Node 1 0 Mediastinal Lymph Node 2 4 Mesenteric Lymph Node 0 1 Head 0 2 3ratn 0 1 Kidney 24 5 Ovary 1 1 Salivary Gland 2 0 Spinal Column 1 0 Subcutaneous Mass 4 0 Abdominal Mass 0 1 Infarction Lung 9 1 Kidney 14 3 Spleen 1 1 Salivary Gland 1 0 E.Tbolis.-.1 Heart 9 0 Kidney 2 0 r . . . . • . . . * r * * * * * • r * . * . . • . Most Probable Lung Infarction --P--Toxemia Pulmonary Insuff=ciency-~- Lause of Ceath 2ena1 Infarction -w--Renal Anoxia Failure 3 Left Atrial Obstruction -i- Congestive Heart Failure Lung data are from 216-3C3F1 mice which died or were killed wnen moribund after 29-67 wks. on test. Primary lung diagnoses were 127 SCC, 57 AC; 31 with both SCC and AC, and 11 with other types of lung lesions. 81 C.TR CElNTRRCTS 029260 11249010 CTR HN 04-4046

F i/wl itepurt TAULE 23 COIItrdCt: CfR-UU3U 0~. -- ..u,l.~.: :i1r -~_ ltbt -- - ..c Q/l)1 - 1«~t~l~l. In6 - - l~ls - 10l - la6s /rt b.. rl UOn,n. ]... rl-4.. - - - l.ns - l.n.rlwi - - -' - 1/10 let 111• let - - - we.r . - • - I/1o 1ot .Ilo as - - i.l•Lllr 0....1 Ie//0 IOa 1/1 loot L/le l.et ./t st f/1 i.tt sts Ltl.rt"/ - - /ts Ilt - - • Olh.e otf oI - - - 114 .s NCA Iw..1 Slts Its O/ts as tltll It IltO 1s o/tt .t .ItI 1./1..rlwe 1112s fIs yts (tt t/t0 tt /Ns It 1/t2 as ./t1 A1...1.e ly ptol.tt. 16129 SSt t0/ts Aet IUte Nt (Ilts ' Ist 1e/tt tst.11/tI AI.NIN w• t.qt.sllq Ir..l.t 6129 as I/ts 61 Iltt t!t 21tr /s )/tt 111 I/27 AIN.I.t.wlt • t.rtn Nt/q ts.1.1.. 0/ts et o/ts a_- I/t. Is Itt . . 11/ I/t2 IIt Iatl Al...l.e.•1c A....c.ecl.r. e/ts et o/ts as elt0 01 Ilte 142 tttt 99 t/2I f~1 LW...e t./l lJJ Mh.esl.tl. Wts as e/ts et Wt0 Ot 1/2e ss e/2t as 1/21 "-1 N wa.... C.11 ~ t1N.1. 0/2s et O/ts et e/2e as ./t0 as Nt2 as ./2I tfcl..s A.w.- ~ - ~ l J t~.t.-.Ie..Iw O Ar...tat1..- __ .. O/ts q ettt 0t pte as Ute as 0/l2 .1 ./2I as o/ts .t Wi Ot 1/te st e/tt es 0/t1 ~~ Air.ro..wt w , t.ec/.+r e/tt et Nas as yt. .1 t/l. ls 4122 Os ettl fV.ra Ic.ll) 0 /yptslal. 0/ts .s O/2s .t o/te a 2/le tt r/22 as o/II _J arw../ /tell! ' ', n1 /t.1.y1../. Itts 21s IS/2s tst e12s ta Itte Ils t/tt ltt tttl Y, sw....c ...- L:~reuH.s tlal.t. ./21 .t ./27 a ytr .t .Itft as .tt2 ut .Itl 0 %.p.r.. t.-%.ec• N .t.. o.+.l. .tt. .1 oits a atr as o/te as oizt an atl n s.r++• r...• S.tJ s1.r ats s_ Olls as ./td tn It2• u Iltt IIt sttl N sM...... l./l (PI tacl.w ltts It ./tt at ./td tll s/te Its Ittt st attl f,^ rt..t .lts as o/ts a .tt. .t 1/te Ilt Ittt tt h/tl IMtIN.It 0/ tte61/s1.NS It1.lAI /111t btwYtS) Atlt. /r/U10AUltt IOOCrtAllOt K I/1[A 4111114 SAtA/flt[) N.RAc 1C -...i: ~u t... • 2-rscli .as ~0:.+rfi - - 1409 S/f l.tt 1/1 IOoI - • 1/1 100s *1 e/10 .t ./1• .t 0/te as Ot e110 as 1/11 tt . e/20 es 101 s/le ut 11/Is IIt I/2e 1ss 119 6/10 IIt NIS IOt 1/20 t01 419 t/l0 Ist luls .Il s/t. 261 st t/10 Ils s/Is IIS 4124 tos st Ol10 Os I/1s tt 1/20 61 as e/10 as 0//s et 0/20 as as e/10 as Ills tt 6124 as as ./10 .1 41S ot O/to *t as 0/10 as • e/lS as 0/to Ot as e/10 as 0/1S O1 elt0 .t as 1/10 a1 ./H as e/te a st 1/10 .s otls as ./te .t rt otl. " • *tl• et e/2e as as ./Ir a ot.s .t .tt. .t Itt .1/te at Ills Ilt ./t. .s . rit e/le It1s tet Nt I/l0 Ist as ./10 a 0/1t as /t/t0 .c../sst at a It I e2 . 02 Mt .a ..t r.t 1/S 20s I/l lert I/t Oa 41s e.1t 0/S q 0/S ./S 00a . 1/S tOt (IS ltt e/1S e/IS es Ot ele ot0 a OI ale 1/10 re 1.' yn yi a et 0/Is as S/e ut t/10 Irs tK 011 (/1L tt 1/0 S.l I/ir let t/S 40% ./I! t/s t/e AJl 2I1e t.t I/s st . I/10 tes I/0 yt 3110 IOt 1N 6" 0/IS as y0 Os 0/le as e/s rs 0/IS i/IS Os ts ty0 NO es Ot e/le et10 Ot et WS ets At u yls ./IS Ot as tV0 0/0 as O1 e/le e/Io as Ot .IS eN a Ot yls .t 0/0 as e/l0 as etS .t e/ls .t- 0/e .t o/1t1 et NS Oc .//s .t e/0 rt .tle as o/1 a ./IS .t .t0 rs .tl. .t •N a ./Ib as ./o q etle .t o/s .1 yls a e/e et e//e as 0/1 .t t/Is IIs 1/e /IL ./1. .s oH ot 10/1ti tts t/e tft Itl. tet MS as ~ ttceti o4 test. ~ Al 11. 16. .AO 22 .eels ur test, 6. 0 .wJ 9/wtcrla Ioot of /[A U.1 Leer g/vco.

Final Report Contract: CTR-0"v30 TABLE 24 DISTRIBUTION OF ALVEOLOGENIC PULMONARY LESIONS AS A FUNCTION OF TIME AFTER MCA TREATMENTSa Weeks on Testb Lesions 12 15 22 24 28 32 36 40 44 48 52 56 60 ,aH 16 20 18 11 10 11 9 11 11 2 5 3 2 ANCN 0 0 7 2 3 3 6 6 13 8 4 3 2 ACN 0 0 1 3 3 10 7 13 14 12 6 6 4 AAC 0 0 0 4 2 2 2 5 14 10 2 7 3 So rma 1 10 1 3 3 0 0 0 0 0 1 0 0 0 `aon-alveoloqenic 4 8 4 5 6 4 4 0 7 3 1 1 0 f Animals 29 29 28 28 22 23 18 15 20 15 8 10 5 3 Total numoer of alveologenic pulmonary lesions observed in a randomly sa.•oled population sacrificed at the Indicated time. MCA was given 9 x 250 ug at biweekly intervals. b Weeks after first :re3t-ent with MCA. ` 7otal nur,.oer of animals sacrificed at the indicated times. 83 . CTR CONTRRCTS 029262 11249012 C ! l O HN •/.I' 4I' 0bP' 0

F1nal Reoort Contract: CTR-C030 TABLE 25 OISTRI3UTION OF SQUAMOUS PULMANARY LESIONS AS A FUNCTION OF TIME AFTER MCA TRE.ITMENTSa Weeks on Testb Lesions 12 16 22 24 28 32 36 40 44 48 52 56 60 SM 8 19 8 3 7 2 1 0 0 0 0 0 0 SN 0 0 6 1 3 5 1 0 0 0 0 0 0 SCC 1 0 6 9 1 8 8 3 7 3 1 1 0 Norra 1 10 1 3 3 0 0 0 0 0 0 0 0 0 Non-squamous 7 9 9 12 11d 9 7 11e 13 11 7 1 5 ! Animals 29 29 28 28 22 23 18 15 20 15 8 10 5 a Total number of squamous pulmonary lesions observed in a randomly sanled population sacrificed at the indicated times. MCA was given 9 z?:0 -49 at biweekly intervals. b Weeks after first treatment with MCA. C Total number of animals sacrificed at the indicated tir,es. d Includes one ani,ral with muco-squamus lesion. e Includes one animal with squamous hyperplasia. 84 CTR CONTRRCTS 029263 11249013 CTR NN 0440SI

Final Report Contract: C'"R-0030 POBLICATI0:1S Henry, C.J., Gayle, T., Florant. L., Dansie, O.R., Creenspan, J., CueYir., M., Holmberg, R., K.K. Xanaqalinqam, and Kouri, R.E, Chronic Inhalation Studies in Mice: I. Facilities and Equipment =or "Vose-On1y" Exposures to Cigarette Smoke. (Submitted) 1994. Hwang, Sonko, 0., Oansie, D.R., Jaqusiak, ;t., Kouri, R.E., and Henry, C..:. Identification of Orinary .Setabolism in Mice of Catechol in Whole Cigarette Smoke. (In preparation), 1984. Lubet, R.A., Herscowitz, H.B., Kanagalingam, K.K., Dansie, C.R., Kouri, R.E., and Henry, C.J. Lack of Significant Immunosuppression Following Exposure of 9C3F1/C:::n Mice to Whole Ciqarette Smoke. (In preparation) 1984. 305 CTR CoNTRACTS 029504 11249254 CT~' ~'"`w~ 04,405Z

Final Report Contract: CTR-C030 TABLE 26 NUMBER OF LUNG LESIONS PER ANIMAL AS A FUNCTION ~ 0~ iIME AFTER 7HE'FIRST MCA TREATMENTa,b (I-100 Serial Sacrifices) Lesions/ Weeks on Test After First MCA Exposure Animal l2 16 22 24 28 32 36 40 44 48 52 56 50 0 10 1 3 3 0 0 0 0 0 1 0 0 0 1 8 13 9 14 13 8 5 1 4 2 1 2 2 2 8 11 12 7 8 11 9 4 3 4 2 6 1 3 1 2 1 3 0 4 4 6 5 5 5 1 1 4 2 2 3 1 1 0 0 4 4 2 0 1 1 5 0 0 0 0 0 0 0 0 3 1 0 0 0 6 0 0 0 0 0 0 0 0 1 0 0 0 0 a' Lung lesfons we~e of%o .n squamous and aveoTogeniclorigin; a tatal of 15 different histological categories were identified. b' 250 ug MCA/0.02 ml gel-saline was given Intratracheally once. every c•,4 weeks for a total of nine times. (Total dose - 225] ag) 85 CTR CQNTRRCTS 029264 11249014 CTR I-IN 04,40*~~ :~

s ! >~rrir ~r f1 r (1 y ^ r ..i ~~ ; C flrf~r r • r ~ ` N r r r ra , 9 C ; U c ii ~u o u o 0 0 0 0 0 0 0 0 0 0 r o o 0 0 o o 0 0 0 o 0 0 0 0 L p O O O O O - O - r O O O 4 J J N t~'O n e N M • O O o O O O O O O O O O O O O O O O O O r O O O O O O O O O O O O o O O O O, O O O O O O O O O O O O O O O O O O J M O O O O O O O r O O - r O O 0 a r « - 0 r O O O O O - - r O ~ fi p r r _ p, p 0 p 0 O p O p Y 0 0 o O o 0 0 o o 0 0 0 0 o r O O 0 O 0 0 0 O O O O O r O « o o p 0 o 0 0 o O o 0 0 - 0 - M N r M - r - "~ - N N Y r r~(ffM ~fffffffffff.fr:'5~~~~T~:..rr~if:ffCfSasR~saAA~~~f Si.~'ir r.y•.rrrrrrr.> yyy~•..M Qa rrrry~rr .~ r e f1 T • 7 7 • ~~ If s ; i=C . . ~.a f1f.l~ ~f~^ifflR~ti~;0 i+ i~ 7~ rr,•y..0i7:~ rr • 7 r a : .n . ~ v 0 00 O 4 O r O O O O O O O O O O O N O O O O O O O O O O O O O O• O O P O O r..y~O O-O O O O O O v 00 O O O O 4 O O 00 O O O ON 4 N O O O O O O O 4 O O O O O O N 4 Or O 00 O-O O w 4 O O O O O V YO O O O O 4 O O O Y O V 4 p r O O N O O Y y Y O 4 V O r0 O O O r YY O N O« rN0 4 O 4 Y iJ Y rw. 00 O-O O O-O O O O r0 O O O O O rr -O O NO O O r0 O O O O 00 O-O -NY O O r rn r0 O• O JO O 00 O O 000 O O 00 O O r O O O O O Np ~•r0 O O/10 O O ON r r0 O ON r-O O-N O O w 00 O O-O - O O ON O O 00 O O O O O O O O MO r0 O O r0 O O-.0- Y N O O OY -O O O O Oa+ O- 000 O 00 O 000 r0 O rrp.. O O O O O O O O r0 O O O O-O OO r«N O OO P O O O O O O O-O OOO O O O O O rrp r O 00 O Y O O O O O O 00 043 « O O p O O O OO r-O O 00 O O O O O-O O O OYr p 00 O OOOO O O OOO~+ O O O O 00 O`'O O r0 O O O O rp0 O 000 OOO O O O O YO O- r.0 O pp O p pp pp O Opp O p p p p pp -p p p«O p p r p ppp O p p rppOp p p p O-O r o-o 0 op o 0 oro 0 0 00 0« O 00 O O O Or O o o0 0 0 0 0 000 o ro 0 4 o u u o 0 0 0-oo 00 O O Op O O Or0 o O O 00 O O 00 O O r r0 O O Y O 00 r O 00 O O 00 O O 00 O O 00 00 - r 000 0 0 0 o p o0 0 0 0 0 0 o r o o u o 00 00 0 o ro 0 00 0 0 00 0 0 0 0 0 0 0 0 o p o- ro 0 .~y~-rrrrrryyrrrrYw M Nrrrrw•Y-r«rNywYYV.14r...~~.Nrrrr•MYr N N ~ ~ or- .N ~ N ci » nt e S

Final Report Contract: CTR-0030 TA3LE 28 SUMMARY OF DATA FROM IN VIVO PASSAGED TUMORS. Diagnosis Numbers of Tumors Growing at Designated Passage P1 P2 P3 P4 PS Alveolar Hyperplasia • 0/1 - - - - Alveolar Hon-Compressing Nodule 0/1 - - - - Alveolar Compressing Nodule 3/8 3/3 2/3 2/2 - Adenocarcinoma 5/10 5/5 3/5 2L3 - Squamous Metaplasia 0/2 - - - - Squamous Neoplasm 0/1 - - - - Squamous Cell Carcinoma 10/20 9/10 6/9 4/6 2/4 Adenosquamous Carcinoma 0/1 uuco-Squamous Carcincrsa 0/1 !!uco-Adenoca rc i noina 0/1 TOTALS 18 17 11 8 2 TOTAL ! iJ"'ORS PASSED - 84 TOTAL i ORIGINAL ANIMALS USED  68 TOTAL i yE'a8oR.`i ANIMALS USED • ti1,500 87 cT'R C.ONTRRCTS 029266 11249016 CTR I-IN 04405E;

Final Report. Contract:.CTR-0030 TABCE 29 SA7V lL rASSACL TUMpI GLN(ALOGT AA1.,.1 sleas 'r.a• ew testi 22 ~ae• er t.erl~ieet '1121/77 Oroa e.ssrlet/.nt ee."n.ei~ e leeelr.. frey~.~wIte ass .n tM i wICressO.lc slp noslsl Sauwroue call sarelnenr I p Sea 0x I Sta 0x I Sex 0x • Sax ax • Sex 0x o0n 009! f McT f KGT 0094 f MOT 009f f SCC 0144 9 vCT 0141 / SCC 0242 f MGT 0244 f Met 0x 026f u 4GT ret • MGT ~`f~ s • MOT 0247 U MOT 0247 f NOT 0244 1 SCC 0470 1 AC rraen 0511 f ASC rreten 0512 f SCC lrozen 024! 9 MGT 04f0 f SCC 0439 1 SCC rreten 0W f MOT OA;1 f GT OW.i f MGT 0wS f MGT 0ub- • MCT 0445 f MGT 01112 f C.o111. Anln.ts OtN O1~ O1N f NGT I Mee ox 0617 f Ice 0660 f Ice rretan pa 0K1 q SCC rros.n 0662 f Ice rrot.n 0K0 V SCC 0700 u see rsnel 6/2/7t rr.e.n 0611 • sce 0654 • SCCt r.aen ~ 06sf u ScC rras+. 06Si u ScC • 0657 u Ice rraen rressw 0659 f Ice rroten 0450 u Ice rreten 061v f AC.0a9 rrot.n 0620 I M.t 04 09~f f On teet T•t 0~2i t On t.ft 0"7 f 0n test 092• • Cn e.st 0611 • rGT 0929 f on tett 0!!0 f 0n test 06" • - MOT 062 9 SCC.N rreten ~ 06S u SCC 0666 f SCC rroten 0687 f MGT 066/ f SCC 0689 9 rGT rresen 0690 f MGT 0691 f MGT 0693 9 MGT 011115 f SCC 3199 • NOT 0196 f Ice r ro e.n 0177 f SCC.od 017! f Ice r~r.sen •• 0~li f SCC =610 f SCC rresen C644b • SCC rreten C64f f SCC rret.w 04 06i6 T f SCC anl...ls tis " 0500 • f NC Ice rreten 0501 f SCC rresow 0503 f SCC.Od rres.n •0501 f MGT 0200 f NGT 0016 4 rGT 0147 f MGT 88 .C.TR CCNTRRCTS Q2925?' 11249017 CTR I-IN 044# 3~.~ E:.p

Final Revort Contract: CTR-0030 CTR-100 FICORES 21-30 89 CTR CaNTE2RCT5 429266 11249018 CTR HN 04'4057

Welght 30 (grams) .~~. 20 ~ I i I I I I I i I i Final Report Contract: CTit-110:10 FIGURE 21 MEIIN BODY WEIGHT OF BC3F1/CUM FEMALE MICE DURING CIIRONIC OR SIIAM EXPOSURE TO 2A1 CIGARETTE SMOKE 50 40 ~ 10 N W N ~ W t UntreateJ xposed LJJ Sham E Smoke Exposed _ I I I I I I l' I t I 10 20 30 40 50 60 70 80 90 100 Wecks on Test I I

Flnal Rcpurt Contrdct: CTR-0030 F I CARtE 22 MEAN BODY WEIGiIT OF BC3F1/CUM FEMALE MICE AFTER MCA TREATMENT ALONE AND WITII CHRONIC EXPOSURE OR SHAM EXPOSURE TO 2A1 CIGARETTE SMOKE J J N 0 ~ :D Ln .o r

I I 1.1 - I I , I PERCENT CARBUXYIIt(10GLOBIN IN aC3F1/CUM HICE AFTER DAILY EXPOSURE TO 2A1 CIGARETTE S140KE f inal Iteport Contract: CTR-003U FIGURE 23 GU 5 c1g k0' ~ ~ N A tD 0 N ~ z m 0 J ~ C) W 30 n N a I u t- 20 ~ ~ z W ~ ~ W ~~. ..r • )0 5 4 jtg 4 9 0 ® Ex 6 5 cig M u I 1 . 1 i I I I : I I Qu W m MER 9 10 11 12 13 14 15 16 17 18 19 20 21 23 25 MUN !I IS ON TES T G NONE a SMOKL R SHAM I

I i, I•• I I Fina) Rcpurt Contract: CTIt-UU7U GU F1Gt1RE 24 I NEftCCNi CAItUUXYIIEMUGLUBIN IN 8C3F1 MICE AFTER TREATMENT WITH MCA 5,cig AN11 UAIIY LXf'OSURE TO 2A I CIGARETTE SMOKE 5 c1g 5Q 40 4 c1g* 6 puff~ 3 ctg Z 0 5 1g N ~ ~ 3~ N W T x a~a 5 19 V 20 4 19 Z 10 ~ N 0 ~ N 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 23 25 MONTHS ON TEST ,=„NONE JfSMUKE 5 c1g 11SNAM

F inal Itepur[ Corkcracc: crlt-oo3u 100 80 J 4 > ~ 60 P N 0 ~ 20 N ~Z~6 - ~0 ~~ GJ FIGURE 25 PERCENT SURVIVAL IN QC3F1/CUM AND C31I/ANF CUM FEMALE MICE TREATED WITH 3-METHYLCNOLANTIIRENE (MCA) 12 20 28 36 44 52 60 INTERVAL (1(EEKS ON TEST)

Final Reoort Contract: CTR-0030 FIGURE 26 ?R03A3ILITIES FOR THE OBSERVATION CF A RESPIRATORY TUMOR AT DEATH IN BC3F1/CUM AND C3H/ANF CUM F=?1ALE MICE TREATED WITH 3-METHYLCHOLANTHRENE (HCA) 1o-~ 201 0 FIGuRE 25. Probabilities for an animal dying of a lung tumor in BC3F1/Cum mice (~ ) and C3H/Anf C= mice (N ) treated with 9 doses of MCA (2250 ug MCA total). Oata are calculated from 374 BC3FI/Cum mice and 113 C3H/Anf Cum mice. Over 95% of the 8C3F1/Cucn and 87% of C3H/Anf Cum ' mice died of lung cancer in these treatment groups. (From: Reference 13). 95 cTR coNTRACTS 029274 11249024 CTR NN 04-4063

Final Report Contract: CTR-0030 FIGURE 27 PROBABILITIES FOR THE OBSERVATION OF A RESPIRATORY TL'MOR AT DEATH IN C3H/ANF FE;+ALE IMICE TREAT© WITH D I FF"cRENT NUMBERS OF ADMIN I$TRATIONS 'OF 3-hET~iYLC;iOLAtiTHRE`IE ,.00-9 0 I• u 4 y f/ wauu orT1ZT /t 7: 10 u Figure 27. Probabilities for an animal dyin of a lun tumor in C3H/ f Cum mice treated with 37 0 ), 67 * ), or 9 ( ~ ) doses of MCA (750, 1500, Or 22S0 ug MCA total dose, respectively). The number of animals per grouo are in Figure . Over 86%, 72% and 87% of the mice died lung cancer in the groups treated with 3, 6 or 9 doses of MCA, respectively. (From: Reference 13). 96 C.TR CONTRRCTS 0292-75 11249025 CT "'' HN 04,4064.

Final Report Contract: CTR-0030 F IGURE 28A PROBABILITIES FOR THE OBSERVATION OF A RESPIRATORY TUMOR AT DEATH IN C3H/ANF FEMALE MICE TREATED 44ITH THREE D I FFcRENT CHE,`1I CALS Figure 2?A. Probabilities for animals dying of lung tumor in C3H/Anf Cum mice treated with 6 doses of MCA (1500 ug MCA total), 5 doses of BP-1.,8-diol (1250 ug BP-7,8-diol total), or 5 doses of QP.(1250 ug BP total). The number of animals are 62. 16, and 85 for the MCA, BP -7,8-diol, and SP treated groups, respectively. (From: Reference 13). 97 cTR coHTRACTS 029276 11249026 C,Tk? I-IN 0'4-4~".~G5

Final Repor,t Contract: CTR-0030 FIGURE 288 x., 3X Y WEEKS ON TEST FIGURE 288. 0istribution of lung cancers in C3H/Anf C;:m mice as as function of time after 3, 6, or 9 doses or 250 ug MCA. There xere a total of 72, 62, and 104 mice in mice treated with 3, 6, or 9 doses of yCA, respectively. (FROM: Reference 13). 98 C.TR CONTRRCTS 029277 11249027 CTR IMIN 044063G

II {I ' ~I I I I I I I I .I F1nal Report Contract: CTR-UU30 I'OSTULATED PROGRESSION OF ALVEOLOGCNIC, SQUA(10US AND DRONCIIOGEN I C NEOPLAS 1 A i N 3-METHYLCHOLANTIIRENE TREATED f1I CE ALVEOLAR AND BRONCHIOLAR EPmlELtiJb ALVEOLAR HYPERPLASIA f 0 t.0 ~ co MINIMAL MODERATE I o ALVEOLOGENIC NON-COMPRESSING NODULE f ALVEOLOGENIC COMPRESSING NODULE f Y AND ' PAPILLAR (?) PLEOMORPHIC CHANGES ALVEOLOGENIC ADENOCARCINOMA MUCUS CELL IIYPERPLASIA Mucus CELL NODULE Q) QRONCHIOGENIC MuclNOUs ADENOCARCINOMA FIGURE 29 MIXED CARCINOMAS SQUAMOUS METAPLASIA _SOUAMOUS METAPLASIA NON-KERATINIZING KERAT NIZING MODERATE t CYST f MODERATE SOUAMOUS SOUAItOUS NON-COMPRESSING NoDULe SOUAMOUS MJIMAL KERATIN z M IN MAL t ALVEOLAR AND BRONCIIIOLAR Eeizt~L~lid HtJCO-SDUAMOUS COMPRESS 1 NG N ODULE 4., CARCINOMA I ADE~OSQUAMOUS I,ARC I NOMA - I M11C0-ALVEOLAR ADENOCARCI-NOMA (~) ~ . . . ` . .ZN . I ( SQUAMOUS `NEOPLAS III)M 1 SOt,~AMOUS CELL LARCINOMA NON-COMPRESSING NOD'1LE SOUIOUS COMPRESSING X NODULE

Fina1 Report Contract: CTR-0030 FIGURE 30 CTR-100 (1-100) INCIDENCE OF INDUCED PULMONARY LESIONS IN BC3F1 CwM FEIIALE MICE AFTER TREATMENT WITii MCA . 100rormal 100rA}1 0 50 50 e 20 40 ; 100 'TNCN 100 rCN ~ c r C 6! u c. i 50{- 100 © VLiC 20 40 - , 11 / 20 40 20 40 50 0 ! \ r /•\-" • 1 20 40 20 40 Weeks 100 CTR COHTRRCTS 029279 11249029 r. 10'01 Non-Alveolo9enic 0- CTR 11H 044'068

CTR CONTE2RCT5 029280 11249030 CTR I-IN 04•4069

Final Report Contract: cTR-oo30 CTR-lO1A Iv. CTR101A. Ce?RONIC EX?OSURE OF BC3F1/CU;i MICE TO 2R1 CIGARETTE SMOKE A. Introduction/Objectives In September, 1978, a long-term chronic cigarette smoke inhalation study was initiated. This second inhalation study was the first designed to use long-term or "lifetime" cironic smoke exposure. Future studies were to address such variables as specie sensitivity, smoke exposure regimen, smoke dose, cigarette type, and duration of exposure. However, after initiation of CTR 101A, it was decided not to perform any other long-term inhalation studies. Thus, the main objective of this one lifetime study was to define the.biological activity, if any, of reference high tar-high nicotine cigarette smoke under one specific set of experimental conditions. The following details summarize the experimental design of this experiment: o Reference cigarette: 2R1-2.8 mq nicotine, 50 mg tar. o Standardized exposure 6-8 puffs per ciqarette, 20 sec conditions:: smoke exposure per minute, one seesion of S cigarettes per day (8 min rist between successive cigarettes), exposures for 5 days per week. o Smoke concentration: 101 o TP"S deposition: approximately 0.2 mg per day per mouse. o Smoke exposure 3,516 HC3F1/Cum female mice for ;roups: smoke-exposed (2053), sham- exposed (1014), and shelf control (449) groups. o Duration of exposure:. . 110 weeks o Duration of study: Lifetime of HC3F1/cum mice. 101 CTR CONTRRCTS 029281 11249031 CTR ~~"I~''''~ ~.~'~ ~~ 0f o

?it+.1 R.port Contract: CTR-0030 CTR-lOlA o 8ap and Smoke Groups: Additional 710 mice assigned for smoke, sham and shelf control groups; BaP (1.2.mq) was given intratracheally 3 times at weekly intervals over 3 weeks; smoke exposures were initiated one week after the last BaP treatment. The experimental design provided for exposures to be performed 5 days/week for 110 weeks, at•which point the animals were held until death. The following specific data were collected and evaluated over the course of the 3 year study: health. Weeks; 1. Daily observations of the animals for signs of ill 2. Documentation of daily 2R1 smoke generation for '_10 3. Documentation of smoke uptake into BC3F1/Cum mice at monthly intervals; 4. Documentation of body weights at monthly intervals; 5. Documentation of the circumstances of death of each animal and construction of survival curves for smoke, sham and shelf control groups; 6. Microscopic evaluation of respiratory tissues and selected other tissues from a random samplinq of the animals t':at• died during the first year; 7. Microscopic evaluation of respiratory tissues and any abnor:ral tissues from all animals that died during the second and third years; 8. Description of the lesions that likely led to death of the animals in the smoke, sham, and shelf control groups; 9. Description of respiratory tract lesions in BC3FL mice associated with treatment with known chemical (i.e. BaP); 10. Microscopic evaluation of respiratory tract tissue and any abnormal tissue from BaP-treated animals and; 11. Description of those lesions that likely led to the death of the animals in 8aP + smoke, eaP + sham, and BaP + shelf groups. 102 CTR CaHTRRCTS 029282 11249032 CTR Nf`] 0`440f 1

Final Report Contract: CTR-0030 CTR-I01A B. Data Collection/!!anagement An Experiment Information Management System on a Hewlett Packard 3000.was used to initiate, manage, and evaluate this long term in vivo experiment. This system provided the Teans for recording and collating the observations and data for each individual animal over the course of the three year period. Standard computer programs were used for scheduling treatments# inventory of animals, gross and microscopic diagnoses, obtaining and recording monthly body weights, recording and documenting daily smoke exposure levels, and final data evaluation. A brief description of the features of this system follows. Animals were randomized, given individual identifi- cation ear tags (National Band and Tag, Knoxville, Tennessee) and assigned to a specific experimental group and treatment schedule. This information was entered into the experiment data base, interpreted by an edit program, and added to the existing data base via an update program. As the experiment proceeded, observations, inventories, monthly body weights, and daily TPN levels were obtained and added to the data base. An automatic-tare Sartorius balance was interfaced with a Hewlett Packard 2644 terminal to collect and record animal weights. At the time of death or sacrifice, autopsy forms were initiated to describe any gross pathological observations and to assign• specific histopathology numbers to each animal. The computer system supports the Systemized Nomenclature of Pathology (SNOP) codes for the interpretation and reporting of the microscopic results. C. Daily Observations 1. Disposition of Animals The disposition of the animals in the six experimental groups is given in Table 30. Because of the large number of animals in this study, mice were grouped and initiated on test at different times. For the smoke, sham, and shelf-control groups (Groups 1, 2, and 3. respectively) , 3 initiation periods were used: September 18, 1978; October 17-18, L978i and February 5, 1979. For the BaP treated groups (Groups 4, S, and 6), 2 initiation dates were used: September 25, 1978, and March 12, 1979. The initial number of animals in each series is given in Table 30, as well as the number of animals alive at various points during the study. A total of 2053, 1014, and 449 animals were initiated on test in the 2R1 smoke, sham, and shelf-control groups, respectively. When exposures were terminated after 110 weeks, there were 315, 222, and 176 animals alive in these three groups, respectively. These mice were observed and held until death. A total of 320, 260, and 130 mice 103 CTE2 CONTRRCTS 029283 11249033 C TR NN ~'.~440'"r"4

Einal R.port Contract: CTR-0030 CTR-101A were put on test for the SaP + smoke, BaP + sham, and 9aP + shelf groups, respectively. Data from these BaP treated groups will be presented in Section IV.I. The following sections describe results from the smoke, sham, and shelf control groups (Group 1, 2 and 3, respectively). 2. Disposition of Animals for Histopathology J The disposition of animals from which microscopic diagnoses were made is given in Table 31. Previous experiments have shown that few effects from exposure to cigarette smoke would be expected during the first year of the expermene. Thus a random number of animals was.selected for microscopic diagnosis during the first year (i.e., those animals that had ear tags endinq in "3"). As shown in Table 31, an average of 15% of the animals that died had respiratory tract tissues (lunq, trachea, larynx, and head) and selected other tissues evaluated. After the first year, non-autolyzed tissues from the respiratory tract and any abnormal tissues were preserved for microscopic evaluation from all animals. During the second and third years, an average of 931 of the animals were evaluated microscopically in the 2R1 smoke, sham, and shelf-control groups. An average of 901 of the animals in the BaP treated groups were evaluated during this time. 3. Clinical Signs a. Response to Smoke/Sham Exposure Mice were observed during and immediately after daily exposure to smoke and sham treataent. For the smoke exposed animals, while a certain amount of adaptation occurred, the animals never accepted smoke exposure without some agitation or struggling in the holder. They occasionally demonstrated some shallow breathing or gaspinq during exposure. Immediately after exposure, mice in the smoke-exposed groups were lethargic, ataxic and hypothermic. The sham-exposed animals also demonstrated agitation and strugglinq while restrained in the holders, but generally appeared normal after exposure. After 20-30 weeks of exposure, certain animals were observed to have reddened skin and worn-away hair around the neck area that fits into the "stock-type" holder. In 30-40% of the cases, these irritated areas progressed to open sores as exposures continued. Polycarbonate inserts to cushion the neck slot were analyzed and found to be quite effective in limiting these neck cuts. However, the scheduled fabrication and use of these inserts wera not carried out because of the decision to terminate long-term inhalation studies. Because of the unavailability of these inserts, the mice had to be rested 104 CTR CCNTRACTS 029284 11249034 C) R 1) l ) 0440f`3-

Oa.1 r- a" # : ~ .. . . ~ . !lnal Report Contract: CTR-0030 CTR-101a periodically during t^e second year of the study. Such rests enabled the neck cuts to heal or become :ess severe. The length of the rests varied from 1 day to 1 week. On September 1980, an exposure-rest schedule was initiated :or those animals that had been on test over 95 weeks. Thus, for those animals on test !rom September 1978, the animals were rested during weeks 101, 104, 106, and 108. For those animals on test from October 1978, the animals were rested during weeks 97, 100, 102, 104, 106, and 108. Smoke exposures were stopped after 110 weeks. For those animals on test from February 1979, the animals were rested during week 82. The solution to the neck cut problem was defined, but not implemented by the Council. It is recommended that such inserts be used for any long-term studies where animals of different ages and/or sizes may be used. b. Observations at Necropsy When animals were sick or could be seen to have visible lesions or tumors, they were isolated and no longer treated. Moribund animals were killed. Such observations were recorded on the individual pathology form that accompanied preserved tissues through histology and pathology. A list of observation codes used in this experiment may be found in Table 32. Mice were observed twice daily for evidence of illness or respiratory distress. In the animals that survived post 110 weeks in the 2R1 smoke, sham, and shelf control groups, moribundity was difficult to predict from clinical signs. This is in marked contrast to those groups in which the animal died from chemically-induced lung tumors, where the animals were found hunched, lethargic, and suffering from respiratory distress (Groups 04, 0S, and 06). • D. Documentation of Smoke Generation 1. Equipment The equipment used to generate, monitor, and deliver 2R1 cigarette smoke to the animals has been described in detail in a previous section of this report (see Section II). Two SEM II machines were required for the large number of animals in these studies, as well as a separate sham-exposure machine that duplicated the smoking machines in all respects, but did not generate smoke. Three animal containment racks were used, one for each smoking machine and one for the sham-exposure machine. Each machine and rack was equipped with monitoring systems and safety 'evices (se3 section II). 105 ICTR CONTRACTS o2s285 11249035 CTR HN 044,074,

F inal Report Contract: CTR-0030 CTR-lO1a 2. Smoke Exposure Regimen • •~ A standardizad smoke exposure regimen was used to generate 2R1 cigarette smoke. For these studies, a nominal 10% -(-9 mg/G, v/v) smoke aerosol was produced. The exposure cycle alternated 20 seconds of smoke with 40 seconds of air each minute for 6 to 8 minutes (6-8 puffs/ ciqarette).These•numbers of puffs per cigarette reduced the nicotine toxicity associated with this cigarette, however, only the first one-half of the cigarette was used in thesea studies. 3. Gevels of TPM Generated Smoke particulates from each exposure session were measured with an on-line optical-type scattering detector. Eight detectors were used and interfaced to 8 strip-chart recorders, that provided a permanent record of the puff-by-puff TPM profile and final integrated values for TPM levels. The integrated TPM values were recorded on a data shaet, along with the date, the experimental group number, and number of animals loaded per exposuro. Five such recordings were made for each group each day (5 cigarettes per day). Lach integrated value was entered on the• Hewlett-Packard data base. A program converted the integrated recorder values to milligram TPM at weekly intervals and provided a cumulative TPM value. The top panel in Fiqure 31 shows the cumulative TP!t generated over the 110 weeks of exposure. In Table 33, the cumulative TPM levels for each 2R1 smoke-exposed group is presented for the 110 week exposure period. The number of mice in each group, the date of first exposu.re, and the date of last exposure are also given in Table 33. The overall mean TPM generated for all 24 smoke-subgroups was 339 + 10 grams. Thus, the inter-group variability in the exposures was less than -3t. This amount of TPM was approximately 271 of the total TPlS generated from the 2R1 cigarette under analytical conditions (Phipps and Bird Smoking lSachine, 11 puffs/ciqarette, smoked to a 23 rsillimeter butt lenqth). 4. T?M Deposition in the Mouse Respiratory Tract As determined from parallel radioactive dosizetry experiments (7) smoke particulate deposition was determined to be approximately 200 micrograms TPM per day per :nouse lung for this exposure regimen usinq 6-8 puffs par cigarette. Sae Henry et al (7) for a description of the methods and results. E. Documentation of Inhalation of Smoke by eC3F1/Cum Mice The amount of smoke inhaled by the animals was monitored by determination of COHb levels. Previous dosimetry studies using radiolabelled smoke particulates had shown that deposition 106 c.TR coNTRacTS o292®6 11249036 CTR NN 0~~ ~• 4".~ " ~

?inzl R.pott Contract: CTQ-0030 CTR-101A of TPM in the lungs using this exposure system depended upon t`:e smoke concentration and total exposure time, and that deposition could be correlated with COHb levels. Thus, at monthly intervals, blood from the retro-orbital sinus was collected from 3-5 mice per group. Vatelson blood collecting tubes (175 ul. Sherwood medical Industries, St. Louis, Missouri) were prepared shortly before use by rinsing the tubes with a drop of sodium heparin (10,000 units/ml, Abbott Laboratories, Baltimore, ,saryland). After blood collection, the tubes were sealed with Critocaps, stored on ice, and analyzed within 30 minutes. The IL-CO-Oximeter (IL-282, Instrumentation Laboratory, Inc., Lexington, Masiachusetts) automatically analyzed the blood sample for percent COHb, grams/deciliter hemoglobin (Hb), percent oxyhemoglobin (oxy-Hb) percent methemoglobin (met-Hb), and volume percent oxygen. The average percent COHb for the 2R1 smoke exposed groups as a function of time on test is shown in Figure 32. Mean values ranged from "91 to 25! COHb. During September and October of 1978, the mice were being gradually adapted to the smoke exposure regimen and thus, were not receiving the entire smoke dose. The overall mean for the smoke-exposed mice was -17.2t over the 110 week exposure period. The mean for the sham exposed and shelf control groups was 1.4 and 1.81 COHb, respectively. The levels of Hb, oxy-Hb, and met-Hb at monthly intervals during the last 13 months of exposure are given in Table 34. Data are from the same blood sample from which COHb was determined. Hemoglobin levels were slightly elevated in the 2R1 smoke exposed mice compared to the sham or shelf control animals (p< 0.05). However, the values are within reported ;anqes for mice (13). The percent oxy-Kb or cnet-Hb was not different between any of the groups. F. Cocumentation of Body Weights 3ody weight analysis of the smoke exposed, sham exposed, and untreated shelf control animals are presented in Figure 33. A computer generated schedule was used to obtain the weights under approximately the same conditions each month. a11 animals were weighed 2 days after their cages had been changed and their °ood replenished. For the smoke and sham exposed animals, weights were obtained at least 2 hours after termination of exposure. The untreated, shelf-control mice gained weight until 50-60 weeks and then gradually declined in weight during old age. The rate of weight gain of the untreated, shelf control animals were significantly greater (p< 0.05) than that of the smoke• and sham exposed mice. At -80 weeks on test, the average weight of the shelf control mice was 50 grams. 107 C.Tt2 CONTRRCTS 029287 11249037 CTR NN 044' Of ~ ~°°'..r

Final Report Contract: CTR-0030 CTA-101A No difference between the mean body weights of the s;ao;ce and sham exposed mice was found over :::e course of the study. Smoke and sham-exposed mice gained weight slowly until -60 weeks on test and then gradually declined. The average maximum weight of SC3F1/Cum :nice in the smoke or sham-exposed groups was 32 grams at about 60 weeks on test. G. Survival The'surviving fraction of mice as a function of time on test is presented in Figure 34. These curves were corrected :or those animals that were randomly removed from the study. T::e survival curves for the smoke and sham-exposed groups decreased more rapidly than the shelf control animals, indicating that smoke or sham exposure caused a small but detectable level of toxicity. Compared to the smoke or sham mice, the shelf group had the steepest survival curve post 80 weeks on test. This is the time when shelf animals were losing body weight very rapidly (see Figure 3,3). Our estimate of cause of death for these animals is presented in Tables 35-37, for the shelf, smoke and sham exposed animals, respectively. During the three year observation peeiod', the shelf controls died of both neoplastic (-60t) and non-neoplastic (-40%) diseases (se• Table 35). The most prevalent cancers in the shelf control mice were hematopoietic tumors (-33%) and fibrosarcomas (-13i), while the most prevalent non-neoplastic diseases were congestion/pneumonia (-8t), nephritis (-4t), and a variety of incidental findings not usually associated with the death of animals (-23%). These same lesions were observed in the smoke exposed (Table 36) and sham exposed mice (Table 37). In general, the most prevalent neoplastic diseases were hematopoietic tumors and sarcomas. Lesser numbers of lung carcinomas, liver carcinomas, and mammary carcinomas were observed. The most common non-neoplastic lesions observed in the smoke exposed mice were congestion/pneumonia, nephritis, otitis media and otitis externa. As observed in the shelf controls, most of these animals (-25ti) died without diagnosis of a major disease. Analysis of these lesions as a function of time on test will be given in the next section. H. Microscopic Evaluation and Analysis The numbers of tissue samples evaluated microscopically for the smoke, sham, and shelf groups are given in Table 38. A total of 987, 659, and 369 mice were examined for the smoke, sham and shelf groups, respectively. Pulmonary tissues from over 96% of these animals were examined. A random sampling of animals that died during the first year was selected for microscopic examination (i.e. those animals that had ear tags ending in "3"). 108 CTR CONTRACTS 029288 11249038 CTTR I-IN 044~'. 71F

:inal Report Contract: CTR-0030 CTR-101a During the remaining _fze, all non-autolyzed tissues from the respiratory tract and all abnormal tissues were examined. A n average of 93% of the animals from the smoke, sha:m, and shelf groups were examined. An attempt was made to determine the cause of death for each cf these animals. At the time of death or sacrifice, the mice were tabulated as to the circumstances.of death. The circumstances are given in Table 39. The major reasons for death were either conditions that randomly removed the animals from the study (i.e smoke or sham exposure-related, holder-related, or documented air or smoke flow factors), or diseases that likely led to the death of the animal. This tabulation allowed us to analyze the data in two ways (see references 12 and 13 :or details). The first way assumed the lesion caused the death of the animal and thus an "actuarial" table was constructed that compared the numbers of animals that died carrying that specific lesion to be analyzed, to the total number of animals alive at the beginning of that time interval. For the actuarial analysis, the total numbers of animals at risk were either those that were strictly defined to have died as a result of their lesions or were generally defined as the total number of animals that had histopatholoqical examinations. In the Tables for this section, (Tables 40-96), these two analyses are designated as "strict. adherence to died or moribund" and "general adherence to died or, moribund, all." The second method assumed that the lesion did not lead to th. death of the animal. Tn this way, the "incidence" of 'a particular lesion at a given time interval was compared to the total numbers of animals that died during that time interval. The numbers of animals in this interval were either those animals that were strictly defined to have been taken off test randomly, or generally defined as the total number of animals that died during that interval. In the Tables for this section (Tables 40-96), these two analyses are designated as "stict adherence to random" and "general adherence to random, all." Statistical analyses were determined according to the zethod of !!antel and Haenszel (see Tables 19 and 20) . The procedure is briefly stated as follows. For 2 groups to be compared, the number of animals with a given lesion and the n=ber of animals which were at risk !or that lesion for each time interval were used to construct 2 x 2 contingency tables. The number of lesions expected and the variance of this number can then be determined for each interval. The sum of the expected values was treated as an approximately normal r*andoa variable with known mean and variance. The Chi-Squace Statistic, corrected or uncorrected for continuity, was then used to determine tse level of significance for the difference between the expected number of lesions and observed number of lesions :or 2~:xperimental groups over any given time interval. 109 C.TR CONTRACTS 029289 11249039 CTR I-IN 0440*1-"E-33

Pinal Report Contract: CTR-0030 CTR-101A . The lesions were analyzed in the following sequence: 1) lung cancers, 2) non-neoplastic respiratory t:act lesions, 3) other non-neoplastic lesions, 4) all malignancies, 5) head and neck fibrosarcomas, and 6) hematopoietic tumors. Because of the statistical methods used, up to 4 different analyses may be cresented for these lesions. Both the corrected and uncorrected "p" values are presented for every 4-week interval (Tables 40-96). The uncorrected value is given in the parenthesis in the tables. Any probabilities Less than or equal to 0.05 are denoted with asterisks. 1. Gung Cancers The incidence of the lung lesions designated avCN and ACN are presented in Tables 40 and 41. A total of 38/940 smoke exposed and 21/630 sham exposed animals were observed to develop these lesions (see Table 40). In those animals defined as randomly removed from test (see Table 41), a total of 11/463 smoke exposed and 3/269 sham exposed animals d.veloped these lesions. Although smoke > sham, the differences did not reach p < 0.05 at any given time lnterval. The comparison of the occurrence of lung carcinomai in smoke exposed and sham exposed mice is presented in Tables' 42-45. All lung cancers were AAC. Under the assumption that the cancers directly led to the death of the mice, the analyses presented in Tables 42 and 43 were generated. A total of 19 luno, carcinomas out of a total of 97• animals observed in the smoke exposed group, while 7 lung cancers out of 651 sham animals were observed (Table 42). The rate of development of AAC was different at p0.14 (uncorrected, p0.08) by 785 days of smoke exposure. Actuarial analysis usinq the animals that were assumed to have died of these cancers (see Table 43), showed that there was no difference between the smoke exposed' and sham controls in terms of the incidence or latency of lung carcinomas. A comparison of the occurrence of lung carcinomas between smoke exposed and sham controls as analyzed by "incidence" is shown in Tables 44 and 45. When all the animals :+ere analyzed (see Table 44), the smoke exposed mice had a higher incidence compared to the sham exposed at p=0.10 (uncorrected: p-0.07) by 757 days of smoke exposure. When only those animals assu{med to have been randomly removed from test are analyzed (see ':able 45), the smoke exposed had a higher incidence compared to the sham at pn0.17 (uncorrected: p•0.07). Thus, in both cases, the incidence and/or latency of lung carcinomas was higher in the smoke exposed mice, but the difference was not less than p0.05. Data discussed in Section LII.D. showed that the lesion defined as ACN possessed the capacity to progress to overt 110 CTR CCh{TRACTS 029290 11249040 CTR HN 04407"'9

rinal Report Contract: CTR-0030 CTR-10171 carcinoma. Thus, we analyzed the occirzence of either ACN or AaC in both the smoke and sham groups. Assumption that these lesions led to death of the animals yielded the analyses presented in Table 46. A total of 49 lesions out of 978 smoke exposed animals :rere observed, compared to 28 lesions out of 651 sham exposed controls. This difference reached p-0.18 (uncorrected; ps0.14) by 813 days of observation. By incidence analysis•(see Table 48), this difference reached p=0.09 (uncorrected; p0•.07) at the sane time. Analysis of data from either those animals strictly defined to have died of their lesion (Table 47), or those strictly defined as not having died of their lesion (Table 49), showed that the smoke exposed group was not different from the sham exposed group. The final method compared the occur:ence of all alveoloqenic lesions (i.e. aNCN, ACN, or )LAC) in both the smoke exposed or sham exposed groups (Tables 50-53). This analysis yielded results similar to those reported in the previous Tables. The occurrence of these lung lesions was slightly higher and appeared slightly earlier in smoke exposed compared to sham exposed mice. The differences never reached p< 0.05. 2. :1on-neoplastic Respiratory Tract Lesions A total of five lesions were analyzed. They were PAMA, congestion, rhinitis, otitis media and otitis externa. The overall incidence of•PAlU1 for the smoke exposed group was 158/978 (Table 54). The incidence of PAMA in the sham exposed groups was 1/651. PAriA was first observed between 309-336 days on test. The number of PAl4As gradually increased during the second year, reaching an average incidence of -35% during the -last 4 months before exposures were terminated at 110 weeks. This incidence was considerably lower than what was observed in the previous CTR-100, described ia Section III. Congestion was defined as excessive blood in vasculature. It can be caused by an active process resulting in lunq irritation, a passive process resulting in reduced flow of blood from the lung, or an artifact usually caused by a delayed death/necropsy interval. A comparison of the incidence of congestion in smoke and sham exposed mice is given in Tables 5S and 56. In both cases, congestion was a rather cc=on lesion and the incidence was actually greater in the sham exposed mice. The incidence of congestion in the shelf controls was" 37/318. The cause of this congestion could not be determined, and it is regarded as an incidental finding. Rhinitis was defined to be inflammation of the nasal cavity. It can be caused by adventitious agents, foreign material, or other nasal irritants. The ineidence of this lesion in both smoke and sham exposed animals is presented in Tables 57 and 58. The incidence was not diffrcent in the smoke or sham exposed mice (14/502 vs. 8/319). 111 'CTR COHTRRCTS 029291 11249041 CTR PIN 0-1'4080

einal Qeport Contract: CTR-0030 CTR-10171 Otitis media was detined to be a relatively severe inflammatory responae in the middle ear. These lesions are usually of bacterial origin. The eustachian tube, Lined by ciliated epitheli.-n can be a portal of entry and is probably aE:ected by the smoke. Extension of the lesion into the brain can result in death of the mouse. The occurrence of otitis media in smoke or sham exposed mice is given in Tables 59 and 60. The total incidence of otitis media was 66/579 in the smoke exposed mice and 35/371 in the sham exposed mice. The latency period for the expression of this lesion was shorter (p < 0.01) in smoke exposed mice (see Table 59). If just those animals assumed to have died randomly were evaluated, both the latency (po0.01) and final incidence- (po0.05) of otitis media were correlated with smoke exposure (see Table 60). A comparison of the incidencs of otitis externa in smoke and sham exposed mice is given in Tables 61 and 62. Otitis externa, an inflammation of the external ear, was usually considered an incidental finding in the mouse. Tnis lesion occurred quite frequently with a total incidence of 140/579 for the smoke exposed and 78/371 in the sham exposed mice. The final incidence was not different between the two groups, but there was an indication that this lesion occurred with an average shorter latency period in smoke exposed mice (i.e. at 561 days the p-0.14 and uncorrected; po0.09). Analysis of the randomly killed animals (Table 62) came to the same conclusion. The Final incidences were not different, but by 561 days there was a 90% probability (94% if uncorrected), that the smoke exposed mice had a higher cumulative incidence of otitis externa, than the sham exposed mice. Tendency of mice to preen themselves following deposition of material about their face may have caused this ear lesion. The incidence of either otitis media or otitis externa in smoke and sham exposed mice is given in Tables 63 and 64. As expected, regardless of the method of analysis, there was a shorter latency period (p<0.01) for the development of these lesions in smoke exposed mice. 3. Other Non-neoplastic Lesions The occurrence of nephritis, an inflammatory lesion of t:ro kidneys, is presented in Tab1eE 65 and 66. This lesion can be caused by adventitious agents, foreign Raterial or other 4zritants. This lesion seemed to be fairly frequent in older age BC3°1/CZtm mice, but smoke exposure had no affect on the incidence or latency of nephri,tis in these mice. It is considered an incidental finding. 112 CTR CaNTRRCTS 029292 11249042 ~ rY" TI 9 1 IN 044081

?inal Report Contract: CTR-0030 CTR-101A 4. Occurrence of all Malignancies A comparison of the occurrence of all malignancies regardless of type or tissue location is given in ':ables 67-70. Assuming these malignancies led to the death of the animal yielded the.analyses presented in Tables 67 and 68. The total incidence of all malignancies was 263/987 for the smoke exposed mice and 189/659 for sham exposed mice. Neither the incidence nor the latency period for development of these malignancies was different between these groups. A-ssuming these tumors did not lead to the death of the animal 'yielded the analyses depicted in Tables 69 and 70. There were early indications of differences between the smoke and sham groups (i.e. corrected, p=0.24= uncorrected, p0.01, 197-308 days), but these differences reflected the observation of only one tumor in the sham group and the relative few numbers of sham animals that had been randomly taken off test up until these time intervals. By 757 days, there was a indication that the cumulative incidence of tumors was higher (p-0.04) in the sham exposed mice than the smoke exposed mice. 5. Occurrence of Head and Neck Fibrosarcomas • A particular ty- pe of tumor, head and neck fibrosarcomaf was observed with a higher frequency in the smoke and sham exposed groups compared to the shelf control group. A comparison of the occurrence of these tumors in the smoke and saam exposed mice is presented in Tables 71-73'. These 'tumors were highly aggressive, and metastatic lesions were often found in tissues far removed from the primary cancer.. A total of 29 head and neck fibrosarcomas were observed in the smoke exposed group and a total of 8 tumors were observed in the sham exposed group (3ee Table 71) . Analysis of data by either actuarial ('"ables 71 and 72) or by incidence (Table 73) showed that the head and neck fibrosarcoma occurred more frequently and earlier in the smoke exposed mice than in the sham exposed control (p< 0.05) . T`:e mechanism by which these tumors developed is unclear- Wherher such factors as localized deposition of smoke particulates in this area and/or the relative severity of neck cuts or abrasions caused by chronic restraint played a role in the-formation of these t::mors is speculation at this time. 113 C.TR CC1NTRRCTS 029293 11249043 CTR MN 0440822

Final Report Contract: CTR-0030 CTR-lOlA 6. Occur:ence of Hematopoietic Cancers :he occurrence of lj:nphosarcomas, reticulum cell sarcomas, or leukemias in the smoke or sham exposed mice is given in Tables 74-77. These cancers were of hematopoeitic origin and are discussed together. Assuming these tumors led to the death of the 3ice -(Tables 74 and 75) showed that these tumors occurred more frequently (p< 0.01) and earlier (by 729 days) in the sham exposed mice compared to the smoke exposed mice. By incidence analysis (Table 76), the occurrence of these tumors in sham exposed mice. (126/6S9) compared to the smoke exposed mice (125/987) was greater at p<0.01. It was obvious, however, that most of these tumors :+era lethal to the mice because :+hen analyzing only those animals that were known to be randomly removed from test (see Table 77) only 29 out of the 125 tumors in the smoke group and 14 out of the 126 tumors in the sham group were able to be analyzed. In this fairly small population, there was no difference in tumor incidence or latency between the smoke and sham mice (see Table 77). 7. Conclusions A variety of malignant and non-maliqnant lesions• were observed in this study. The total occurrence of these lesions was quite similar in both the smoke and sham qroups; but the relative distribution of lesions in these two groups of mice was quite different. Lesions around the head, ear and respiratory tract such as, head/neck fibrosarcomas, otitis media, otitis externa, and lung lesions (AVCN, ACN, and AAC) were all higher in smoke exposed mice. On the other hand, the occurrence of laukemias, lymphosarcomas, and reticulum cell sarcomas was higher in the sham exposed animals. The results would seem to be a classic example of competing risks, where the occurrence of a specific lesion was altered by the simultaneous expression of another pathological change that removed the animal from the study. The mice died with approximately the same average latency period in both the smoke and sham groups, but the types of :esions found at death would seem to be influenced by the daily exposure to 2R1 cigarette smoke. I. Effect of 2R1 Cigarette Smoke on Mice Pretreated with BaP 1. Rationale Previous studies from this laboratory have shown that the respiratory tract of 3C2F1 :nice is sensitive to MCA or 114 CTR caNTRACTS 029294 11249044 Cf f'~'e f f N ti..A ~°f440"83

Final Report Contract: CTR-0030 CTR-lOlA • BaP-induced lung cancers (see Section III of this report and References 12 and 13). Study CTR-100 showed that daily exposure to 2A1 cigarette smoke had little effect on type, latency or incidence of MCA-induced lung cancers. It was reasoned that because the MCA induced lung cancers occurred so rapidly, (i.e. <40 weeks), that any potential influence of daily smoke exposure was not detected. Thus, for this study, a weaker chemical carcinogen was used, SaP, and doses were selected so that tumors would be expected only after a 12-18 month latency period. Therefore, the BaP treated mice could be exposed to 2R1 cigarette smoke for at least 12-18 months before large numbers of lung cancers would be expected to occur. This approach should allow the evaluation of the potential co-carcinoqenic and/or promotinq effects of daily 2R1 cigarette smoke exposu:s. 2. Daily Observations A summary of numbers of animals on test as a function of time after initiation of smoke exposure was given in Tables 2 and 3. A total of 320, 260 and 130 mice were put on test for the BaP + smoke, BaP + sham, and BaP + shelf groups, respectively. For these groups, a total of 225, 202, and 10J animals were diagnosed for evidence of lung cancer. As observed for the groups exposed to smoke alone (see previous section), after 20-30 weeks of exposure, -20t of the animals were observed to have reddened skin and worn-away hair around the neck area that fit into the holders. Many of these lesions progressed to open sores by 40-60 weeks on test. By 50 weeks, many of the animals had evidence of respiratory distress and by 80 weeks, greater than 801 of the remaining animals were observed to have pulmonary problems. Microscopic evaluation of these animals will be discussed later in Section 1.6. There were no obvious differences in clinical signs among the BaP + smoke, BaP + sham, or BaP + shelf groups. A comparison of body weights of these animals as a function of time on test is given in Figure 35. The BaP + shelf groups doubled their weight between initiation of the study and approximately 60 weeks on test. The BaP + sham and BaP + smoke groups failed to gain weight as rapidly as the shelf controls. Theae data were quite similar to that for the non-BaP-treated mice presented in Figure 33. Daily exposure to smoke or sham treatment suppressed normal weight gain of these mice and by 60 weeks on test, the smoke-or-sham exposed animals weighed an average of -32 q, while the shelf controls weigh an average of 50 g. All three groups of animals tended to lose weight from 60 weeks to termination at 120 weeks. 11S . CTR CONTRRCTS 029295 11249045 W./' ! / 7 ! MI ! I 044084

Final Report Contract: CTR-0030 CTR-l0U 3. Documentation of Smoke Generation Smoke generation was documented as described in Section IV.D. ?or the BaP-treated group, an average o~ 200 ug TPa was esti3ated to be deposited per day of smoke exposure. Thus, for every week on test, the pulmonary tissue of these mice were exposed to approximately one mg TPM. 4. Documentation of Inhalation of Smoke At monthly intervals, blood gas analyses were performed to ascertain percent carboxyhemoglobin (t COHb) in these three BaP-treated groups. The procedures were described in Section IV.D. A comparison of the % COHb in the BaP-treated and non-treated animals for the first year of smoke exposure is presented in Figure 36. BaP-pretreatment had no effect on the ability of these mice to inhale the smoke aerosol. 5. Survival The surviving fraction of animals as a function of' time on test is presented in Figure 27. These curves were correc ted for those animals that were randomly removed from the study. The survival curve for the BaP + smoke group decreased more rapidly in the first 20 we.ks of exposure than did the Bap * sham group. In turn, the survival curve for the BaP + sham group decreased more rapidly than the BaP + shelf group. Some toxicity related to smoke or sham exposure was occurring.. By 70-80 weeks on test all three groups had attained similar levels of survival and in fact, "t::e eaP + shelf group had the steepest decrease in survival post 80 weeks. Our estimate of cause of death for these animals is presented in Tables 78-80, for the BaP + shelf, BaP + smoke, and BaP + sham groups, respectively. (Note: These animals were found dead in the cage or were moribund. A11 animals that died durinq exposure, in the holders, or of a known accident are not included in this Table but will be discussed later). Approximately 801 of the animals died of hematopoietic cancers, fib Cosarcomas, congestion,and otitis externa. About 85% of these lung cancers were AAC, 10% were poorly differentiated carcinomas, and S: were SCC, or ASC. Analyses of these lesions as a function of time of smoke or sham exposure will be given in the next section. 116 CTR CONTRRCTS 029296 11249046 rW' I R I IN 044,0855

Final Report Contzact: CTR-0030 CTR-101A 6. rticroscopic Evaluation and Analysis A total o: 223 BaP + smoke and 202 HaP + sham animals were examined microscopically for evidence of respiratory lesions. An attempt was made to determine the cause of death for each of these animals, as previously described in Section IV.H. At the time of death or sacrifice, the animals were tabulated as to the circumstances of death. These circumstances are presented in Table 81. The major reasons for death were either conditions that randomly removed the animals from the study (i.e. test sacrifices, death on smoking machine, or death in holders), or diseases that' likely led to the death of.the animals. In the case of BaP-treated animals, most of the animals that lived past one year after chemical treatment died with pulmonary carcinoma. Statistical analysis of the data from these BaP-treated animals was performed as described in Section IV.H. The incidence of PAMA in the BaP + smoke and BaP + sham exposed animals is presented in Table 82. The overall incidence was 115/22S or 311. The first PAMAS were observed between 57-84 days on test and were observed in approximately SO• of the animals throughout the study. This was a contrast to the incidence of PAlU1a inn non-BaP-treated animals (see Table 54), where the final incidence was lower -(20t) and the time of first occurrence was later (309-336 days on test). It would seem that BaP pre-treatment modified the macrophage response resulting in higher incidence of these pigmented cells. The incidence of ANCU and/or ACDi are given in Tables 83 and 84. Analyzing the incidence of those lesions at 4 week intervals compared to either those animals dying randomly (strict adherence to random - Table 84), or all animals dying in that time interval (Table 83), showed that at no time was the BaP + sham group different from the BaP + smoke group. The incidence or latency of these alveologenic lesions was not different between smoke and sham groups. Analyses of occurrence of alveologenic lesions (i.e. aVCU, ACN) or neoplastic tumors (i.e. AAC, SCC and POC) are presented in Tables 8S to 96. Three sets of analyses are given. Tables 8S to 88 present a comparison of lung carcinomas in the 3aP + smoke to that of the BaP + sham groups. Table 89 to 92 present a comparison of lung carcinomas, plus the ACL1 lesion for botti the BAP + smoke and BaP + sham groups. Tables 93 and 96 present a comparison of all alveoloqenic lesions for 9aP + smoke and BaP + sham groups. 9aP-induced carcinomas (i.e. AAC, POC, and SCC) appeared slightly faster in the sham exposed group, although statistical differences were observed only when analyzed as described in Table 87. By 477 days of exposure, the smoke exposed mice had fewer SaP-induced carcinomas than the sham exposed controls. This observation may result from the fact tha: 117 CTR CC]HTRRCTS 029297 11249047 CTR HN 044C-1860

Final Report Contract: CTR-0030 CTR-101A a much higher Yercentage of smoke exposed anisals died during the first 200 days of exposure co:apared to the sham exposed animals. These animals were taken off test before they could develop any BaP-induced cancers. Comparing the SaP + smoke exposed mice to the 9aP + sham exposed,csice t::roughout the observation period of -900 days showed that the relative frequency of occurrence of lung carcinomas andlor ANCN or ACN was similar in the two groups, (see Tables 89-96). Daily exposure to 2R1 cigarette smoke failed tc alter the incidence or latency of eaP-induced alveologenic lesions or pulmonary carcinomas. 118 cTR coHTRRCTS o29~sa 11249048 CTR HN 04408'1-r`

Final Report Cotstsaett CTR-0030 CTR-101A TABLES 30-39 119 C.TR CaNTE2RCTS 029299 11249049 CTR f IN 044(D88

i fln.l Report Coanct: CTR-00i0 TABLE 30 CNNOtliC 1N11ACATION OF 2R1 CIGARETTE SNORE IN 8C]fl/CUM fElIALE NICE: OISIOSlT10N Of ANINALS NUMBER 0f ANINAIS CeEMICAL E1P0StNE DATE Of FIRST Elli'OSURE INITIAL NUIBER AIiYE AFTER I TEAA ALIVE AFTER 2 TEARS ALIVE AT III YlERS OATE ERPOSWiE TERNINATED None 2R1 Seiote 9/18/18 860 208 96 78 10/24/00 10/17-18/78 840 383 ISI 126 11/21/80 2/5/19 3S] 211 12S lil 3/20/81 TOTAL 205] 86S 382 315 N ' None SAe. 9/18/78 430 210 65 58 10/24/80 10/ 17-18/78 410 250 101 87 11/21/80 ~ ~.. 2/S/19 17/ 139 96 77 3/20/81 ~ TOTAL 101/ 599 262 222 ° None Shelf-Control 9/18/)8 26S 247 143 98 N/A ~ ~ ~ (MosR) 10/17-18/18 /SS 1S2 71 62 - 2/S/79 29 28 20 16 ~ 0 TOTAL 449 627 237 176 OAI 2Rl S.ot. 9/25/78 200 29 1 s ~ 3/12/79 120 100 9 _ - ~ ~ - TOTAL 320 129 !0 - •pr. ,,.~ 8AY S11/41 9/25/18 200 1]] 8 _ - ~ (Npnc) 3/12/19 60 37 8 _ ~-^~ `w~ TOTAL 260 170 16 - ~ 8Ap Shel(-Control 9/25/78 100 96 - - N/A e N - 3/12/19 30 22 2 = ~ TOTAL 130 IIB 2 0 0 e Nut .ppllc.ole. 6 s were Soute d os 8AP d d 6 g~( eapose . yruup or s cm e.p e •treae Anlwls le the

floal Report Coatract: C1R-0010 I TAt4tE 11 CIWONIC 11YUU1TIOM 0f 2R1 CIGARETTE SMDRE IN Klfl/CIM NICE DISPOSITION Of.AtlINILLS fOR MISTOPAi110lOGY ~ ~ YEAR 1 YEAR 2 fEAR 3 CuMUlAT1YE o h / OM IE/D. N01 c NO NECAOPSr NO NECMOPSY ND MUttOPSY 11E1D, NOT NO NECROPSt CIEIIICAI ElPOSuRE TEST DIAGNOSED p10CESSED OR DIAGNOSIS DIAi110SED OR DIAGMOSIS DIAGNOSED C4 DIAGNOSIS DIAGNOSED pNOCESSED OR DIAGNOSIS None 201 Soot. 20S1 1S4 59 97S 42S 21 406 1 995 59 1009 Non. Slw. 1014 .)h 14 325 111 10 277 6 6S9d 14 340 Non. Nm. 449 3 0 19 129 40 237 21 369 0 e0 Rap 2R1 5.ot. 320 10S 2 84 117 6 9 1 227 2 91 llap Sha. 260 52 1 31 13S 19 16 D' 203 1 56 liap No.. 130 2 0 lo 100 16 2 - 104 0 ' 26 letr.tr.che.l Lst/llatlon once per week for three reeks prlor to SiJI 11 .aposuro; 1.2 0s Renzo(a)pyren. (Oap) In 0.02 .1 of 0.21 gel.tln-sallne was lL.a per Lst/ll.t/o.. Co.dillons for s.oke eaposure conslsteJ of 101 201 clyarelte s.oke; 20 seconds of s.ok. alternatl.g wlth 40 seconds of air for a perlod of 6-fi .Iiwtes. flr. swch e.pos.res wer. 9/ren each day. SAad aposurr w.s tb sar" without s.ok.. A+r1.9 the first sl. .onths o/ the eperirent, appro.l.clely 201 of the anl.als (anl.al numbers endlny In ] or tl) Md their tissues saved at .ecropsy. Only 101 (anlwal w.m•ers cedl.y 1• 3) were processed f.r hlstepalh.loglc dlynos/s. Contalns 2 anb.els that went leto the /ourth lear.

Fina1 Report Contract: CTR-0030 TABLE 32 08SERVATIOfl COOES A Accidental Death a 8 Blind b i.t. death C Lethargic c Overdose death 0 Ceath (spontaneous) d E E-nnaci ated e In holder before loading F Fighter, Injury f On module G Good 9 After exposure H Harvested h Twisted neck/broken neck 1 Dehydration/starvation 1 Improper loading J Culture i Fel l o ff modul e/dropped K Killed (moribund) k L Leukemia k M Moribund m Power failure N Respiratory ailment n Neck cut ~ 0 Heel cut P Pathology p flose cut Q Test sacrifice q R Runt r Cage and/or Feeder accident S Skin disease s Water accident T tuoor (Gross only) t After eye-bleeding U Sick u Bled for COHb V Kyphosis v Bled for seralogy W Papilloma w X Experiment terminated x Y Dysentery y Z Necrotic (Gross only) z i Cannibalized ? Lung observation ~ t!achine accident • Missing (Assumed deao) + Path taken (Norm.al) : Rx death [ Sent for external testing S Terminatior, of ;roups iu CTR CQNT RRCT5 029302 11249052 CTR NN 044,091

Final Report Contract: CTR-0030 TABLE 33 SU11VARIZED DOCUMENTATION FOR SMOKE EXPOSURES OF BC3F1/CUM MICE GROUP SERIES # f OF MICE OF FIRST EXPOSURE OFOFIRST SMOKE RECORD OFOLAST SMOKE RECORD (110 WEEKS) TPM 1XP SURE PER GROUP (a) 0181Ai 1 85 9/18/78 3/23/79 10/24/80 345 01G18• 2 65 9/18/78 3/23/79 10/24/80 346 018ZA4 1 10 9/18/78 5/03/79 10/24/80 347 0184Ai i 65 9/18/78 5/03/79 10/24/80 346 01848j t 75 9/18/78 S/03/79 10/24/80 349 0184Ca 1 90 9/18/78 5/03/79 10/24/80 345 01840' 1 75 9/18/78 3/23/79 10/24/80 340 01850 2 75 9/18/78 3/23/79 10/24/80 342 01858a 2 85 9/18/78 3/23/79 10/24/80 342 0185Cll 2 90 9/18/78 3/23/79 10/24/80 342 , .850a 2 90 9/18/78 3/23/79 10/24/80 342 01850 2 55 9/18/78 5/03/79 10/24/80 346 01 !15 F s 3 to0 10/17/78 4/06/79 11/21/80 336 0185G b 3 35 10/17/78 5/03/79 11/21/80 346 01B6Ab 3 100 10/17/78 3/23/79 11/21/80 337 01868b 3 100 10/17/78 S/03179 1 1 /21'/80 342 C185Cb 3 100 10/17/78 3/23/79 11/21/80 339 01860y 3 100 t0/17/78 S/03/79 11/21/80 338 0196Ey 3 100 10/17/78 3/23/79 11/21/80 334 0186Fy 3 105 10/17/78 5/03/79 11/21/80 337 0197ay 4 100 10/18/78 3/23/79 11/21/80 349 0190Ay S 117 02/05/79 3/23/79 3/20/81 312 01608~ S 117 02/05/79 5/03%7~ 3/20/81 319 01s0C` S 119 02/05/79 3/23/79 3/20/81 318 a No smoke exposures were given during weeks 101, 104, 106, tnd. 108. No s.moke exposures were given during weeks 97, 100. 102, 104, 106, and 108. No smoke exposures werz givern during week 82. 123 CTR CONTRRCTS 029303 11249053 t,.x f f'"'ti i i N 044,092

I i I I I: I, I~, I I, I I 'I I I i I I I I I I I I I i I I I I, N O } J En ~ ~...1 A 7u ~ { N P Ftn..i Report Contract: CIR-0030 TABLE INCIDENCE Of TOTAL ItEMOGL08IN (118), PERCENT OXYiIEMOGLOBIN (X 0211B) AND PERCENT METNEMOGLOBIN (X MET HB) IN 8C3F1 MICE DURING LONG-TERN EXPOSURE TO 2111 CIGARETTE SMOKEa . HB G/DL Z 02HB X MET H B DATE MONTH SMOKE SHAM SHELF SMOKE SHAM SIIELF SMOKE SHAM SHELF 0 '79 1 18.11.6 16.31.8 151.8 45.2:6.8 3S.4s4.2 48.3s5.5 I.Is0.1 0.50.2 1.110.7 .1 '80 2 16.55s.37 13.212.1 12.61.9 49.1s1.7 41.9s9.5 51.5s3.4 0.910.2 0.5:0.3 1.1:0.4 f '80 3 16.6s.5 15.5=.9 14.41.4 43.5s3.3 36.4s9.7 36.4:9.7 1.2:0.2 0.510.2 1.110.4 N '80 4 16.011.1 15.21.8 14.3s.0.9 48.3s4.5 45.5s6.1 47•5t3.4 1.4to.4 0.8:0.3 1.110.3 A '80 5 15•9s.4 15.41.6 14.0s.7 43.4s10.1 37.516.2 45•7s6.2 0.8:0.4 0.90.2 1.51.41 K '80 6 15•9s1.0 13.oi.6 12.811.4 40.2110.3 37.1:2.6 45.214.5 0.4:0.4 0.8s,23 1.210.5 j '80 7 15•9s•2 11.4:1.0 10.5s2.3 32.2t3.3 47.812.9 48.os5.5 1.0410.3 1.11.04 1.110.4 j '80 8 14.6s1.8 io.8s1.9 10.7s2.6 49.8t3.5 ti3.8s6.1 48.2s4.1 0.90.2 0.710.1 0.810.2 A 680 9 15.111.4 10.9s2.1 1o.1s1.3 47.9s2.3 45.7:4.4 44.8s3.8 0.9=0.5 1.os.33 2.611.4 S '80 10 12.21.8 10.3s1.4 10.8s1.3 42.7s2.9 40.6:2.2 40.5s3.9 1.1:0.2 0.930.3 1.1s0.8 0 '80 11 13.6s1.9 11.9se.8 11.010.9 52.9s4.3 31.5s4.0 38.4s6.3 0.910.4 0.710.3 0.710.2 N '8o 12 13.7s1.3 1o.4s1.8 10.6s1.7 45.3s4.2 43.6s4.6 0.4:0.3 0.3t0.3 0.7:0.2 0.8:0.3 0 '80 13 13.6:1.8 11.5:1.4 12.812.2 46.5s4.5 45•1s4.8 43.7:4 0.9:0.6 0.6:0.4 0.610.2 15•2st.6 12.812.2 12.3s1.8 ti5.3t4.8 4o.9s4.9 4y.6s4.4 0.910.3 0.810.2 1.110.6 a Data are from the last 13 months of smoke exposure from Sertes•I and 2 antmals.

TAOLE 35 INCtOENCE Of 1ES10tIS TIIAT LIKELY CAOSEO TNE OEATN Of SNEIf COMTMOL RClf1/CUM NICE PEM100 OF 06SERYATION YEAR 1 lifE OF tESION Mu1s1ER 1 tyrpbsarcora / (33) Congestloa 1 (3)) Mo major disease autod 1 (33) YEAR 2 ifPE OF LESION NutBEN NEOPtASTIC l~.p1wsarco.a, retlcul~ 4) c- al l sarca.e. laekodas Sarcros ~ fl6rosarcows Nead. esck 2 Other 2 lung carcinoma 4 Llvor carcinoma 4 Namwary carcinoma 3 OtAor carcinoma 3 NOM-NEOPtASTIC MepMlt/s, cystltls, f Conyest/on. pae.oo./a, 7 iwg inflai.atary los/ons No .aJor dlseau aoted, but 36 rltA Lcidental fl.dfogs of oatraoedullery Mrstopolesls, otltlt .od/a and estoroa, uterlae Aydro.etra, lwy granulora, IuM Ne.orrAago spleen and liver ..crosls, thyroid AYperpiaslo TEAR 3 I TTPE Of EESION NtaBER i MEOPLAS IC (31) t1.pbsarcm, r.tlwlu. ~ (28) (6) «ll sarcm, leole.las Sarcomas g (1) f/erosarcu.as f~j o ~:~ ~~ 22 f;~ (3) lway carclam 14 (6) (3) Liver carcinoma 12 (5) (2) Naary carclno u 9 (4) (2) Other carcinoma 2 (1) NON-NEOPEASIIC (4) IlepArttfs, cystltls 6 (~) (6) Congestton. pneu.onla. 22 (9) (28) lung intla+.atorr loslons Adanms of lung. llver, 13 (5) p/tutarr. thyroid. .a..arr gland No major disease notod, but SS* (23) with tncld.atal flndings of a~lonlc hyperplatla luny thro.posls. liver necrosls. liver Newanglo.a. lung granulo.a, alveolar .acro- pAage accu.ulatlon. o.tra- ardullary penat.potesis, ot/tis media and osterna.. ovarian necrosis. enterltls.hep.t/tlt } . TOTAI NtRNER Of ANIMALS 3 121 . 230

I I I flnal Rcport Contract: C1R-0070 1EAR I TYPE 0f tES10N f /drosarca.a Con9estloa No major disoasa noted. bet with locldeata) flodlags of alveolar •yperplasla aM 1uq Merorrhaye 39 (21) NON-NEOPtASi1C Nepbrltts, cystltts, 11 ttdney /aftltrat.s Conyestloo. poei.oola 21 1uq INfl.ratory loslons Alveolar bIrperplasla. 3 alveolar aoo-t:dqrossioa and co.prass/oy Nodulas Ot/tls oodla or eatarsa 10 No major disease noted. ]2 but rlth lacldeatal f/od/nqs af olseors Mta- plasla, liver aacrosls, estraasdrllarr Nemato- polesif,hep.tltls, ear .Ites, adeaouas of lung and liver. IOIAI faMtllR Of Is11NAL1 10 • PER100 0f OBSERYATION iAOIE 36 INCIUENCE 0f lES1UNS TWT LIKtIY CAUSED 1HE DEATM 0f t<UF1/CW1 NICE OURING CtMUN11C EfIPOSuRE T0 2R1 CIiARETTE SftOtf YEAR 2 NuraE R T YEAR l iYPE OF LESION MlM6ER I iYPE OF LESION NtN1tlER S I I I 1 (5) NEOPIAS C 3 (17) l sarco.a, r.tlculu. 14 (n) c. 1 sarcos. Iwko.las Sarco.as f/prosarco.as tlead• neck . Other lung carclno.a Liver carcls,o.a Ilr.rr carcinoma ~ Other carcl.o.a 1S0 ()) (17) NEOPIAS IC tr, sarcw; reticulu. m (10) co 1 sarca.as, lauk..fas Sarco.as 11 (5) flprosarco.as Other mock 32 1491 lun9 carcinoma f (1) Liver carcinoma 10 (3) Il...arr carcinoma s (2) Other carcinoma 2 (1) NON-NEOPIASrIC Napbrttls, c~s tltls. 25 (1) tldnar Isflltrates Congestlo.. pneu.oola ]9 (IJ) lung lafla..atory loslons (2) Alveolar Nlrpuplasta, 1) (4) (6) (20) alveolar sne-coepresslnn aad co.pr.ssly tadries Adeaosas of llver t thyroid 7 (1) 0titls media or estenu 41 (12) No major dlseas. .oted, 'i! (20) but with lac/d.ntal flndleys gf utra- aedoillary araatopo lesis. liver aacrasls. Aeptlt/s, telaagl.ctK11 ear .Ites, uterlac .etritis, thyroid cyst. rh1/tls, spioslc ectasla. l.ng AcaorrWge 343 0

Ilnal Report coolract: CIR-0070 lAtllE 3) InC10lNCE Of LESIONS 11Y1I LlKEL1 LEAD TO THE DEATH Of K3fl/CIRI MICE ouRIRi CMRORIC SILAII EtlOSURE PER100 Of OBSERYAIION T E A R I I E A R 2 TTPE Of LESIOM MtMOER I 1tPE 0/' lESIOM IoUMBER I MEO/tAi C Maoetr9 carclaos 1 (33) Ll.plasarco.., rot/culu. 36 (32) Mu w Jor dlseaso .otod 2 (61) call tarwt, loula.lao Sarcooas ) (6) f /broterca.as M d ] ea , oock OtMr 6 1~1 . luag urclnoae 2 (2) N A Liver carclno.a I (1) tD O tn "A It.asrr carc/.a.a Other oeoplasu 1 1 (1) (1) V ?U bOM-K 1L NepAr/tta, cystltls 1 (6) n n N Rld.q latlitratu Coagettlo., pneusoala, 20 (1)) -4 iw fl l / --4 z ae.a y a ory loslo.s Alveolar byperplasta, S (4) C Y • oIveolar w-co.pr.sstoy 843 co.pretsip oodulea 0tttlt .edla or eelatwa 4 (3) ~ ~x l J No .aJor dlseases ootod, 26 (22) "42.` but rllA /aclde.tal .r.- 9 Ln 0 (lod/n's wch as liver oecrosls, luy' ranr- lo.., o.tra.edullary a..alup~ Ltls, u/erloe 6,vperplas/a.osseous rta- .~. N plasia, perltonltls. eooslos/s, iaourrMye "!.6 W ~ 101A1 MaOER Of AMIW1lS ] NJ 120 ;sJ raft s ! Y E A R 3 1tPE Of 1ES10M Ru1bER 1 RE0Pl1lSf (C lt.pbosarco.a, reticulum 13 (28) coli sarco.a, loutoe/as Sarcoers l0 (4) f 10rosarcve,at 0~:;~° 10 l;~ tung carclw.a S (2) Liver carcinoma e (3 1tro.ary carctnou 6 (21 OtMr oeoplas.s 3 (1) R0M-REOPI/ISilt Mepbr/t1s; c)itltts Is (7) kidney /otiltrates Coagestiow, poaeaoola, Is (6) luag Intlaa.atory lesloa Alveolar Ayparpl.s/a. !< (3) alveolar oo.-co.prass/oy aod co.prestlM bodeles 0tltls eiedla or eaterna 32 (12) No maJor dlseases noted, •67 (26) but wItw Inclde.tal t/odlags tuch as eatra- .edrllary ettasla, uter/ne endo.etrltlt, uterloe Oyp.r- platla, avarlaa bwto- cytt, realogllit. liver edeaora, bepatltls, leleo- 9lectaslt, ear sites. ra/altls, 1lardarlao yland adeno.., eustacA/tts, hewrrlu9/ 25!

Final Report Contract: CTR-0030 TABLE 38 NUMBERS OF TISSUE SAMPLES EVALUATED HISTOPATHOLOGICALLY a,b NUMBER OF SAMPLES SMOKE SNAM SHELF Lung 978 651 356 Trachea 601 376 196 . Ear 287 174 94 Nose 280 196 75 Head/Neck 488 334 188 Back/Shoulder 15 9 6 Heart 171 107 75 Liver 282 180 171 Kidney/Bladder 251 163 114 Uterus/Ce.rvix/Ovary 212 156 99 Stomach/Small and Large Intestine 178 104. 90 Mammary Gland 18 13 14 Adrenal 188 117 69 Spleen 271 179 146 TOTAL ANIMALS 987 659 - 369 b a Tissue sa.mples that were diagnosed as "autolyzed°; "tissues not submitted"; or "no diagncsis" are not Included. Lungs, all abnormal tissues, a random sampling of animals that died throughout the study, and all animals that died post-termination of smoke exposure were necropsied. 128 CTR CQNTRRCTS 029308 11249058 CTR I-IN 04409 .7

T1na1 R•pott Cont=act: C;R-0030 CTR-10 U , TABGES 40-53 GQNC CANCER INCIDENCE/aCTQARIAL 130 . CTR CaNTRRCTS 029310 11249060 CTIR HV# 0440c..19

TABLE 40 INCIDENCE OF ALVEOLaR NON-COMPRESSING NODULES OR COMPRESSING NODQLES I:J BC3F1/CQM MICE DURING LONG-TER.'4 EXPOSURE TO 2R1 CIGaRETTE SMOKE (GENERaL ADHERENCE TO RANDOM, ALL)a,b THU., JAN. 5, 1984 SMOKE SHAM -------------- ---------------- Da.YS ON TEST PRESENT aBSENT PRESENT ABSENT PROBAaIL:TY -------------------------------------------------------------- 1-28 0 9 0 5 1.0 29-56 0 17 0 7 1.0 57-84 0 8 0 3 1.0 85-112 0 16 0 C 1.0 113-140 0 6 0 2 1.0 141-168 0 7 0 3 1.0 169-196 0 11 0 2 1.0 197-224 0 15 0 2 1.0 225-252 0 10 0 5 1.0 253-280 0 13 0 5 1.0 281-308 0 5 0 4 1.0 309-336 0 8 0 7 1.0 337-364 0 24 0 29 1.0 365-392 0 29 0 29 1.0 393-420 0 28 0 24 1.0 421-448 1 23 0 14 1.0 (.45) 449-476 0 23 0 25 1.0 (.45) 477-504 1 21 1 21 1.0 (.67) 505-532 0 28 1 21 i.0 (.85) 533-560 1 36 1 20 1.0 (.70) 561-588 0 28 1 18 .67 (.41) 589-616 1 29 1 29 .70 (.47) 617-644 4 30 1 27 1.0 (.92) 645-672 1 34 0 30 .94 (.73) 673-700 0 60 0 20 .94 (.73) 701-728 1 41 1 23 . 1.0 (.86) 729-756 4 59 0 26 .64 (.48) 751-784 2 37 0 33 . .39 (.29) 785-812 3 28 2 23 .39 (.29) 813-840 4 31 3 20 .49 (.38) 841-868 2 37 3 29 .69 (.S7) 869-896 1 28 1 25 .70 (.59) 897-924 2 3i 1 26 .64 (.53) 925-952 3 31 2 17 .70 (.59) 953-980 3 25 0 13 .51 (.43) 981-1008 1 22 1 21 .53 (.44) 1009-1036 1 37 1 15 .61 (.52) 1037-1064 1 8 0 2 .58 (.49) 1065-1092 0 5 0 3 .58 (.49) 1093-1120 1 2 0 2 .51 (.43) 1121-1148 0 0 0 0 = 940 630 ---------------- -------------------------------------------- -- a Defined as the total number of animals with histopatholoqic examination. b The total number of animals in a qivea time interval is the sum of the number of animala in the Present and Absent col=ns. 131 CTR CaNTRRCTS 029311 11249061 CTR VIN 044100

TABLE 41 INCIDENCE OF ALV80LAR NON-COHPFESSING NODOLES OR ALVEOLAR COMPRESSING NODDLES IN BC3F1/CQM NICE DURING LONG-TERM EXPOSURE TO 2R1 CIGARETTE SMOKE (STRICT ADHERENCE TO RANDOM)a,b TKQ:, JAN. 5, 1984 SMOKE SHAM -------------- ---------------- DAYS ON TEST ?RESE:IT aBSENT PRESENT ABSENT PROBABILITY -------------------------------------------------------------- 1-28 0 a 0 3 ' 1.0 29-56 0 16 0 7 1.0 57-84 0 7 0 3 1.0 85-112 0 16 0 0 1.0 113-140 0 6 0 2 1.0 141-i63 0 7 0 3 1.0 169-196 0 10 0 2 1.0 197-224 0 14 0 2 1.0 225-252 0 10 0 5 1.0 253-280 0 13 0 5 1.0 281-308 0 4 0 4 1.0 309-336 0 5 0 7 1.0 337-364 0 22 0 28 1.0 365-392 0 26 0 28 1.0 393-420 0 21 0 19 1.0 421-448 1 19 0 13 1.0 (.42) 449-476 0 20 0 21 1.0 (.42) 477-504 0 16 0 18 1.0 (.42) 505-532 0 23 1 IS 1.0 (.78). 533-560 1 25 0 12 1.0 (.89) 561-588 0 19 1 11 1.0 (.61) 589-616 1 11 0 18 1.0 (.93) 617-644 2 9 0 7 .77 (.49) 645-672 1 24 0 15 .58 (.36) 673-700 0 31 . 0 6 .58 (.36) 701-728 1 19 0 4 .51 (.31) 729-756 2 31 0 2 .48 (.29) 757-784 2 18 0 4 .36 (.23`, 785-e12 0 1 0 0 .38 (.23) 913-840 0 0 0 0 .38 (.23) 841-868 0 0 0 0 .38 (.23) 869-896 0 0 0 0 .38 (.23) 397-924 0 0. 0 0 .38 (.23) 925-952 0 0 0 0 .38 (.23) 953-980 0 0 0 0 .38 (.23) 991-1008 0 0 0 0 .38 (.23) 1009-1036 0 12 L 5 .66 (.46) 1037-1064 0 0 0 0 .66 (.46) 1065-1092 0 0 0 0 .66 (.46) 1093-1120 0 0 0 0 .66 (.46) 1121-1148 0 - 0 0 - 0 .66 (.46) 11 ~~ ~ _--- ---------------- -------- a a Defined as those animals taken ott test randomly. b The total number of animals in a qiven time interval is the sum of the number of animals in the Present and Absent columns. 132 CTR CONTRRCTS 029312 11249062 CTR PIN 0-1,4101

TABLE 4 2 ACTUARIAL ANALYSIS OF 8C3F1/CUM MICE DYING OF LUNG CANCER DURING LONG-TER.`i EXPOSURE TO 2R1 CIGARETTE SMOKE (GENERAL ADHERENCE TO DIED OR MORIBUND, ALL)a,b MON., JAN 2, 1984 SMOKE SHAM ------------------ ------------------ DAYS ON TEST D/W TUMOR AT RISK 0 W/TUHOR AT RISK PROBABILITY ------------------------------------------------=---------------- 1-28 0 978 0 651 1.0 29-56 0 969 0 469 1.0 57-84 0 962 0 639 1.0 85-112 0 944 0 636 1.0 113-140 0 928 0. 636 1.0 141-168 0 922 0 634 1.0 169-196 0 914 0 631 1.0 197-224 0 904 0 629 1.0 225-252 0 889 0 627 1.0 253-280 0 879 0 622 1.0 281-308 0 866 0 617 1.0 309-336 0 861 0 613 L:0 337-364 0 853 0 606 1.0 365-392 0 829 0 577 1.0 393-420 0 800 0 548 1.0 421-448 1 772 0 524 1.0 (.41) 449-476 0 748 1 510 1.0 (.79) 477-504 0 724 0 486 1.0 (.79) 505-532 0 703 0 463 1.0 (.79) 533-560 0 67S 0 441 1.0 (.79) 561-588 1 638 0 420 1.0 (.81) 589-616 1 610 0 401 .92 (.54) .617-644 0 580 0 371 .92 (.54) 645-672 2 546 0 343 .47 (.26) 673-700 1 511 0 313 .34 (.19) 701-728 1 451 1 293 .48 (.29) 729-756 2 409 G 269 .25 (.16) 757-784 1 346 C 243 .19 (.11) 785-812 1 307 0 210 .14 (.08) 813-840 3 277 1 185 .11 (.07) 841-868 0 241 2 162 .33 (.24) 869-896 0 202 C 130 .33 (.24) 897-924 0 173 0 104 .33 (.24) 925-952 1 140 1 77 .43 (.31) 953-980 2 106 0 58 .29 ( 20) 981-1008 1 78 0 45 .24 (.17) 1009-1036 0 55 0 23 .24 (.17) 1037-1064 1 17 0 7 .20 (.1S) 1065-1092 0 A 0 5 .20 (.15) 1093-1120 0 3 1 2 .29 (.21) 1121-1148 0 0 0 .2^ ( 21) -r~- ~ . --------------------------------•------------••------------------ a Defined as the total number of animals that had histopathologic examination. b The nu3xber of animals that died of the lesion or tsmor. • 133 CTR CCNTRRCTS 029313 11249063 CTR HN 0441042?

TABLE 4 3 ACTUARIaL ANALYSIS C? 9C3F1/CUH MICE DYINC OF LUNC CANCER DURING LONC-TE4.`S EXPOSURE TO 2R1 CIGARETTE SMOKE (CENERAL ADHERENCE TO DIED OR MORIBUND, aLL)a,b MON., JAN 2, 1984 SMOKE SHAH ------------------ ------------------ DAYS ON TEST D/W TUlSOR aT RISK D W/TUMOR AT RISK ?ROB.IBILITY ---------------- --------------------------------- ----------------- 1-28 0 504 0 379 1•.0 29-56 0 503 0 377 1.0 57-84 0 502 0 377 1.0 85-112 0 501 0 377 1.0 113-140 0 501 0 377 1.0 141-168 0 501 0 377 1.0 169-196 0 301 0 377 1.0 197-224 0 500 0 377 1.0 225-252 0 499 0 377 1.0 253-280 0 .4.99 0 377 1.0 281-308 0 499 0 377 1.0 309-336 0 498 0 377 1.0 337-364 0 495 0 377 1.0 365-392 0 493 0 376 1.0 393-420 0 490 0 375 1.0 421-448 0 483 0 370 1.0 449-476 0 479 1 369 .90 (.26) 477-504 0 476 0 365 .90 (.26) 505-532 0 470 0 361 .90 (.26) 533-560 0. 46S 0 355 . 90 (.26) 561-588 1 454 0 346 1.0 (.86) 589-616 0 44S 0 339 1.0 (.86) 617-644 0 427 0 327 1.0 (.86) 645-672 0 404 0 306 1.0 (.86) 673-700 0 394 0 291 1.0 (.86) 701••728 1 365 1 277 1.0 ( 79) 729-756 0 343 0 257 1.0 . (,79) 757-784 1 313 0 233 1.0 89) ( 785-a12 i 294 0 204 .96 . (.65) 813-840 3 264 1 179 .67 (.45) 841-868 0 229 2 156 1.0 (.99) 869-896 0 190 0 124 1.0 (.99) 897-924 0 161 0 98 1.0 (.99) 925-952 1 128 1 71 1 0 ( 88) 951-980 2 94 0 52 . 1.0 . (.84) 981-1008 1 66 0 39 .89,(.70) 1009-1036 C 43 0 17 .89'(,70) 1037-1064 1 17 0 7 .79 (.61) 106s-1092 0 fi 0 5 .79 (.61) 1093-1120 0 3 3 2 .97 (.79) 1123.-1148 .0 - 0 0 - 0 .97 (.79) „T 2 '- -- ---------------------------------------------------------------- a Det:ced as thQ total ncaber of animals that had histopathologic examination. The n-=ber of animals that died of the'lesion or t=or. 134 C.TR CONTRRCTS 029314 b 11249064 Cf f`s: NN 044103 ~

' TABLE 44 INCIDENCE OP MALIGNANT TUMORS IN THE RESPIRATORZ TRACT OF 3C3F1/CDH MICE DURING LONC-TER*t EXFOSURE TO 2R1 CIGARETTE SMOKE (GENERAL ADHERENCE TO RANDOM, ALL)a,b MON., JAN. 2, 1984 SMOKE SHAM -------------- ---------------- DaYS ON TEST ?RESENT ABSENT PRESENT ABSENT ?ROSABILITY -------------------------------------------------------------- 1-28 0 9 0 5 1.0 29-56 0 17 0 7 1,0 57-84 0 8 0 3 1.0 85-112 0 16 0 0 1.0 113-140 0 6 0 2 1.0 141-168 0 7 0 3 1.0 169-196 0 11 0 2 1.0 197-224 0 15 0 2 1.0 225-252 0 10 0 5 1.0 253-280 0 13 0 5 1.0 281-308 0 5 0 4 1.0 309-336 0 8 0 7 1.0 337-364 0 24 0 29 1.0 365-392 0 29 0 29 1.0 393-420 0 28• 0 24 1.0 421-448 1 23 0 14 1.0 (.45) 449-476 0 23 1. 24 1.0 (.88) 477-504 0 22 0 22 1.0 (.88) 505-532 0 28 0 22 1.0 (.88) 533-560 0 37 0 21 1.0 (.88) 561-588 1 27 0 19 1.0 (.73) 589-616 1 29 0 30 .77 (.42) 617-644 0 34 0 28 .77 (.42) 645-672 2 33 0 30 .32 (.16) 673-700 1 59 0 20 .26 (.13) 701-728 1 41 1 23 .41 (.25) 729-756 2 61 ' 2 26 .27 (.16) 757-784 1 38 0 33 .18 (.10) 785-812 1 30 0 25 .13 (.07) 813-840 3 32 1 22 .10 (.06) 841-868 0 39 0 30 .30 (.20) 869-296 0 29 0 26 .30 (.20) 897-924 0 33 0 27 .30 (.20) 925-952 1 33 0 le .39 (.28) 953-980 2 26 0 13 .28 (.19) 981-1008 1 22 0 22 .20 (.14) 1009-1036 0 38 0, 16 .20 (.14) 1037-1064 1 8 0 2 .19 (.13) 1065-1092 0 5 0 3 .19 (.13) 1121-1148 0 3 1 1 .29 (.?.0) 0 0 0 0 .29 (.20) 19 T 6rr -------------------------------------------------------- ------ - a Defined as t::e total number of animals with histopatholoqic b examination. The total number of animals in a qiven time interval is the sum of the number of animals in th• Present and Abs.nt colusns. 135 .CTR CQNTRRCTS 029315 11249065 CTIR 1-11-4 0'441 104

TABLE 4 5 INCIDENCE OF MALIGNANT TUMORS IN THE RESPIRATORY TRACT OF BC3F1/CUa MICE DORING LONG-TyRji EXPOSURE TO 2R1 CIGARETTE SMOKE (STRICT ADHERENCE TO RaN00M)a,b MON., JAN. 2, 1984 SMORE SHAM -------------- ---------------- DAYS ON TEST PRESENT ABSENT PRESENT ABSENT ?ROBABILITY -------------------------------------------------------------- 1-28 0 8 0 3 1.0 29-56 0 16 0 7 1.0 57-84 0 7 0 3 1.0 85-•112 0 16 0 0 1.0 113-140 0 6 0 2 1.0 141-168 0 7 0 3 1.0 169-196 0 10 0 2 1.0 197-224 0 14 0 2 1.0 225-252 0 10 0 5 1.0 253-280 0 13 0 5 1.0 281-308 0 4 0 4 1.0 309-336 0 5 0 7 1.0 337-364 0 22 0 28 1.0 365-392 0 26 0 28 1.0 393-420 0 21 0 19 1.0 421-448 1 19 0 13 1.0 (.42) 449-476 0 20 0 21 1.0 (.42) 477-504 0 16 0 18 1.0 (.42) 505-532 0 23 0 16 1.0 (.42) 533-560 0 26 0 12 1.0 (.42) 561-588 0 19 0 12 1.0 (.42) 589-616 1 11 0 18 .48 (.16) 617-644 0 11 0 7 .48 (.16) 645-672 2 23 0 15 .20 (.08) 673-700 1 30 0 6 . .18 (.07) 701-728 0 20 0 4 12 (.07) 729-756 2 31 0 2 .17 (.07) 757-784 0 20 0 4 .17 (.07) 785-812 0 1 0 0 .17 (.07) 813-840 0 0 0 0 .17 (.07) 841-868 0 0 0 0 .17 (.07) 869-896 0 0 0 0 .17 (.07) 897-924 0 0 0 0 .17 ( .07) 925-952 0 0 0 0 .17 (.07) 953-980 0 0 0 0 .17 (.07) 981-1008 0 0 0 0 .17 (.07) 1009-1036 0 12 0 6 .17 (.07) 1037-1064 0 0 0 0 .17 (.07) 1065-1092 0 0 0 0 .17 (.07) 1093-1120 0 0 0 0 .17 (.07) 1121-1148 0 0 0 0 .17 (.07) T =4 o 272 --------------------------------------------------•------------- a b Defined as those animals taken o!f test randomly. The total numb.r of animals in a qiven time interval is the sum of the number of animals in the Ptesent and Absent columns. 136 C.TR CONTRRCTS 029316 11249066 CTR t-IN 0,44 1 OEZ5

T3lBLE 4 6 dCTUAFLUL AN.II,YSIS OF BC3F1/CUM MICE DYING OF LUNG Ca..vCER OR ALVEOLaR COMPR:SSZNG NODULES DURING LONG-TER.K EXPOSURE TO 2R1 CIGARETTE SMOKE (GF.NERAh ADEERENCE TO DIED OR MORIBIIND, ALL)a,b TBU, JAN. 5, 1984 SMOIQr S811M ---------- -------- ------------------- D:,YS ON TEST D W/TQaOR AT RISK D W/TU:SOR AT RISK PROBABILITY ---------------------- ---------------- ----------------------------------- 1-28 0 978 0 651 1.0 29-56 0 969 0 646 1.0 57-84 0 952 0 639 1.0 85-112 0 944 0 636 1.0 113-140 - 0 928 0 636 1.0 141-168 0 922 0 634 1.0 169-196 0 915 0. 631 1.0 197-224 0 904 0 629 1.0 225-252 0 889 0 627 1.0 253-280 0 879 0 622 1.0 281-308 0 866 0 617 1.0 309-336 0 861 0 613 1.0 337-364 0 853 1 606 .87 (.24) 365-392 0 829 0 577 .87 (.24) 393-420 0 800 0 548 .87 (.24) 421-448 1 772 0 524 1.Q . (.79) 449-476 0 748 1 510 .7S .(.37) 477-504 2 725 1 485 .95 (.64) 505-532 0 703 0 463 .95 (.64) 533-560 1 675 0 441 1.0 (.89) 561-588 1 638 1 420 1.0 (.80) 589-616 2 610 0 401 1.0 (.80) 617-644 4 580 0 371 .44 (.30) 645-672 4 546 2 343 .43 (.31) 673-700 . 3 511 0 313 .23 (.16) 701-728 2 451 2 293 .34 (.25) 729-756 3 409 1 269 .27 (.20) 757-784 2 346 3 243 .:7 (.38) 785-812 4 307 2 210 .42 (.34) 813-840 7 276 1 185 .18 (.14) 841-868 0 241 3 162 .41 (.34) 369-896 0 202 1 130 .52 (.43) 897-924 0 173 0 104 .52 (.43) 925-952 4 140 4 77 .75 (.66) 953-980 4 106 0 58 .51 (.44) 981-1008 2 78 1 45 .50 (.43) 1009-1036 0 55 2 23 .75 (.66) 1037-1064 2 17 0 7 .66 (.57) 1065-1092 1 8 1 S .69 (.61) 1093-1120 0 3 1 2 .78 (.69) 1121-1148 0 0 0 0 .78 (.69) 49 2C ------------------------------------------------------------------ a Detined as the total number of animals that had histopatholoqic ezamination. b The nu:mber of animals that died of the lesion or tumor. 137 CTR CaNTRRCTS 029317 11249067 C'-*T~~' 111"41 04'4106

TABLE 4 7 ACT0ARIN+ ANALYSIS OF 3C3F1/CL'a MIC^., DYING OF L'J:1G C.LVG::R OR C0MPRESSING :IODQLES DORI:TG LC:1G-TERki EXPOSURE TO 2R1 CIGAeL~.T:'r S:".CZS (STRICT ?.Ds:.eL'-.T1CE TO DIED OR MORI3G':JD)a,b TSU. .;~+.'V 5, 1984 SaO1CE S HAM ------------------- ------------------ DAYS ON T:ST D W/TLRIOR .~T RISX D W/TL'MOR AT RISX PROBABILITY --------------------------------------------------------------------------- 1-28 0 504 0 379 1.0 29-56 0 503 0 377 1.0 57-84 0 502 0 377 1.0 85-112 0 501 0 377 1.0 113-140 0• 501 0 377 1.0 141-168 0 501 0 377 1.0 169-196 0 501 0 377 1,0 197-224 0 500 0 377 1.0 225-252 0 499 0 377 1.0 253-280 0 499 0 377 1.0 281-308 0 499 0 377 1.0 308-336 0 498 0 377 1.0 337-364 0 495 1 377 .89 (.26) 365-392 0 493 0 376• .89 (.26) 393-420 0 490 0 375 .89 (.26) 421-448 0 483 0 370 • .89 (.2b) 449-476 0 479 1 369 .37 (.11) 477-504 1 476 1 365 .45 (.20) 505-532 0 470 0 361 .45 (.20) 533-560 0 465 0 355 .45 (.20) 561-588 1 454 0 346 .77 (.46) 589-616 1 445 0 339 1.0 (.75) 617-644 3 427 0 327 .79 (.55) 645-672 1 404 1 306 .88 (.65) 673-700 1 394 0 291 .69 (.49) 701-728 1 365 2 277 1.0 (.80) 729-756 1 343 1 257 1.0 (.88) 757-734 2 313 2 233 1.0 (.98) 735-812 4 294 2 204 1.0 (.85) 313-840 7 264 1 179 .46 (.36) 341-368 0 229 3 156 .89 (.77) 369-396 0 190 1 124 1.0 (.92) 397-924 0 161 0 98 1.0 (.92) 925-952 4 128 4 71 .93 (.32) 953-980 4 94 0 52 .97 (.37) 981-1008 2 66 1 39 .95 (.B5) 1009-1036' 0 43 0 17 .95 (.85) 1037-1064 2 17 0 7 .84 (.73) 1065-1092 1 8 1 5 .89 (.73) 1'.93-1120 0 2 I. 2 .99 (.89) 1121-1148 0 0 0 0 .99 (.89) 35 23 ---------------------------------------------------------------- b a Dafined as thoae animals that died or were killed when moribund. The number of animals that died of lesions or tumor. 138 C.TR CaNTRaCTS 029318 11249068 CTR twIN 044107

TABLE 48 INCIDENCE OF ALVEOLAR COHPRESSING NODULES OR LUNG C.LVCER IN BC3F1/CQ:S MICE DURINC LONG-TERM EXPOSURE TO 2R1 CIGARETTE SMOKE (GENERAL ADHERENCE TO RANDOM, ALL)a,b T'fU., JAN. 5, 1984 SMOKE SHAlS -------------- ---------------- DAYS ON TEST PRESENT r\BSENT ?RESE:IT ABSENT , PROBABILITY ---------------------------------------------------------- ---- 1-28 0 9 0 5 1.0 29-56 0 17 0 7 1.0 57-84 0 8 0 3 1.0 85-112 0 16 0 0 1.0 113-140 0 5 0 2 1.0 141-168 0 7 0 3 1.0 169-196 0 11 0 2 1.0 197-224 0 15 0 2 1.0 225-252 0 10 0 5 1.0 253-280 0 13 0 5 1.0 281-308 0 5 0 4 1.0 309-336 0 8 0 7 1.0 337-364 0 24 1 28 1.0 (.37) 365-392 0 29 0 29 1.0 (.37) 393-420 0 28 0 24 1.0 (.37) 421-448 1 23 0 14 1.0 (.90) 449-476 0 23 1 24 .94 (.51) 477-504 2 20 1 21 1.0 • (.96) 505-532 0 28 0 22 1.0 (.96) 533-560 1 36 0 21 1.0 (.82) 561-588 1 27 1 18 1.0 (.94) 589-616 2 38 0 30 .71 (.50) 617-644 4 30 0 28 .20 (.13) 645-672 4 31 2 28 .16 (.10) 673-700 3 57 0 20 .10 (.06) 701-728 2 40 2 22 .19 (.13) '29-756 3 60 1 25 .19 (.14) 757-784 2 37 3 30 .33 (.25) 785-812 4 27 2 23 .26 (.20) 813-940 7 28 1 22 .09 (.07) 841-858 0 39 3 29 .25 (.19) 369-896 0 29 1 25 .32 (.26) 397-324 0 33 0 27 .32 (.26) 925-952 4 30 4 15 .53 (.44) 953-960 4 24 0 13 .35 (.28) 98L-1003 2 21 1 21 .30 (.25) 1009-1036 0 38 2 14 .50 (.42) 1037-10c4 2 7 0 2 .45 (.38) 1065-1092 1 4 1 2 .49 (.41) 1093-1120 0 3 1 1 .59 ( .51) :121-1148 0 0 0 0 -Tr- _QT3_ In - -n-r- a DeEined as the total number of animals with histopathologic examination. b The total auber of animals in a given time interval is the sum of the number of animals in the Present and Absent columns. 139 CTR CaNTRRCTS 029319 11249069 CTR 11N 0,44108

TABLE 49 INCIDE:ICE OF ALVEOLAR COMPRESSION NODULES OR LU:IG CaNCERS IN 3C3F1/C7!! :4ZC: DQRI:IG LO:IG-TERH EXPOSIIRE TO 2R1 CIC;~RE TTE SJSOR E (STRICT ADHERENCE TO R<1.`100M)avn THU. Ja.~I 5, 1984 SMOKE SHAM ---------------- ---------------- DaYS ON T_ST PRESENT ABSENT PRESE:IT a8SE:7T PROBABILI'_Y ---------------------- ------------------------------------------------- 1-28 0 8 0 3• 1.0 29-56 0 16 0 7 1.0 57-84 0 7 0 3 1.0 85-112 0 16 0 0 1.0 113-140 0 6 * 0 2 1.0 141-168 0 7 0 3 1.0 169-196 0 10 0 2 1.0 197-224 0 14 0 2 1.0 225-252 0 10 0 S 1.0 253-280 0 13 0 5 i.0 281-308 0 4 0 7 1.0 309-336 0 S 0 7 1.0 337-364 0 22 0 28 1.0 365-392 0 26 0 28 1.0 393-420 0 21 0 19 1.0 421-448 1 13 0 13 1.0 (.42) 449-476 0 20 0 21 1.0 (.42) 477-504 1 15 0 18 .55.(.19) 505-532 0 23 0 16 .55 (.19) 533-560 1 25 0 12 .38 (.15) 561-588 0 19 1 11 .89 (.52) 529-616 L 11 0 18 .50 (.27) 617-644 1 10 0 7 .35 (.18) 645-672 3 22 1 14 .29 (.17) 673-700 2 29 0 6 .23 (.13) 701-728 1 19 0 4 .20 (.12) 729-756 2 31 0 2 .19 (.11) 757-784 0 20 1 3 .42 (.28) 785-812 0 1 0 0 .42 (.28) 813-840 0 0 0 0 .42 (.28) 341-a68 0 0 0 0 .42 (.28) 869-696 0 0 0 0 .42 (.23) 897-924 0 0 0 0 .42 (.28) 925-952 0 0 0 0 .42 (.23i 953-980 0 0 0 0 .42 (.28) 961-L008 0 0 0 0 .42 (.28) 1009-1036 0 12 2 4 .98 (.77) 1037- 1064 0 0 0 0 .9E (.77) 1065-1092 0 0 0 0 .98 (.77) 1093-1120 0 0 0 0 .98 (.771 1121-1L48' 0 0 0 0 .98 (.77) 13 461 5 267 ----------------------------------------------•---------------- a b Defined as those animals taken off test randomly. The total niaber of animals in a qivea time interval is the sum of the number of animals in the Present and Absent columns. ' CTR CONTRRCTS 029320 , 11249070 CTR NN 044,109

TaBLE S 0 ACTUARIAL ANALYSIS C: BC3c 1/CL^I rsE DYING OF AL-,,-r.OLCGF`•lIC LC:IG LESIOUS (ANCN, AG`7, OR .ZACi DURING LO:JG-TEZSS _X?OSUR: TO 2R1 CIGARETTE SMOKE (CENERaL ADHERENCE TO DIED OR HORIBUND)arb FRI., JAN. 6, 1984 SMOKE SHAlt ------------------ ------------------ DAYS ON TEST D/X TUMOR AT RISK 0 W/TOHOR AT RISK ?ROBABILITY ----------------------------------------------------------------- 1-28 0 978 0 651 1.0 29-56 0 969 0 646 1.0 57-84 0 962 0 639 1.0 85-112 0 944 0 636 1.0 113-140 0 928 0 636 1.0 141-168 0 .922 0 634 1.0 169-196 0 91S 0 631 1.0 197-224 0 904 0 629 1.0 225-252 0 689 0 627 1.0 253-280 0 879 0 622 1.0 281-308 0 866 0 617 1.0 309-336 0 861 0 613 1.0 337-364 . 0 853 1 606 .87 (.24) 365-392 0 829 0 577 .87 (.24) 393-420 0 800 0 S48 .87 (.24). 421-448 1 772 0 524 1.0 (.80) 449-476 0 748 1 S10 .75 (.37) 477-504 3 725 2 485 .85 (.50) 505-532 0 703 1 463 .56 (.36) 533-560 1 675 1 441 .51 (.33) 561-588 1 638 1 429 .47 (.31) 589-616 2 610 1 491 .48 (.42) 617-644 5 580 1 371 1.0 (.91) 645-672 4 546 2 343 1.0 (.99) 673-700 3 511 0 313 .80 (.67) 701-728 2 451 2 293 .93 (.79) 729-756 7 409 1 269 .46 (.37) 757-784 4 347 3 243 .51 (.42) 785-812 5 307 2 210 .40 (.33) 813-840 7 276 3 185 .30 (.26) 841-868 2 241 5 162 .64 (.56) 369-896 1 202 1 130 .68 (.60) 397-924 2 173 1 104 .67 (.5S) 925-952 5 140 4 77 .81 (.73) 953-980 5 106 0 58 .55 (.48) 981-1008 3 78 1 45 .49. (.43) 1009-1036 1 55 2 23 .65 (.58) 1037-1064 3 17 0 7 .55 (.48) 1065-1092 1 8 1 5 .58 (.51) 1093-1120 1 3 1 2 .60 (.53) 1121-1148 0 0 C 0 69 . -TB- -----------------------------------------------------•----------- a Detined as the total number of animals that had histopatholoqic examination. O The nsmber of animals that died of the lesion or tumor. 141 CTR CONTRRCTS 029321 11249071 C TFZ W-4 04,4110

T.A3LE 5 L ACTOARIAL ANALYSIS OF 3C3F1/CUM MICE DYINC OF .ALVEOT-CGF':IIC LUVG LESICJiS M:VCN, ACN, OR AAC) DUfiING LCaG-TER4 S:S?OSUR.. TO 231 C:CA2-rTTE S:40KE (STRICT AaHERE*tCE TO DIED OR MORI9DND)a•z FRI., JAN. 6, 1984 SMOKE SFiAH ----------------- ----- ------------- OAYS ON ':EST 0 W/TUMOR AT RISK 0 W/TUHOR AT RISK PROBABILITY -------------------------------------------------=--------------- 1-28 0 504 0 379 1.0 29-56 0 503 0 377 1.0 57-84 0 502 0 377 :.0 85-112 0 501 0 377 1.0 113-140 0 501 0 377 1.0 141-168 0 501 0 377 '_.0 169-196 0 501 0 377 1.0 197-224 0 S00 0 377 1.0 225-252 0 499 0 377 1.0 253-280 0 499 0 377 1.0 281-308 0 499 0 377 1.0 309-336 0 498 0 377 1.0 337-364 0 495 1 377 .0 (.26) ~ 365-392 0 493 0 376 .89 (.26) 393-420 0 490 0 375 .R9 (.26) 421-448 0 483 0 370 .89 (.26) 449-476 0 479 1 369 .37 (.11) 477-504 2 476 , 2 365 .47 (.26) 505-532 0 470 0 361 .47 (.26) 533-560 0 465 1 355 .27 (.14) 561-598 1 454 0 346 .47 (.28) 589-616 L 44S 1 339 .46 (.29) 617-644 3 427 1 327 .82 (.62) 645-672 1 404 1 306 .78 (.59) 673-700 1 394 0 291 .95 (.7G) 701-728 1 36S 2 277 .70 (.55) 729-756 3 343 1 257 .96 (.79) 757-784 2 313 1 233 .80 (.73) 785-812 5 294 2 204 '_.0 (.93) 813-840 7 264 3 179 .90 (.73) 841-868 2 229 5 156 .36 (.74) 369-996 1 190 1 124 .91 (.70) 397-924 2 161 1 98 .85 (.74) 925-952 5 128 4 71 .70 (.61) 953-980 5 94 0 52 1.0 (.95) 981-1008 3 66 1 39 1.0 (.97) 1009-1036 1 43 0 17 1.0 1.92) 1037-1064 3 17 0 7 .87 (.78) 1065-1092 1 8 1 5 .90 (.82) 1093-1120 1 3 1 2 .93 (.35) 1121-1148 0 0 0 0 51 32 ---------------------------------------------------------------- a Oeticed as those animals that died or vere killed when moribund. b The number of animals that died of lesions or tumor. 142 C.TR CONTRACTS 029322 11249072 CTR HN 044111

TABLE 52 :VCIDE.`tCE OF ALVECLOGENIC (?-*IC.J, aC:1, DR ACC) :.U:1G LESI0:7S I:7 BC3e^1/CUM lSICE DURI:JG LONG-TERH E:CPOSUR_r TO 2R1 CIGARETTE SMOKE (GENERAL :.DH-zR~~JCE TO RANDOM, ALL) a.b. FRI., JAN. 6, 1984 DAYS ON TEST SMOKE SHAM -------------- ---------------- PRESENT. aBSENT PRESENT ABSENT . PROBABILITY -------------------------------------------------------------- 1-28 0 9 5 1.0 29-56 0 17 7 1.0 57-84 8 3 1.0 a5-112 ~ 16 0 1.0 113-140 0 6 2 1.0 141-168 0 7 3 1.0 169-:.96 0 11 2 1.0 197-224 0 15 2 1.0 225-252 0 10 6 1.0 253-280 0 13 6 1.0 281-308' 0 5 4 1 0 309-336 0 8 7 . 1.0 337-364 0 24 28 1.0 (.37) 365-392 0 29 29 . L.0 (.37) 393-420 0 28 24 1.0 (.37) 421-448 1 23 14 1.0 (.90) 449-476 0 23 24 .94 (.44) 477-504 3 19 20 L.0 (.96) 505-532 0 28 21 .94 (.67) 533-560 1 36 20 .80 (.58) 561-588 1 27 18 .74 (.53) 589-616 2 28 29 .96 ( 77) 617-644 5 29 27 .79 . (.62) 645-672 4 31 29 .59 (.46) 673-700 3 S7 20 .42 (.32) 701-728 2 40 22 .58 (.46) 729-756 7 56 25 .35 (.27) 757-784 4 35 30 .35 (.28) 785-812 5 26 23 .23 ( .13) 813-840 7 28 20 .18 (.l4) 841-868 2 37 27 .40 (.34) 869-896 1 28 25 .42 (.35) e97-924 2 31 26 .38 (.32) 925-952 5 29 is .61 (.44) 953-980 5 23 13 .32 (.27) 981-1008 3 20 21 .24 (.20) 1009-1036 1 37 14 .36 (.30) 1037-1064 3 6 2 .30 (.26) 1065-1092 1 4 2 .33 (.28) 1093-1120 1 2 1 .36 (.30) 1121-1148 0 0 0 0 ---------------- ----------------------------------------------- a Defined as the total number of animals with histopatholoqic examination. b The total aumbez of animals in a qiven time interval is t::e sum of the numbez of animals in the Present and Absent columns. 143 CTR Cah4TE2ACT5 029323 11249073 C TR i-IN 04,4112-

TABLE 53 IaCIDc,:CE OF ALVEOLCGEYIC LUNG LESIOVS (A.~1C:1, AC:1, OR AAC) .•7 3C3F1/CU24 MICE DURING LONG-TERIi EXPOSURE TO 2R1 CIGARETTE SMOKE (STRICT ADHERENCE TO RANDOH)a,b FRI., JAN. 6, 1984 SMOKE SHAlt -------------- ---------------- DAYS ON TEST PRESENT ABSENT PRESENT ABSENT. PR081BILITY --------------------- ----------- ----------------------- ------- 1-28 0 8 0 3 1.0 29-56 0 16 0 7 1.0 57-84 ' 0 7 0 3 1.0 85-112 0 16 0 0 1.0 113-140 0 6 0 2 1.0 141-168 0 7 0 3 1.0 169-196 0 10 0 2 1.0 197-224 0 14 0 2 1.0 225-252 0 10 0 5 1.0 253-280 0 13 0 5 1.0 281-308 0 4 0 4 1.0 309-336 0 5 0 7 1.0 337-364 0 22 0 28 1.0 365-392 0 26 0 28 1.0 393-420 0 21 0 19 1.0 421-448 1 19 0 13 1.0 (.42) 449-476 0 20 0 21 1.0 (.42) 477-504 L 15 0 18 .55 (.19) 505-532 0 23 1 1S 1.0 (.70) 533-560 1 25 0 12 .88 (.51) 561-588 0 19 0 11 1.0 (.97) 589-616 1 11 1 18 .91 (.5a) 617-644 2 9 0 7 .50 (.30) 645-672 3 22 0 14 .39 (.25) 673-700 , 2 29 0 6 .31 (.20) 701-728 1 19 1 4 .29 (.18: 729-756 4 29 0 2 .25 (.16) 757-784 2 18 0 3 .40 (.27) 785-312 0 1 0 0 .40 (.27) 813-840 0 0 0 0 .40 C .27! 841-868 0 0 0 0 . 40 (.27) 869-396 0 0 0 0 .40 (.27) 897-924 0 0 0 0 .40 (.27) 925-952 0 0 0 0 .40 (.27) 9g3-980 0 0 0 0 .40 (.2"1 981-1008 0 - 0 0 0 .0 (.27) 1009-1036 0 12 2 4 .89 ( . 70) 1037-1064 0 0 0 0 .89 ( .'07 1065-1092 0 0 0 0 .89 (.70; 1093-1120 0 0 0 0 .8!- (.70, 1121-1148 0 0 ` _ 0 0 - .89 .70: 18 T V 6 266 ------------------------------------------ ---------- --------------- a Dsfinsd as those animals tak.n off test randomly. b Th• total numb.r of animals in a given time int.rval is the sum of th• number of animals in the Present and Absent columns. 144 C.TR COh4TRRCTS 429324 11249074 CTR VIN 0-1.4113~

rlnal deport Contract: CTR-0030 CTR-10U1 TABGES 54-64 NON-NEOPGASTIC RESPIRATORT TRACT :.ESIONS 145 CTR CaNTRRCTS 029325 11249075 C1 R HN 044114,

:?3LE =.4 I;iCIbENCS OF PICHENTEb xL7?OLAR MACROPHAGE ACCQ:SULAT.O;J bQRIaC.LONC-TER?S EX?OSURE T0 2R1 CIGARETTE SMOKE • (ALL A2IIHALS) a,b `:04. ,:aN. 2, 1984 ---------------- CAYS C:7 ^. EST ?RESE:7T A3SEN? ------------------------------------------------------------ 28 o 9 -- 29-56 0 1.7 57-84 0 3 85-112 0 16 113-140 0 6 141-168 _ 0 7 i69-196 0 11 :97-224 0 15 225-252 0 10 253-280 0 13 281-308 0 5 309-336 1 7 337-364 2 22 365-392 0 29 393-420 2 25 421-448 0 24 449-476 2 21 477-504 0 22 505-532 3 25 533-560 5 32 561-588 7 21 589-616 7 23 617-644 5 29 645-672 5 30 673-700 22 39 701-728 15 27 729-756 20 43 757-7a4 12 27 '35-a12 9 27. 313-840 7 29 341-a6a 4 35 369-896 6 23 397-924 3 25 925-952 5 29 953-930 3 25 )31-1008 4 :g 1~39-1036 3 35 :037-1064 1 0 :065-1092 0 5 1093-1120 0 3 1121-1148 0 0 13'- 's 2 0 --------------------------------------------------------------- 1 :efined as the total nuaber of animals with histooat::oloq:c eranization. 5 T`:e :otal numb.r of aniaals 'a a qiven tims interval is t~e s~ of the nuaber of animals~in the Present and Absent co:=.ns. 146 C.TR COHTRACTS 029326 112#9076 C T TR NN 04 4 11 E.5

TABLE 5 5 INCIDENCE 0F CONGESTION IN 8C3F1/CUH MICE OURI:IC LONC-TER:4 CHRONIC EX?OSURE TO 2R1 CICARETTE SMOKE (CENERAL ADHERENCE TO RaNDON, ALL)a'o '!ON., JAN. 16, 1384 SMOKE SHAM -------------- ---------------- OAYS ON ':ES': ?R:SE:JT ASSENT PRESENT ASSENT ?R08ABILITY -------------------------------------------------------------- 1-28 3 6 3 2 .70 (.36) 29-56 : 12 2 5 .33 (.56) 57-84 5 2 2 1 .94 (.68) 85-112 5 10 0 0 .94 (.63) 113-140 2 4 1 1 .32 (.60) 141-168 4 3 1 2 1.0 (.84) 169-196 2 9 1 1 .33 (.64) 197-224 4 11 0 2 1.0 (.82) 225-252 2 8 1 4 1.0 (.84) 253-280 2 11 1 4 .96 (.79) 281-308 3 2 3 1 .36 (.69) 309-335 4 4 5 2 .53 (.30) 337-364 3 1.6 16 13 .19 (.15) 365-392 11 18 14 15 .14 (.10) 393-420 6 22 8 16 .08 (.06) 421-448 5 19 5 9 .35 (.04) 449-476 10 13 12 1.3 .05 (.04) 477-504 8 14 9 13 .05 (.04) 505-532 7 21 7 15 .04 (.04) 533-550 11 26 5 '-5 .J8 (.06) 561-538 12 -6 9 :0 .03 1.06) 589-616 4 26 14 16 .01* (.01) 617-644 4 30 4 24 .01* (.01) 645-672 3 32 8 22 <.01*<(.01) 673-700 13 47 3 17 .31* (.01) . 701-729 7 35 4 20 * .01* (.01) 729-756 3 60 0 26 .31* (.01) 757-734 2 37 1 32 .02* 1.01) 735-312 3 28 2 13 * .02* (.01) 313-340 4 31 3 41 0 .32' :.02) 341-a68 1 38 2 30 .02* !.01) 369-896 2 27 2 24 .02* (.31) 397-924 3 30 2 =5 .02* !.02) * 325-y52 2 32 1 '_3 .02* !.C21 353-930 1 27 2 1: .02* :.'1) 331-1008 2 21 1 21 •0 2* \.J21 1009-1036 5 32 2 1-4 .02* !.32) . 1037-1064 1 8 1 1 .02' (.02) 1065-1092 1 4 1 2 .02• ;.32) . 1093-1120 0 3 0 2 .J2* :.J21 * 1121-1148 0 0 0 0 .02* !..12) ' = ~ ~ ----- =~'----- 1-5 8 -----3: ~----------------- a 3efined as t::e =ota1 number of animals with histaQat::olog:c ezaQination. T!:e :otal number zf animals in a given tiae interval is :^e 3U= of the nuab.r sf animals ia the Present and Absent c31-ns. r 147 CTR CONTRACTS 029327 11249077 CTR 11N 04'411 G

T:.BLF. 56 INCIDENCE 0P CONCESTICa ru SC3e 1/C'JH HICE DURI!!C LONG-TERM EXPOSURE TO 2eZ1 =:C.1RP.TTE SMOKE (STRIC': ADHERENCE TO RANDOM) ':UZS.. :AN. 17. 1984 SMOKE SHAM -------------- ---------------- CaYS C?7 ':EST PRESENT A3SE:JT ?RESENT ABSENT ?ROBd3::.I'Y -------------------------------------------------------- ---- 1-28 3 3 0 .27 ;.0?) 29-56 5 11 2 5 .57 (.35) 57-34 6 1 2 1 .32 35-112 6 10 0 0 .32 (.Sol 113-140 2 4 1 1 .71 (.3?) 141-16a 4 3 1 ~ '' .96 74) 169-196 2 3 L 1 .76 :97-224 4 10 0 2 .97 ( 77) ~ 225-252 2 3 L 4 .97 73) ,. ' 253-280 2 1 4 .91 (.7:) 281-308 3 1 3 1 .92 (.7~) 309-336 3 2 5 2 .32 (.5c) 337-364 8 14 '-6 12 .30 !.23) 365-392 11 15 14 14 .24 ' 13) 393-s20 5 16 6 13 .10 (.15) 421-448 4 16 5 8 .12 (.C9) 449-476 9 11 12 9 .03 477-504 7 9 8 1C .09 t.: 91 505-532 7 16 7 9 .07 ..G:.• 533-560 8 18 5 7 ,e. . • 561-5a8 12 7 5 7 .12 ~9) 589-616 1 ,1 13 6 .02• • 517-644 1 10 3 4 <.01• . . M - 645-672 2 23 5 10 < A1•~<.J-) • .~ 573-700 10 21 2 4 701-728 5 1s 3 1 <.01 ^1)' . V "29-756 0 33 0 2 <.01 -57-734 0 20 0 4 <.01"<.01) ' "35-a12 0 1 0 0 3:3-a40 0 0 0 0 341-36a 0 0 0 0 <.OL';<.Ci) ~ 369-a96 0 0 0 a <.ol 397-924 0 0 0 0 <.J1',<. ,-) ' 925-952 0 0 0 0 f; =l f <.O1 <.J 953-990 0 0 931-1008 0 0 0 0 0 0 <.01" :009-1036 4 8 1 5 <.01 ';<.C_: ~ 1037-1064 0 0 0 0 < 0 1 . .<.J ~ 1065-1092 0 0 0 0 <.01';<.J1l ' 1093-1120 0 0 0 0 <.01';<. 1121-1148 0 0 0 0 136 333 124 1ie --------------------------------------------------------------- a 7etlned as ehose animals taken ot: test randomly. °he total ze=ber of aniaals ia a given tiae interval :s ::e sua of the number o: ani=als ta the Present and Absent columna. 138 CTR CCHTRRCTS 029323 1124907s ~` CTR NN 041411-

TABLE 37 INCIDENCE CF RHI:7I:'IS IN BC3F1/CUH MICE DURING LCYC-TERH EXPOSURE TO 2R1 CIGARETTE SMOKE (CE:fERAL ADHERENCE TO RaNDOH, ALL)a1b :HU., .:aN. 5, 1984 SMOKE SHAM -------------- ---- ------ --- DAYS CN TEST ?3ESENT dBSENT PRESENT ABSENT ?R03A3ILI:": -------------------------------------------------------------- 1-28 0 4 0 3 1. a 29-56 3 13 0 7 1.0 57-84 0 1 0 2 1.3 85-112 0 0 0 0 1.0 113-140 0 0 0 0 1.0 141-168 0 0 0 0 1.0 169-196 0 0 0 0 1.0 197-224 0 0 0 0 1.0 225-252 0 0 0 0 1.0 253-280 0 0 0 0 1.0 281-308 0 0 0 0 1.0 309-336 0 1 0 0 1.3 337-364 0 1 0 0 1.0 365-392 0 17 0 22 1.0 393-420 0 14 0 8 1.0 421-448 1 4 0 4 1.0 .37) 449-476 1 5 0 3 .72 (.23) 477-504 0 17 0 16 .'2 (.28) 505-532 3 19 0 17 .72 ;,28) 533-560 0 3: 0 15 .72 .23) 561-saa 0 22 0 15 .'2 ;.23) 589-616 _ 22 0 23 617-644 : 17 1 16 ~1 ;.27) 645-672 3 25 0 19 .31 ;.27) 673-700 2 36 0 10 .36 '.19) 701-728 1 23 0 14 .26 (.14) 729-756 0 36 1 13 .53 (.36) 757-784 1 24 0 15 .44 ;.27) 795-312 0 13 0 5 .44 .27) 313-a40 J 11 0 3 .:4 (.27) 341-868 3 15 0 11 .44 ;.27) 369-395 1 12 0 9 .32 ;.:9) 397-924 ~ :5 0 12 .32 (.19) 925-952 0 17 1 7 .59 (.40) 953-980 : 14 1 8 '2 (.521 981-1008 1 13 4 12 :.00 (.93) 1009-1036 2 31 0 12 1,03 (.38) 1037-1064 0 6 0 2 1.00 (.38) 1065-1092 J 4 0 2 1.00 (.38) 1093-1120 1 2 0 1 .97 (.79) 1121-1148 0 0 0 0 .97 (.79) --------------------------------------------------------------- a Detiaed as the :otal ,u-mber of animals with histopat: o:oqic ezamination. 5 :ye total nu=ber a5 aniaals iz a qiven eiae iaterval is the sum of the n-b.r of acizals ia the Present and Absen: co1::3cs. 149 CTE2 CoNTE2acT5 029329 11249079 CTR I-IN (34-1• 116

TA3LZ .93 INCIDENCE OF RHINITIS I:J 3C321/C:.H MICE DURING LONC-TE?4 EXPOSURE TO 2R1 CIOaRET':S SMOKE (STRICT ADHEREaCE TO RANDOM)a,b rHU., :arr. 5, 1984 SMOKE SHAN --------------- -------------- :aYS Ca :SST ?RESE:IT :,dSE:f;' ?3ESE:7T X3SENT PROBa31L::Y ----------------------------------------------------------------- :-23 0 4 0 3 1.0 29-56 0 -2 0 7 1.~ 57-34 0 0 2 1.3 35-ii2 0 % ~ 0 1.0 ::3-140 0 0 0 _.0 _;1-i63 0 7 0 0 1.0 :59-196 0 : 0 0 1.0 197-224 0 : 0 0 1.3 225-252 0 7 0 0 1.0 253-2a0 0 0 0 1 0 231-30a 0 ; a 0 . 1.0 309-335 0 ~ 0 0 1.0 337-364 0 0 0 1 0 365-392 0 .~ 0 22 . 1.0 393-i20 0 =1 0 4 1.0 421-448 1 4 0 4 1.0 (.37) 449-476 1 S 0 2 .77 '.29) 477-504 0 :2 0 14 .77 (.29) 505-532 0 17 0 15 .77 ( ~9) 533-560 0 21 0 11 .77 ( 29) 561-583 0 0 11 .77 ! =31 539-015 0 0 14 .77 : :31 517-514 1 3 1 3 1.0 545-672 0 .9 0 12 1.0 ":) 573-7C0 2 20 0 4 .91 (. 5) '0:-'23 0 :5 0 4 .31 (.:51 19-755 0 23 1 1 1.0 :57-"34 1 13 0 3 1.3 ! 3'_) '35-312 0 ~ 0 1.0 (.31) 313-340 0 ~ 0 0 :.0 (.3~) 33:-303 0 ~ 0 0 :.0 ?:1 359-33~ 0 ~ 0 0 1.0 337-324 0 ~ 0 0 :.0 2 0 0 0 11.0 . , 133 -~30 0 7 0 0 1.0 ~ ?~:-:J03 0 ~ 3 0 '_.0 ( :) ::09-1036 0 11 0 6 1.C :337-i064 0 n 0 0 1.0 ( ?'_) :365-1092 0 0 0 C 1.0 (.j1) 1393-1120 0 0 0 0 1.0 (.?:) _:21-1143 0 0 0 0 1.0 (.31) 6 :s 2 4=2 --------------------------------------------------------------- s :etlned as t::ose aaiaals taicen off test randomly. :'h . :ota1 a~er af ani=a1s ia a given tiae iaterval is :~e a;3 0: :::e n=ber .3f ani;ala !a the ?rea.nt and Xbsanc col=ns. 150 CTR CONTRRCTS 029330 1'245090 CTR NN 04,4119

TABLE 59 I:ICIDEyCE OF OTITIS MEDIA IN BC3F1/C'JM MICE DURING LCNC-TERM EXPOSQRE TO 2R1 CICARET':E SMOKE ;OENELAL ADHERENCE ';0 RANDOM, :.LLl i,b FRI., ,TAN. 6, 1384 SMOKE SHAlS ---- -------------- ------------ OA'lS ON TES'T ?RESE:lT a3SENT PRESENT A3SENT ?ROBaBILI;Y ----------------------------------------------- =-------------- 1-2a ~ 3 0 2 1, 0 29-56 9 13 0 7 1.~ 57-a4 9 1 0 2 a5-112 0 0 0 1 :.0 113-140 0 0 0 0 1.0 141-168 0 0 0 0 1.0 169-196 0 0 0 0 1.0 197-224 0 0 0 0 1.0 225-252 0 0 0 0 1.0 253-280 0 0 0 0 1.0 281-308 0 0 0 0 1.0 309-336 0 1 0 0 1.0 337-364 0 1 0 0 '_.0 365-392 5 19 0 23 .37 (.02)~ 393-420 0 15 0 9 .07 (.02)! 421-448 1 S 0 5 .04• f.02)! 449-476 0 7 0 3 .04* (.02)• 477-504 2 16 1 15 .3 5• 505-532 3 21 0 17 .J2• 533-560 1 33 0 15 .O1• ;61-588 1 22 0 15 Oi• (<.31) • Sd9-616 J 24 0 23 .J1• (<.01) • 517-644 0 19 0 21 .01• :<.01) • 545-572 1 31 0 23 .01• (<.01) • 573-700 3 41 2 14 .03• (.02)• 701-729 0 27 2 15 .15 (.09) 729-756 1 51 1 15 .23 (.16) 757-734 2 29 1 20 .23 (.1-6) 735-312 3 13 5 7 .54 .43) 313-340 5 11 3 1o .39 (.311 341-368 5 18 2 1.5 .23 (.21) 359-896 3 11 2 13 .'2 ( 17) 397-924 3 '_ 4 '- 1 2 :7 (.i31 325-952 ~ 11 3 5 .:9 (.:5) 353-9a0 4 11 3 5 .25 (.201 981-i008 2 12 7 9 .5a (.S0) 1009-1036 9 24 1 :1 .36 ( .30) 1037-1064 2 4 1 1 .39 (.33) 1065-1092 0 4 0 1 .39 (.34) 1093-1120 2 1 0 1 .34 (.23) 1121-1148 0 0 0 0 .34 :.23) ~6- --------------------------------------------------------------- ~ Defi^ed as t*-e :otal ::uaber of animals with 4istopatholoqic ezamination. b "he =oti1 ::aber -,: aniaals in a given time interval is =ae sL= of the num5er o: aniaals in the Present and Absent col.:rns. 151 .CTR CONTRRCTS 429331 11249081 CTR HN 044•12-10

L.`iC:I;r:ICS OF OT:T:S :1SD:A :;; 3C3F1/CJ:S KZCS DURZaG LONG-:ERIi EX?OSU?E TO 2R1•~C:GaRETTE SMOKE (STRICT ACHERE:JCZ TO R.1N00K)a•b ':HU.. :A:7 5. 1984 S:SOKE SHAM -------------- ---------------- :aYS :a .SST ?RESENT :.dSE.1T ?RESEaT aBSE:1T PROBd32L::Y -- ----------------------------------------------------------- __23 0 3 1 2 1.0 23-:6 0 :2 ) 7 1.0 57-34 0 0 ~ 2 1.0 35-_:2 0 0 ~ 0 1.0 _:3-:30 0 0 ~ 0 1.0 :41-:53 0 0 ~ 0 1.0 :69-'36 0 0 ~ 0 1.0 :97-224 0 0 ~ 0 1.0 225-252 0 0 ~ . 0 L.0 253-230 0 0 0 0 1.0 231-309 0 0 ~ 0 1.0 309-336 0 0 7 0 1.0 337-364 0 , ~ 0 1.0 365-392 4 17 0 23 .10 (.03)• 393-420 0 12 0 4 .13 (.33)' 42.-448 1 5 0 5 .06 ( :2) • 349-475 0 6 ~ 2 .C6 ;.'.2)' 477-504 2 11 : 13 .06 .:2)' :05-532 2 19 ~ 15 .02• 533-500 1 23 0 11 .02' 0 18 ~ 11 .02• 539-5:6 0 12 ~ 14 .02• 517-044 0 9 ~ 6 .02' 545-072 1 23 J 14 .01' 573--;,0 1 25 0 6 .01' 70 1--'3 0 18 0 4 .01' 729--:6 0 32 J 2 .04• 57--33 " 2 :7 ,. _ 3:---= 3 1 ~ 0 .04• 313-e:0 0 0 J 0 .04' 341-~53 0 0 J 0 .04' 359-3?5 ^ J 0 7 0 .v4• ~ • .3--..; ~ 0 ~ 0 04• ._. • • ?25-i52 0 0 ~ 0 . .04• ?53-?30 0 0 ~ 0 .04' ?31-iJ09 0 0 J 0 .04' :009-1036 2 9 - 5 .05• 1037-1064 0 0 0 0 .05• ( ~3) • :065-1092 0 0 0 0 .05• -093-1120 0 0 0 0 .05• 1121-1138 0 - ~ 0 3 0 .05• T :, 3 149 --------------------------------------------------------------- a :e:iaed as those animals taken of: test randomly. b :'.`.e _atal aumber of aa:=als ia a gi•.ren time interval i s =he a-= of the nu=b.c of animals :a the Present and ;Lbsent col::mna. 152 CTR CCNTE2RCTS 029332 11249082 CTR HN 0`44121

TABLE 61 INCIDENCE OP OTI:':S EXTERNII I;J 8C3F1/CUH M:CE JURIaC L0:7C-TE?14 EX?OSURE TO 2R1 CIC.IRET'TE S:!ORE (CE!7EeZAL ACHERENCE TO RANDOM, ALL)3,b ':':.. JAN. 5, 1384 S SOxE SH&H -------------- ---------------- OaYS ON T=S: ?R:SE;IT A3SENT ?RESENT a3SENT ?R03A3:LITY -------------------------------------------------------------- 1-28 0 • 3 0 2 :.0 29-56 0 _3 0 7 57-84 0 1 0 2 :. 0 a5-112 1 0 0 0 :.~ 113-140 0 0 0 0 :.0 141-168 0 0 0 0 1. 0 169-196 0 0 0 0 1'.0 197-224 0 0 0 0 '-.0 225-252 0 0 0 0 1.0 253-280 0 0 0 0 1 .0 281-308 0 0 0 0 1.0 309-336 0 1 0 0 1.0 337-364 0 1 0 0 :.0 365-392 5 1-9 1 22 .22 (.10) 393-420 2 1-3 1 8 .27 (.15) 421-448 0 6 1 4 .50 (.31) 449-476 ' 6 0 3 :40 (.25) 477-504 2 1-6 1 15 .33 (.21) 505-532 3 21 2 15 .38 (.27) 533-560 7 27 1 14 .13 (.12) 561-5a8 3 20 1 14 .1.4 (.,.I9) 599-616 2 22 5 19 .43 (.34) 617-644 2 :7 6 13 .39 (.76) 645-672 6 26 4 19 .36 (.75) 673-700 6 38 4 12 1.3 ;,95) 701-728 3 24 1 16 ~.~ .93) 729-756 :3 39 2 14 .74 (,55) 757-784 9 22 5 16 .55 (.56) 795-312 5 :2 3 9 .60 (.53) 313-a40 5 11 5 3 .56 (.53) 341-a6a 7 :6 5 12 .56 (.53) 959-a96 3 :1 4 1.2 .70 (.53) 997-924 5 -2 6 7 .39 ;.32) 325-352 :3 7 2 5 .53 (.57) 953-980 3 7 5 3 .5o i.60) 391-1008 9 5 4 12 .35 ;.30) 1009-1036 :6 :7 6 5 .38 (.33) 1037-1064 2 4 1 1 .40 (.36) 1065-1092 3 1 1 0 .42 ;.33) 1093-1120 2 1 1 0 .:5 (.110) 1121-1148 0 - - 0 0 - 0 .45 (.:0) r3 0 ~~ -WM 2 r a Oefiaed as the totsl nuber of animals vith h:stooat::oloqic ezamination. 5 The totsl zumber o: sn:=als in a given time intetval :s the sua of the number af aaiaals iz the ?reaent and absent tolumns. 153 CTR CCNTRACTS 029333 11249083 C ~m~~' ~`~~~~ 04412.2.

TABLE 62 -NCIZENCE OF OTITIS P.XTE?.:1A Ia dC3F1/CUI'i t:ICE DUR::JC LONC-TERH EXPOSURL TO 2R1 CIGARETTE SMOKE (STRICT AOHE?E.4CE ': RANDOM)&,b THU., .::,Y. 5, 1384 SMOKE SHAM -------------- ---------------- DaYS C'! ;'EST ?RESENT ASSENT ?RESEYT A.BSENT • ?RO3a3:L:-': -- ----------------------------------------------------------- 1-23 0 3 0 2 1.0 29-55 0 12 0 7 1.0 57-34 0 0 0 2 1.,0 35-112 0 0 0 0 1.0 113-140 0 0 0 0 1.0 141-153 0 0 0 0 1.0 i59-195 0 0 0 0 1.0 197-224 0 0 0 0 1.0 225-252 0 0 0 0 1.0 253-230 0 0 0 0 1.0 281-303 0 0 0 0 1.0 309-335 0 0 0 0 1.0 337-364 0 1 0 0 1.0 365-392 4 17 1 22 .30 (.14) 393-420 1 11 0 4 .24 (.:1) 421-443 0 6 1 4 .52 (.29) 449-475 L 5 0 2 .42 (.24) 477-504 1 12 1 13 .46 (.29) 505-532 3 13 1 14 .32 (.20) 533-560 7 17 1 10 .13 (.J9) 561-533 3 15 1 10 .L0 (.06) 539-61-5 1 11 4 10 .33 (.24) 517-644 1 3 3 3 .68 (.55) 545-672 4 20 2 12 .66 (.54) 573-7.'0 5 21 2 4 .35 (.71) 701-723 3 1s 0 4 .71 (.59) 729-"55 3 24 : 1 .33 (.7:) %57-"34 8 '-1 1 3 .70 (.39) 735-312 0 1 0 0 .70 (.59) 313-340 0 0 0 0 .70 (.59) 341-353 0 0 J 0 .70 (.59) 359-595 0 7 0 .70 ~.59) 3971-324 0 0 0 0 .70 (.59) 325-952 0 0 0 0 .70 (.591 953-980 0 0 0 0 .'0 11.59) 9Ift-1008 0 0 J 0 .70 (.59) L009-1036 4 7 3 3 .34 (.72) 1037-1054 0 0 0 0 .84 (.72) 1065-1092 0 0 0 0 .34 (.72) 1093-1120 0 0 0 0 .84 (.72) 1121-1148 0 0 0 0 .34 ( 721 ~- -2T- ~~ -T3 0 . --------------------------------------------------------------- 3 De:iaed as those aniaals taicen of: test tandomly. ne :o:al ::L=ber o: aniaals :a a qi7en time izterval is the sum o: the numbec of ani;als ia the Pcesent and A.Sseac columna. b 154 'CTR CONTRACTS 029334 ) 124sCS-s CTR I~IN 0-14121-3

::.3L-r 5 3 :NCIDE:7CE OF OTITIS MEDIa OR G^.IT:3 EXTERNA IN BCJFL/CCY M:CE DQRI:JG LONG-TER."i EX?rJSOZE -.'J 2R1 CICARETTE SHORF. (CENEZAL ADHERENCE '0 RANDOM, ALL)d,b THO., .:AN. 5, 1984 S:!oeTE Si{A21 -------------- ---------------- 7JAYS 0;1 .=S:' ?RESEYT aBS-s:1T ?R?SE:lT ABSENT ?ROBABI:.'-': -------------------------------------------------------------- -2a 0 3 0 2 1.0 29-56 0 :3 0 7 1.3 57-a4 0 : ~ 2 =.0 35-112 0 ~ 0 0 '-.0 113-140 0 1.0 :41-15a 0 ~ 0 0 1.0 169-196 0 0 0 0 1.0 197-224 0 0 0 0 1.0 225-252 0 0 0 0 1.0 253-280 0 0 0 0 1.0 231-308 0 0 0 0 1.0 309-336 0 1 0 0 1.0 337-364 0 1 0 0 1.0 355-392 ~7 1 . 22 .07 ! ^: • .~ ~ 393-420 2 '3 1 8 .10 421-4f8 , 5 1 4 .15 ;.03) 449-476 1 6 0 3 .12 477-504 4 '4 1 15 .04• !.C2)' 505-532 5 :8 2 15 .02• 533-560 7 27 1 14 561-5a8 4 '_9 1 14 539-616 2 22 5 18 517-644 2 :7 6 15 .19 645-672 7 25 4 19 .19 673-700 9 35 6 10 .44 "01-723 3 24 3 14 .57 !.S3) ?29-756 :i 38 3 13 .45 ' :3) 'S7-784 10 21 6 15 .42 :5) '95-312 7 :0 7 5 .59 313-340 10 7 6 7 .s8 i31-o6d 10 13 7 10 .48 369-896 5 9 6 10 45 .i?1 397-924 9 9 5 7 .35 ?25-952 13 4 5 3 .39 :.?1` •:3-i30 == ~ 3 1 .43 ?91-:J08 10 4 10 5 .32 33; :J09-1036 23 -J 6 6 .29 . .:) :J37-1Q64 4 2 ~ 0 .33 :?) :J65-1092 3 1 : C .35 :093-1120 3 ~ 1 0 .35 :121-1148 0 - 0 0 0 .35 ..3:I Irr 392 :J6 265 --------------------------------------------------------------- a :e:ined as t::e cotal ^=ber a: aniaals vith histooecholoy:.. °_YaIIination: b :'.`.e total :uaber ot aniaals :n a qiven time interval is :=e s;rz of ;he zu=ber of lni3als in t`:e Present and Absent col"'9ns. CTF2 COHTRRCTS 029335 11249085 CTR NIA 044 124

''=ABLE 64 INCIDEaC CF OTIT--S 3'EDIA AND OTITIS E'LTEe't.VR I:r 3C3e 1/CL'a :SICi DURS:IG LONG-TER'4 EX?OSUeZE TO 2eZ1 CZGi.RET'TE S.40KE (STRICT .1DHERENC TO RANDOM)a.b TRU. JAN 5, 1984 SMOKE S'.l-.%.'1 ----------------- ----------------- ;.aYS C;i T_ST 7?Z- SZaT A3SEaT PRESE;2T :,BSE;JT 2ROBA3I:._:Y ------------------------------------------------------------------------ 1-28 0 3 0 2 1.0 29-56 0 12 0 7 1.0 57-34 3 0 0 2 1.3 85-112 0 0 0 0 1.0 113-140 _ 0 0 0 0 ?.3 141-158 0 0 0 0 1.0 169-196 0 0 0 0 1.0 197-224 0 0 0 0 1.0 225-252 0 0 0 0 1.0 253-280 , 0 0 0 1.0 281-308 0 0 0 0 1.0 309-336 0 0 0 0 1.0 337-364 0 1 0 0 1.0 365-392 6 15 1 22 .08 (..13)• 393-420 1 11 0 4 .07 (.02)' 421-448 1 5 1 4 .12 (.06) 449-476 1 5 0 2 .09 (.J5)' 477-504 3 10 1 13 :04'(:02)• 505-532 5 16 1 14 .02•(<.O1) 533-560 7 17 1 10 <.Oi•(<.01) 561-588 3 15 1 10 <.Oi•(<.01) 589-616 1 11 4 10 ~3•( .32)* 617-644 L 8 3 3 645-672 5 19 2 12 673-700 6 20 2 4 ._7 (.:3) 701-72S 3 15 0 4 .13 (.i0) 729-756 3 24 1 .1 . =9 (..4) 757-734 9 10 2 2 .20 (..5) 735-312 0 1 0 0 .20 (..51 313-340 3 0 0 0 .20 (.:5) 341-363 0 0 0 0 .20 369-d96 0 0 0 0 .20 (.:5) 397-924 3 0 0 J .20 (..5) 325-352 3 0 0 J .-3 1..51 953-330 0 0 0 0 .20 (..5) 331-1JC 3 0 0 0 3 .20 (..5) 1009-1036 6 5 3 3 .20 (.:5) 1037-1064 0 0 0 3 .20 ( _5} :065-1092 0 0 0 0 •.20 (..5) '093-1120 0 0 0 0 .20 (..5) 1:21-1148 0 0 0 0 .23 (..51 66 223 23 :29 --------------------------------------------------------------- a b Dafined as those animals taken ott test :andcmly. The total ::c:m5er of animals in a qi7en time interval is the sc= of the numb.r of aniaals in the P:es.nc and absent calu=ns. 156 C.TR CCNTRRCTS 429336 11249086 CTR I-IN 044' 1'5-1

?Snal Report COnt:ac:: CT3-0070 STR-101K Taa:.=s 65-66 OTHER NON-N?.OPLASTIC LESIONS :57 CTR COHTf2RCTS 029337 4.1-2490-5' CTR MN 04• 4.12G

:'ABLE 63 I:1CIDE:7CE CF VEPHRITIS IN BC3F1/CCM MICE DURING LCDtC-TERl4 ZX?OSURE TO 2R1 CIGARETTE SMOKE (CENERAL ADHERENCE TO RANDOM, ALL)a,b FRI., JAN. 5, :384 SHORE SHAM -------------- ---------------- DAYS CN T.'.S'. ?RES E:)T ABSENT PRESENT n3SENT ?ROBd3ILI^Y ------------------------------------------ ------- - 1-23 0 7 0 3 :~ 56 23 - 0' 14 0 7 1.3 57-a4 L 0 2 L J 35-112 C 0 0 . 1 iiJ-i:0 _ 0 0 0 141-153 0 0 0 0 1.0 169-135 197 2 0 0 0 0 1.0 - 24 0 0 0 0 1.0 225-252 0 0 0 0 1.0 253-230 8 0 0. 0 0 1.0 231-30 0 0 0 0 1.0 309-336 0 1 0 0 1.0 337-364 0 1 0 0 1.0 365-332 2 2 3 1 ;,0 (.50) 393-420 0 1 0 1 1.0 (.50) 421-448 7 0 0 0 1 1.3 (.50) 449-4 5 0 1 0 0 1.0 (.50) 477-504 5 0 3 : 0 ( 5 505-532 ~ • . 0) 533 560 4 1 1 .65 (.321 - ssl Sas 1 6 1 1 .37 (.1-8) - 1 8 0 L 539-615 1 .34 (.22) 517-644 6 2 3 .24 (.:2) 645-672 5 0 4 .40 (.24) 573-700 1 6 0 5 .59 (.39) 701 723 2 7 0 5 .35 (.72) - 7 7 1 5 4 5 .56 (.40) 29- 56 3 9 1 3 757-734 1 .59 (.44) 735 812 5 1 7 .66 (.50) - 313 840 0 5 1 2 .48 (.35) - 331 363 4 6 0 5 .92 (.76) - 396 369 7 6 2 .29 (.21) - 397-923 1 0 6 7 1 0 3 4 .26 (.19) ?:5-952 333-330 5 1 1 .20 (.15) i31-i003 a 2 2 1 1 •i5 (•10) .1009-1036 2 3 0 6 .30 (.23) 1017-1064 0 11 2 5 .24 (.13) 1065-1092 3 0 1 .24 (.:3) 1093-1120 - 3 2 0 1 .29 (.21) 1121-1148 1 1 0 .29 (.21) 0 - 0 0 .29 (.21) ' ~ 2 6 3 4 ----------------------------------------------------------- a Oetiaed as :^e :otal numb.r of aniaals vith histopatholoqzc ezacinat:on. 5 The total -=ber o: aniaals iz a given :i=e '..^.terval :s =-e sum of :be a.::ber of aniaala in the Ptas.nt and Absent co1::3ns. 158 CTR CONTRACTS 02933e 11249088 CTR NN 044,12;~

TABLE 66 IaCIOENCE OF NEPHRITIS IN 8C321/CIIH MICE OORINC LONC-TEZH EXPOSURE TO 2R1 C:CIIRETTE SMOKE (STRICT ADHERENCE ':0 RANDOK)a,b FRI., jA!i. 5, 1984 SHGRE SHAM ---------------- --------------- :.a'i3 Ca '3ST ?RESE:JT a3SENT PRESE:IT ASSENT ?R08A8ILI:'Y ------------------------------------------------ +---------------- 1-28 0 7 0 3 1.0 29-56 0 13 1 7 1.0 57-34 7 0 7 2 1.0 35-112 0 0 1 0 1.0 113-140 0 0 0• 0 1.0 141-138 _ 0 0 0 0 1.0 159-196 0 0 0 0 1.0 197-224 0 0 0 0 1.0 225-252 0 0 0 0 1.0 253-280 0 0 1 0 1.0 281-309 0 0 0 0 1.0 309-336 0 0 0 0 1.0 337-364 0 1 0 0 1.0 365-392 2 2 3 1 1.0 (•.S0) 393-42^4 0 0 ~ 1 1.0 (.50) 421-44a 0 0 0 1 1.0 (.50) 449-476 0 0 0 0 1.0 (.50) 477-504 0 2 0 2 1.0 (.50) 505-532 1 3 1 1 .73 (.38) 533-560 1 4 1 0 .35 (.16) S61-588 0 6 0 1 .35 (.~6) 539-615 0 3 1 2 .20 (.09) 617-644 0 3 0. 2 .20 (.J9) 545-672 1 4 3 L .28 (.:4) 673-700 1 3 0 0 .28 (.i4) 701-723 0 4 1 1 .14 (.06) 729-755 1 4 0 0 .14 (.06) 757-734 1 3 3 2 .24 (.13) 795-812 0 0 3 0 .24 (.13) 313-843 0 0 3 0 .24 (.13) 941-363 0 0 3 0 .24 (.1-3) 369-395 0 0 0 0 .24 (.13) 997-924 0 0 1 0 .24 (.13) 325-992 0 3 : 0 .24 (.-3) 353-i3J 0 0 : 0 .24 (.13) 99:-1003 0 0 3 0 .24 (.13) 1009-LJ36 1 8 2 4 <.01•(<.011• 1337-LC64 0 0 3 0 <.O1•(<.01)9 1065-1092 0 0 0 0 <.01•(<.01)• 1093-1120 0 0 0 0 I.:21-1148 0 0 - 13 0 9 7 0 9 31 --------------------------------------------------------------- s Ze:lned ss :noe• ani3als :aken o:- test randomly. The :oca1 -:rzb.r ot animals ia a qiven tim• izterval is :he s= o! the nc:mber of anisals :1 the Present and A.b..n: rol-=ns. ~ 5 C.TR CaNTRRCTS 429339 11249089 CTR Nf _i 04412-B

?inal Report Contzact: C:R-~;030 CT2-101a TABLES 67-70 aLL N.ALIG:UNCIES INCLDDED THE POLLOWI:JC: ADENOSQQAMOOS CARCINOMA ALV'EOLAR ADENOCARCI:IOMA CARCI:70SARCOMA ?IBROSARCOMA CRANULOSAL CELL TUMOR iE.w.ANGIOSARCOMA :{ZPATOCELLOW\R CARCINOMA LEICMYOSARCOMA LEQET-MIA, ALL TYPES :.Y'SPHOSARCOMA !SYSOSARCOltA .4EOPLASM, TYPE UNDETERNINED CST!OSARCOMA ?OORLY DIPPERENTIaTEO CARCI`IC`4A R8:7AL CARCINOMA RETZCZLCM CELL SARCOMA SaRCC?U1 SQOAMOOS CELL CARCINOMA STRC!!AL TUMOR °O:SOR, TYPE 0:7DETER.`SI:IED 160 CTR coNTRacTS 029340 11249090 CTR NN 044,1: "~

T?LeLF. 5 7 AC''.1RZ.1L A.vALYSIS OF 3C3F1/C~^4 :,!:CE DYING OF ALL :IALZGaaNCZ_S DURING LOYG-TERM EXPOSG?W '. 2R1 CIGARETTE SM~1~ (GENERAL ADHERENCE TO DIED CR I~tORIBUND, ALL) ' TQE., J.1.v 3, :384 S:SOFCF. S c'iAH ------------------ ------------------- DAYS :.N T EST 0'1/TUMOR AT RISK 0 il/TUNOR AT RISK PR08118iLi^_': ------------------------------------------------- --------------------- -- - 1-28 0 987 1 659 1.0 29-56 0 978 654 1.~ 57-84 0 961 3 647 1.0 85-i12 0 953 1 644 1.3 113-140 0 937 3 644 L.0 141-158 0 931 3 642 1.0 169-196 0 924 3 639 1.0 197-224 0 913 1 637 .37 (.24) 225-252 0 898 ~ 635 .37 (.24) 253-280 0 988 ~ 630 .87 (.24) 281-308 0 875 .1 625 .87 (.24) 309-336 1 970 3 621 1.0 (.3:) 337-364 0 362 : 614 .77 (.33) 365-392 1 338 3 585 1.0 (.0:) 393-420 0 309 1 556 .69 (.40) 421-448 1 781 1 532 .63 (.39)• 449-476 1 757 2 517 .37 (.22) 477-504 5 734 3 491 .:0 (.29) 505-532 2 711 2 469 .35• 533-560 6 682 4 447 .43 (.33) 561-588 10 645 426 .98 (.35) 5a9-616 14 616 :~ 407 1.0 (.9-) 517-644 13 586 :3 377 i6 (._Q3) 645-672 10 552 349 .35 673-700 25 517 7 318 1.] (.9:) 701-729 9 457 :: 298 .64 (.59) 729-756 21 415 3 274 .45 (.4J) 757-734 12 351 :3 248 .:6 (.:4 735-312 17 311 215 .20 (.:3) 313-3i0 21 280 3 190 .42 (.3?) 341-35d 15 245 :3 166 .34 (.2:i 369-395 17 204 1-3 133 .30 (.:3) 397-924 '-9 174 :~ 104 .35 925-952 16 131 :3 77 .34 (.3:? 953-930 8 106 5 58 .33 (.3~) 991-: JC3 10 79 5 45 .33 (.331 '_009-i036 - 6 55 5 23 .26 (.:3) 1 037-1063 3 17 J 7 .30 (.Z31 :065-1:92 2 3 5 .29 (. :) :393-1120 0 3 : 2 .27 (. 5) 1121-.-48 0 0 3 0 .27 (..,-) 265 139 3 b ---------------------------------------------------------------- Dofi^ad as th• total numb.r of ani=als that had tiistopstholoqic e:amination. :"s• nu=b.r of animals that diad of ;h• la.ion or tumor. 161 ' CTf2 C0NTRRCTS 029341 11249091 C7 R ) 1! 1 O444.h. ,30

TABLE 63 ACT'Jll<iIAL ANALYSIS OF BC3F1/CUM aZCE DYI::G OF .1.LL YALIG,tA,tC.~S DURI2IG LO.iG-TERH EXPOSURE TO 2R1 CIC.n.e2ETT.°. SMOKE (STRICT A,OHEREBICE TO DIED OR :SORI3ClID)a,b TUES., .IJ4H 3, 1984 .~. SMOKE 5 i',j,.K ------------------- -------------------- OaYS :~:f '.'•:ST J il/:_':!flR AT RISiT. 0 i7/TU;:OR AT RISt''C ?R08A3ILI.Y --------------------------------------------------------------------------- 1-28 0 510 1 387 L.0 29-56 3 509 0 335 1.0 57-34 1 508 J 335 :.~ 85-112 ~ 507 0 385 1.3 113-140 ~ 507 0 335 :.0 141-168 3 507 0 385 1.0 169-196 0 507 0 3a5 1.0 197-224 0 506 0 3a5 L.0 225-252 0 505 0 3a5 1.0 253-280 0 505 0 385 L.0 291-308 0 505 0 3a5 l.o 309-336 1 504 0 385 1.0 (.39 337-364 3 501 1 335 :.0 (.86 365-392 3 499 0 334 :.3 (.d6 393-420 0 496 1 383 .33 (.42 421-448 0 489 1 378 .3S (.21 449-476 1 485 1 376 .37 (.26 477-504 3 482 2 371 .57 '(.36 505-532 2 476 2 367 .62 (.45 533-360 6 470 4 361 .30 (.65 561-588 7 459 1 352 .73 (.65 589-616 9 449 6 345 .7J (.59 517-644 :2 431 12 333 :.0 (.94 645-572 3 408 7 312 .63 (.61 673-700 :7 398 7 296 .35 (.38 701-729 3 369 11 232 .37 (.30 729-756 :5 347 17 262 .56 (.51) 757-7a4 3 317 16 233 .13 (.16) 785-312 :7 298 11 209 .23 (.201 313-3y0 21 268 8 134 .51 (.471 341-368 :5 233 13 160 .:Z (.331 369-396 :7 192 13 :27 .33 (.33• 397-924 :9 162 10 33 ..5 (.~L• 325-352 :5 129 :J 7: ..: (.39 353-330 3 94 5 52 (.?3~ 331-i008 :0 66 6 39 .41 (.33i 1009-'_036 3 43 2 1.7 .33 (.216i :037-1064 3 17 0 7 .33 (.30) :065-1092 2 8 2 5 .41 (.38) :~93-1120 0 3 1 2 .33 (.361 :121-1148 0 0 0 0 .33 (.361 225 170 ---------------------------------------------------------------- a Deti^ed as t::ose anfaals that died oc were kilLed when aoribuad. 5 Tye nu=ber a: aniaals that died of iesions o: t:::aor. 162 c.TR CONTRRCTS 029342 11249092 CTR VIN 04,41 '41

'T33LZ 59 :.ALIGt7.1NCIZ3 ::7 3C3F1 /C::K MICE DURINC LCNC-T=?u ='/.?•^,Sz:k. TO 2R1 C.:.:.isTTE SMOKE (GE:IEPAL ~dE?~iCZ TO RANOOM, ALL) a, b :*JE., J.11i 3, 1984 SMORE S HAM IAYS O2•I TEST ------------- ?RESENT a3S ---- ----------------- E.`(: ?P.rSENT ABSENT PRC3A3:I::Y ---------------- ---------------------------------------------- ------- :-23 0 9 0 5 11.0 29-56 0 17 0 7 1 .3 57-34 0 3 1 3 '_.3 33-:12 0 i5 ~ 0 :.~ :13-140 0 5 1 2 i;1-163 0 7 3 3 L.~ 159-196 0 11. 1 2 '-.0 137-224 0 15 1 1 .24 (.01) ' 225-252 0 10 0 5 .24 (.01)' 253-230 0 13 0 5 .24 (.01)' 231-308 0 5 0 4 .24 (.01)' 309-336 1 7 0 7 1.0 (.49) 337-364 0 24 : 28 .54 (.23) 365-392 1 29 7 29 :.0 ".59) 393-420 0 28 23 421-448 1 23 1 14 449-476 1 22 2 24 .49 (.31) 477-504 5 18 4 18 .75 (.5d1' 505-532 2 27 2 20 .53 (.53) 533-560 . 6 31 4 17 .53 '.50) 351-Sa8 10 19 1 18 .-3 i.51) 539-616 14 16 1.o 20 .39 '.3's) 5:?-54s 13 21 13 15 .55 .53) 545-672 10 25 '1 20 .39 '.30) 373-700 25 35 7 13 .73 .3J1 7J1-728 9 33 ': 13 .34 i."7) 729-756 21 43 13 8 17 (.'5) 757-734 12 28 13 15 .34'1.74)' 735-312 17 :3 11 14 .09 (.131 3:3-340 21 14 9 16 .29 (.251 341-363 15 25 13 20 .: 3 (.25) 359-396 17 3 13 16 .43 i.39) 337-923 19 4 i0 17 .'2 (.i 7) 325-?52 16 -9 1-0 9 ii .5 0 1 ?43-330 8 20 5 8 .:3 ~.:31 i39 10 13 5 16 .'5 (.'0) :; 39-10 36 6 32 5 11 .59 i.5 51 :737-1064 3 6 3 2 .64 1.501 :365-1092 " 2 3 2 1 .50 (.56) 13 .93-1120 0 3 1 1 .55 i.5i1 1 :-_-1138 0 0 0 0 .55 i.5:1 265 722 :39 470 --------------------------------------------------------------- 3 5 =a::aQd as t::e total :::--%ber of iniaals with histooatholog:t aza.=i.^.ation. :"e tatal ==bar of aniaals :n a given t:me interval is t`e 9-= o! the number of animals in ttse Present and Absent tol::ans. CTR CQNTRACTS 029343 1!249093 C T R- HN ~".44. 13"

r:rc::,~ rc~ ~.7 TABLE 70 ALL MALIGNANCI33 IN 3C3F1/CzI<i MICE DURI:rG SXPOSURE TO 2R1 CIGARETTS SHOKE (STRICT ADEERENCE TO RANDOM)a.b TVE., JADT 3, 1984 SMOKE SHAM ---------------- ----------------- DA7; :a :~.._ ?RES.-~w1T A3SG^.JT ?RES'G.`IT .13Se.NT ?ROBA3I:."': --------------- -------------- --------------------------------------- 1-23 0 8 0 3 1.~ 29-5i 0 16 0 7 57-34 0 7 0 3 35--?2 0 16 0 0 1:3-::v^ 0 6 0 2 131-:5a 0 7 0 3 1.~ 1 0 10 0 2 197-224 0 14 1 1 ,25 225-252 0 10 0 5 ,26 (,3 :). 253-2a0 281-308 0 0 13 4 0 0 5 4 .26 .26 309-336 0 5 0 7 .25 337-364 0 22 0 28 .26 (.011* 365-392 1 25 0 28 1.0 (.55) 393-420 0 21 0 19 :.3 r,5;) 421-44a 1 19 0 13 1.0 (.95) 449-476 0 20 1 20 1.0 (.63) 477-504 2 1s 2 16 1. 0 (.77.), 505-532 0 23 0 16 1.C (.77) 533-560 0 26 0 12 1.0 (.77) 55i-:33 3 16 0 12 .37 (.52) 539-515 5 . 7 4 14 .11.0 (.23) 617-644 1 10 1 6 .;3 (.?5) 545-572 7 18 4 11 573-?-70 3 23 0 6 701-723 0 20 0 4 ,29 (.2:) 729-7:5 5 29 1 1 .42 (.33) 757-734 4 17 2 2 .49 (.56) 735-3'_2 0 1 0 0 ,69 (.56) 3'_3-340 0 0 0 0 .39 (.56) 341-353 0 0 3 0 .59 (.55) 359-396 0 0 0 0 .53 (.s6) 397-924 0 0 0 0 .59 (.'s5) 925-952 3 0 0 0 .53 (.:6) 953-g30 0 0 0 0 ?31-.:03 0 0 0 0 .59 (.:61 iJ09-:035 3 9 3 3 .?5 (.33) :~37-i064 0 0 0 0 .36 (.331 '_465-1092 0 0 0 0 .36 (.33) 1393-1120 0 0 0 0 .96 (.331 ::21-1148 0 0 0 0 .96 (.33) :0 437 19 253 --------------------------------------------------------------- 3 5 2e::-ed as :hose animals raken off test randomly. :'Se :z:al ncmber of animals in a given tiz• interval Ss the scm of tna nusber of animals in the Present and Xbsent cal=ns. 154 CTR CaHTRACTS 029344 11249094 CTFZ f f N 044• 13,,3

?inal Report Coctract: CTS-0030 CTR-LOla ,X3LZS 71-73 ?IBROSaRCOMA 165 CTR CONTRRCTS 029345 11249095 CI R I N .Y• 4I YL L...A 4

TASLE 72 aCT:A2IA1 aNAL'tSIS OF BC3F1/COM :SICZ DYING OF HEAD AND NECK ?I3ROSARCOHAS DURING LONG-TZRM EXPOSURE TO 2R1 CIGaRE^TE SMOKE (BTR:C:' ADHERENCE TO DIED OR HOR23aN0, SHOULDER FS 4OT 14 T:HOR C-.L)a,5 MON., JAN. 2. :334 SMOKE SHAM ------------------ ------------------ :AYS ::r 'T.'aT ^./S'1 `.?`SOR AT RISR 7 i7/TC:40R AT RISe'C ?R08A31Li:': ----------------------------------------------------------------- 1-23 3 510 1 387 1.0 29-55 0 509 1 335 1.p 57-34 385 1.0 85-11. 2 0 507 ~ 385 1.0 113-140 0 507 0 395 1.0 141-16a 0 507 0 385 1.0 169-L96 0 507 0 38S 1.0 197-224 0 506 0 385 1.0 225-252 0 505 0 38S 1.0 253-280 0 505 0 385 1.0 281-308 0 50S 0 385 1.0 309-336 1.' S04 p 385 1.0 337-364 0 501 a 385 1.0 365-392 0 499 0 384 1.0 393-420 0 496 0 393 1.0 421-448 0 489 0 378 1.0 449-476 0 485 0 376 1.3 477-504 1 482 0 371 1.0 (.:3) 505-532 0 476 p 367 p r 331 1 533-560 2 470 0 361 „ , .35 (.:;) 561-5d9 L 459 0 352 .22 (.:3) 539-615 = 449 0 345 , 517-s44 2 431 1 333 i7 (,:3) 545-672 0 408 0 312 ~7 r„ :?) 673-7~0 3 398 1 296 ~3 70:-723 3 369 1 282 , ~9 729- 35 1 347 1 262 , :3 (.:3) '57-734 1 317 0 238 , .39 (.:51' 735-312 4 298 0 209 313-340 4 268 0 134 34:-363 2 233 '] 160 c 0!•!<.::;• 359-3y5 3 192 1 127 , 397-924 2 162 38 925-952 353-980 0 0 129 94 _ p 0 71 52 i ' 931-1008 2 66 0 39 ) 1009-1036 1064 :037 0 43 0 17 <,O - 0 17 0 7 < 01•(<.J L065-1092 0 8 p 5 . 1093-1120 0 3 0 2 1121-1149 0 0 0__ 0 29 6 ---------------------------------------------------------------- 3 CaEi^ed as those animala that diad =or:bucd. 5 or :rere killed vhen :.`.e n=ber a; aainala that died of leeiona or t:mor. ;67 ' CTR CONTRRCTS a29346 11249096 CT~.'~' HN 3, ~

Ta3LE '3 ZYCIDENCE OF 'ric'1D A:1D NECR F:9ItOS.LACC:4AS IN SC3F1/CC:! HZC: LONG-TE?.i :iQOSUcZE TO 2R1 CIGARETTE SMO)CF. (GzNZ?.u. :Z3ERZNCE TO RANDOM, ALL) a °~ TQ:.'. :A.*J 3, 1984 S:!C5--. . ;-b.•' SSm ------------- --- ----------------- OU;tI:7G 3AYS ON TEST ?RESE:JT j.3SE:)T PRESENT ABSENT ?ROBASI :Z TY ---------------------------------------------------------------------- --28 0 9 0 5 1.0 23-56 0 17 0 7 1.0 57-34 0 a 0 3 1.0 35-112 0 15 0 0 1.0 113-140 0 ~ 0 2 1.3 141-168 0 7 0 3 159-196 0 11 0 2 '.~ 197-224 0 15 0 2 1.0 225-252 0 10 0 5 1.0 253-230 0 13 0 5 1.0 281-308 0 5 0 4 1.0 309-336 0 a 0 7 1.0 337-364 0 24 0 29 1.0 363-392 0 29 0 29 L.J 393-420 0 28 0 24 1.0 421-448 J 24 0 15 :.D 449-476 0 23 0 25 1.3 477-504 1 22 0 22 1.0 (.33) 505-532 0 29 0 22 1.0 (.33) 533-560 2 35 0 21 .;0 (.16) 561-538 I 28 0 19 .26 (.10) 589-616 = 29 1 29 .36 (.13) 517-544 z 32 1 27 .32_ (.:9) 545-672 0 35 0 31 .32 (.:9) 573-700 3 57 1 19 .39 (.:5) 701-728 3 39 1 23 .33 (.22) 729-756 1 63 2 24 .71 (.54) 757-7a4 1 39 0 33 .57 (.42) 735-312 4 27 0 25 .20 (. _4) a13-340 4 31 0 24 .37 (.3s)• 341-368 2 39 0 33 .04•(.32)• 359-d96 0 30 1 28 .06 (.C:)• 397-924 2 31 1 26 .:o' (.03)• 925-352 0 35 0 19 .35 (.03)• 953-9a0 3 29 0 13 .:5 (.~3) • 981-i0C8 2 21 0 :2 J3•(.:2)• :009-1036 0 38 0 :6 .03•(.321 • :J37-1064 0 9 0 2 1J55-1092 0 5 0 3 .33•(.:2)• '_J93-1120 0 3 0 2 i121-1148 0 0 0 0 29 958 8 651 --------------------------------------------------------------- a Cetiaed as the tatal ::ub.r of animals with histopatioloqir ezamination. 5 '"he total auaber of aniaals in a given time int.rval ia the sum of the number of an:mala in the Present and Absent ro1.:3as. 168 CTR CaNTRRCTS 02934~ 11249097 CTTR PIN 0441,36

?izal 2eport Contract: CTR-0030 C:R-101A '"ABLES 74-96 HEV.A:C?OIZTIC :7MORS 159 CTR CONTRACTS Q2934c3 i i Z, Y3 0 9 8 CTR HP-4 0,44137

TaiBLL 74 aCTUaRL\t. A-`IAL'LSIS OF 3C3F1/CLlM MICE DYING OF Hr'.M.'lTOFOIE:'I: :'.^•;035 DURING LONG-Trs't.*f F.X?OSURE TO 2Rl CIC.IRET':E S.40KE (GE:YEcZAL .1DHr-R...°'*IC : TO DIED OR MORIBUND, a1.L)a,b 'rTED., JAN 4, 3.984 SMOKE S cW.`S ------------------- ------------------- ::,YS ON TEST ] ir/ . U.".Oe2 AT RISK D W/TU:20R AT RISK ?RDB.I3ILI:'`L ------------------------------------------------------------------------- 1-29 0 987 0 559 1.0 23-56 0 978 0 5:4 1.0 57-34 0 961 0 647 1.0 35-112 ~ 953 0 544 1.0 113-140 0 337 0 544 1.0 i41-163 0- 931 0 542 :.0 :69-196 0 924 0 639 1.3 =97-224 0 913 1 637 .37 (.24) 225-252 0 898 0 635 .87 (.24) 253-280 0 888 0 630 .37 (.24) 231-308 0 375 0 625 .37 (.24) 309-336 0 870 0 621 .37 (.24) 337-364 0 362 0 614 .97 (.24) 365-392 . 338 0 595 1.0 (.30) 393-420 ~ 309 1 556 .76 (.371 421-448 0 781 1 532 .38 (.17) 449-476 1 757 1 517 .3.8. (.20) 477-504 1 734 3 491 .12 (.06) 505-532 1 711 1 469 .13 (.07) 533-560 4 682 4 447 .12 (.07) 551-588 6 545 1 4426 .50 (.39) 539-616 3 615 7 407 .39 (.31) 617-644 7 586 10 377 .11 (.09) 545-572 5 552 5 349 .10 (.J81 673-700 14 517 6 31.3 .27 (.22) 731-728 4 457 8 298 .39 (.071 729-756 13 415 14 274 .04•(.03) 757-73i 5 351 13 248 735-312 6 311 6 215 913-340 9 280 6 :90 341-368 3 245 5 359-396 3 204 8 397-924 7 174 6 :04 315-952 5 !'41 5 i53-930 3 106 3 931-'_008 3 78 5 :5 `(<.0 1009-1036 4 55 4 23 :037-1064 - 1 17 0 ; :065-1092 0 3 1 5 1093-1120 0 3 1:21-1148 0 0 0 125 126 ---------------------------------------------------------------- a Uetiaed as the total a:zber of animals that ^ad tiistooatholoqic ezamilation. b Tho nL=ber a= aciaals that 3:ed of the l.sion or t::aoc. 170 CTR CONTRRCTS 029349 ;1:49099 CTR W4 0441 '38

:'A3LZ 7 5 ACT.'asZLP.L :..1IAYLSIS OF SC3F1/C::M HICE D'IIVG OF Yr.•SATO?02ET:C DDRIaG LONG-TBR.'•i a.?OSuRE TO 2R1 CIGARETTE SMOKE (STRICT '..3''d'arZt.."\C: TO DIED OR MORI3CArD) a,b %tL'D., .7:,X 4, 1984 SMOKE SZm -------------------- ------------------- = ::LOP.S :AYS C:7 :::ST D i7/TU:10? AT ZIStZ 7 W/ TU:SOR AT RISK ?R03?3IL:: ----------------------------------------------------------------------- !-23 0 5:0 0 387 29- » 0 5:9 0 385 !.J 57-33 0 ::8 0 385 :.^. 35-1:2 0 5:7 0 385 :.3 l:3-1S~^, 0 :17 0 385 ' . ~ :31-I53 0 577 0 385 :.~ 159-i35 0 507 0 385 !,~ 197-224 0 506 0 385 1.0 225-252 0 505 0 385 :,0 253-280 0 505 0 385 1,0 231-308 0 505 0 385 11•0 309-336 0 504 0 385 !•0 337-364 0 501 0 385 !.3 355-332 0 439 0 384 :.: 393-420 0 496 1 383 .30 (.: 421-448 0 3a9 1 378 .37 (.. 449-476 1 485 0 376 .33 (.- 477-504 0 482 1 371 •35 (.: 505-532 1 476 1 367 .47 (.: 533-560 4 470 4 361 .45 (.: 561-5a3 4 459 1 352 .91 (.' :89-616 4 ::9 4 345 •'7 (.• 517-544 6 431 9 333 .32 (.: 545-572 1 408 2 312. 573-7;,J a 338 6 236 .3J (. 701-723 4 359 a 282 •=1 (• 729-755 9 347 14 262 757-734 3 317 12 238 -35-312 6 298 6 209 313-340 9 268 6 134 341-353 3 233 5 160 369-396 8 132 a 127 397-924 7 !52 6 93 9ZS-.=2 5 :29 953-900 3 94 3 52 <,~:•(< 931-1:;03 3 56 5 39 < J1'(< !,~09-1036 2 43 2 1337-1064 1 1.7 0 7 1065-1092 0 8 1 5 1093-1120 0 3 1 2 :121-1133 0 0 0 0 < JI•l< 97 112 ---------------------------------------------------------------- a Je:::ed as rhoae aa Laals that died or .ere killed when =o:i5und. 5 :`:e cu=ber of aniaals that died of leiions or tuaor. 171 CTR coNTRacTS 029350 11249100 CTR HN 0441,,`, 9

TA.BLE 76 IYCI.7F`,IC: TVHORS I;I BC3e 1/C'J.4 :I:C-s :;:?:`iC .'.C:IG--.X?OSURE ;,^r 2R1 CIG.4ZETTE S:40KE ' (Gi.aERAI. ADHERc^JCE TO RANDOM, .l:.L) 3, b TT., .Ta.`i 3, 1984 SMC `= S iiAM ----------------- ---------------- :.1YS ON TEST ??=SZ.YT i,35EaT ?RESENT .13SE:IT ?ROHA3I:.ITY ------------------------------------------------------------------- 1-28 0 9 0 5 1.0 29-56 0 17 0 7 :.0 57-34 7 3 0 3 .0 a5-112 1 :5 0 ~ -.'.J 113-140 1 5 0 ~ 131-16a . 3 7 0 3 169-196 3 =1 0 2 :.~ 197-224 3 15 1 1 .24 (.01)• 225-252 0 10 0 5 .24 (.01) • 253-280 0 13 0 5 .24 (.01)• 281-308 3 5 0 4 .24 (.01)` 309-336 0 9 0 7 .24 (.01)• 337-364 3 24 0 29 .24 (.01)` 365-392 : 23 0 29 1.0 (.521 393-420 : 28 1 23 .59 (.2:) 421-448 0 24 1 14 .25 (.10) 449-476 1 22 1 25 S0 (.20) 477-504 1 22 3 19 . :3 ( . 10 )' 505-532 '_ 28 1 21 .:9 (.11) ' 533-560 4 33 4 17 . 13 (.08) 561-588 5 23 1• 13 .SS (.:1) 589-616 3 22 7 23 .7; (.521 6i7-644 7 27 10 13 .34 (.27) 645-672 5 29 5 26 .:J (.34) 673-700 :i 46 6 14 .32 (.27) 701-724 4 39 8 4 15 • .~3 (.~5) 729-756 :3 51 14 12 757-733 5 35 13 20 785-312 5 25 6 19 313-340 9 26 6 3s1-36a 3 33 5 369-396 3 22 3 397-924 7 26 6 925-952 5 30 5 ii 353-390 3 25 3 iJ ?31-1)08 3 20 5 :/ ::09-1036 4 34 4 12 <.0 Ji)• :037-1064 _ 9 0 2 1.065-1092 0 5 1 2 1-14,33-1120 0 3 1 :121-1138 0 0 0 :25 962 126 533 --------------------------------------------------------------- a Cet:::ed as the total n=ber of animals with histopatholoqic azamination. °4e total number a: animala in a qiven time inte:val is the 5 sum of the nu=ber ot animals in the Present and a5sent =o1_.ns• 172 CTR CONTRRCTS 029351 ''2:9101 CTR MH 04,4140

':ABLE 77 IYCIDEYCE 0£ HS.V11bP0IZT:C TQHOItS IN 3C3£1/CCH !lICE DURI:IC GONC-T=Pr! EX?OSURE TO 2R1 ~C_ CdRET':E SHORE !S':RIC': a:.HERE;7CE ':0 RwGC!4) a.b jaN. 5, 1984 S?SORS SHAtt -------------- ---------------- DAYS 0:J :ES^ ?RESE4^. n35ENT ?RESENT Ae35E:7T ?R03.13iLITY -------------------------------------------------------------- 1-28 0 3 0 3 29-56 I 15 0 7 ? ~ 57-84 : 7 0 3 .~ 35-112 0 15 0 0 :.~ 113-140 5 0 2 1.0 141-i63 0 7 0 3 1.0 169-196 0 10 0 2 1.0 197-224 0 14 1 1 .26 (.01) • 225-252 0 10 0 5 .26 (.01)• 253-280 0 13 0 5 .26 (.01) • 281-308 0 4 0 4 .26 (.0L) • 309-336 0 5 0 7 .20 (.31) • 337-364 1 22 0 23 .25 (.01)• 365-392 1 25 0 28 _.3 (.56) 393-420 0 21 0 19 1.0 (.56) 421-448 0 20 0 13 1.0. (.56) 449-476 0 20 1 20 .66 (.28) 477-504 1 16 2 1E .49 (.26) 505-532 3 23 0 16 .:9 (.25) 533-560 3 26 0 12 .49 (.26) 561-588 2 17 0 12 .93 (.59) 589-616 4 3 3 15 .93 (.70) 617-644 1 10 1 6 -.0 .31) 645-672 5 20 3 12 _.0 (.35) 673-700 6 25 0 6 .70 f.56) 701-728 0 20 0 4 .70 (.56) 729-755 4 30 0 2 .64 ;.50) 757-7a4 2 19 1 3 .80 :.50) 7a5-312 0 1 0 0 .3o !.50) 313-a40 3 0 0 0 .30 .50) 341-a68 7 0 0 0 .30 (.5:i) 369-836 3 3 0 0 .30 i.50) 397-924 3 J 0 ~ .30 ;.50) 925-952 3 0 0 0 .30 :.bJ) 953-980 0 0 0 0 .30 ;.SJ) 981-1008 3 0 0 C .30 ;.50) 1009-1036 2 10 2 4 1.0 (.37) 1037-1064 0 0 0 0 :.0 ;.37) 1065-1092 0 0 0 0 i.0 :.3%) 1093-1120 J 0 0 0 1.0 (. 37) 1121-1148 0 - 0 0 0 1.0 •: . 37) ~ 3i9 i4 .53 --------------------------------------------------------------- a b Detined as those animals taken off test :andoaly. The total nuaber of aniaals in a given tiae interval is the st= of the n::a5er of animals in the Present and Absent col=ns. 173 CTR coNTRacTS 029352 11249102 C,TR VIN 0-44-141

11nc1 kepurt Cootr.ct: C1R-0010 tI1St0L06iC W/SlkYAIIUItS III YC]ft/CUM ftMNC nlCt AfltR 1ktA11LNf ttIT11 Iutl1u(A)PrRCMC (RAP) Pt.RtODS Of ObURVe1tOM 1EAR 1 YtAR 2 1EAR 3 (1PE Of lESION Mt1MItR I fYPE Of tfSION KrrtER I 11PE Of IESIaM kuwt.R I lcuscawlc 1 (10) Lung ccrclnu.. Itl (80) lung c.rclr..c '! (100) ~ N 4~ ALeul.r eury.resting 1 (40) (y.ptaslreu.e, 2 (2) <1) •t.Ju14 cct/calw c.ll O W "-1 ~ s.rcu.. Other neopl.s..s 2 (2) 4 1 0 r h Cunyestlon 2 (2) Alveolar no.-cu..µresslny 1] (l3) t •nJ co.pnsslwg ~odules ?U 33 No n.Jor Jlieases aot.0 1 (1) ^ ..~..~y1.. Mr " 1 Ln TOIAL IwwtR Of ANIW1tS 2 9N 2 ~ w ~ Cil (A) 1

11r..1 Mcporl tonts.cl: C1N-0US0 1AlNl 19 INCIIYItL (M Ilillx/i IIIAI l IKII V CAVfID ll/t ItAIII IN YCII l/LiRI MICI IUItUMIfN: •lN1U(A)fTNLNt 1N1AIItN1 AtN IIrOSINtF 10 2A/ CIGMf11f YIt4N(t PtN1U0 U1 UtlSINYAiIUM 4J C) `~ 0 v an _ ~~ w k "a"'~ "7* ~ .w. ..~ ~ ""i Ln ~ ~ afi.k .~ N ~ (A) ~ Ul -A .~ ~ T E A N I 11Yt W ttS10M NIYritN I 1tPt 0f t[SIUN luny c.rclnw s S Lw ..Jor dlse.ses aoteJ, 2 Wt rlth Incld.at.l fladlaps of p1y..a1eJ alveolar ..croyh.y. .ccur.l.tloo ..d alveolar eon-ca.yress- Ly .odula (/1) Lung c.rcitwm. 1 L A N 2 (29) ll+.(N~os.rcuu. ntlculur cell urco.. Ilbrus.rcu.. Otlwr c.rc/na.. Nu .rJur d/scats .oteJ. but with lacldent.l flndlnyt of alveolar aon-cuspretslas and co.press/ny aorules. hep.tltls, nephr(tis, ot/lls, eitero. lUlAl Nufllt(1 l1f ANIMAtS / NpM/lN 1 1 l: A N 3 ITPE 0f LtS10N NUrbfN i 00 (l9) Lung c.rclnur. ) (lUU) ~ (4) 1 (1) 1 (1) 10 (14) )l )

f/n.l Meport TAetE AO Contr.ct: CIR-0030 INCIOCMLE Of 1ES10MS IIIAT lIttET G1USf0 TME OEATN Of BClfl/CUt MICE fOLE0YIMG REN10(A)PTRENE 1PEARfEMI AtID SMdMI EtPaSURE n~ P/R1W 0f OUStNYATION fEAR I • TEAR 2 TEAR ] 1tPE 0f lESlON Nu18ER I TrPE 0f 1ES10N M1M8ER f TrPE Of IESIOM 1rMER s lrn9 carclno.. S (S0) luny carclno.a 1S (R0) luny carclno.a 10 (OI) No rJor dlsease eoted, S (50) l1.pAosarco.a 3 (4) lyepAosarco.e 1 (B) but r/tA l.cideatal tlw/ays of alveolar f/orosarco.a 1 (1) eorrcoepresslay aa6 co.pressl.y .odrles Mo erJor disease .otN, 10 (14) No ..)or disease noted. 1 (e) but .Ith /ecldental but with lecld.ntal ' flnd/iys of alveolar f/edle9s of alveolar eoa-coryressle9 oM ooo-co.presslef aed co.press Uy wrules, ca.presslny .odrles etitls etterM + V .~ 33 S .Wr ~~ l J Ln lOfAi Mu1IKR Of ANIMALS 10 69 12 Ry»J rinl Q N Ww ~ Ln `4

'.' -f,-oTi7,f7O NW NUO soLstrz<< 9SE6Z0 S13bN1N00 S10 - ut t = IiE EE E: EE F: ... .. .. .. .. ... ... ... ... ... ... ... ... ... ... ... ... ..- --- --- ... ... --- •-- ... :.. --- __: ~iSt' ... C.. .::-~ :.• ... tl -. .. iL..: ... ... ~.:.~ ... ... ..1 ... ... 1 1 . . ... 1 . ... i...= :...i i.._: :.... i-- ~...: i•..i :...~ E...~ ....: ... ... ... .. .. .. .. St 1 't t' t 333t•f+ ... .. .. .. ... .. .. .. .. ... .. -. .. .. ... .. .. .. .. .t ... .. .. .. .. ... .. .. .. .. ... .. .. .. .. . .. .. i: ... .. . t ... .. .. .. .. ... .. .. .. .. ... .. .. .. .. t ... .. .. .. .. ~'t ... .. .. .. .. z ~i t ... .. .. .. .. ... .. .. .. .. ... .. .. .. .. ... .. .. .. .. t= i ... .. .. .. .. `

TABLE 32 IvC20ENCE OF PIGMENTED ALVEOLAR MACROPHAGE aCCCKULaT:ON AFTER BAP TREATMENT AND LONG-TERli EXPOSURE TO 2R1 CIGARETTE SMOKE (ALL ANIMALS)a,b THU., .:AN. 26, 1984 ~. BAP + SMOKE 8AP + SHAM -------------- ---------------- DAYS ON T:S T ?RESENT ABSENT PRESENT ABSENT -------------------------------------------------- 1-28 0 2 0 3 29-56 0 18 0 3 57-84 24 9 0 0 85-112 11 0 0 1 113-140 2 1 0 1 141-168 2 2 0 5 169-196 2 6 0 5 197-224 2 4 0 5 225-252 2 0 0 2 253-280 5 1 3 2 2B1-308 5 0 0 5 309-336 2 1 0 8 337-364 0 4 0 9 365-392 0 2 0 S 393-420 8 2 0 12 421-448 3 1 2 10 449-476 1 3 0 11 477-504 4 4 0 14 505-532 6 2 0 B 533-560 8. 7 0 16 561-588 10 10 0 13 589-616 7 1 0 10 617-644 3 4 1 12 645-672 3 8 0 5 673-700 2 6 0 7 701-728 1 4 0 7 729-756 1 5 0 5 757-784 0 2 0 2 785-812 0 0 0 5 813-840 0 0 0 2 841-868 0 L 0 2 869-896 1 0 0 0 897-924 0 0 0 1 925-952 0 0 0 0 953-980 0 0 0 0 981-1008 0 0 0 0 1009-1036 0 0 0 0 1037-1064 0 0 0 0 106S-1092 0 0 0 0 L093-1120 0 0 0 0 1121-1148 0 0 0 0 -- -_--- -________ L1 5 110 6 196 3 -----~--------~ ------ ------------------- Detined as the total number of animals vith hfstoQatholoqic examination. b The total numb.r of animals in a qiven tim• interval is the sUM ot the numb.r of animals in the Present and Absent cal;:mns. 178 CTR COHTRACTS 029357 11249107 ~ TR NN 044146

:ABLE 83 INCIDENCE OF ALVEOLaR NON-COMPRESSING NODULES OR A.LVEOLAR COMPRESSING NODQLES IN BC3F1/CQa MICE AFTER TREATMENT WITH 8AP aND LONG-TERM EXPOSURE TO 2e21 CIGARETTE SMOKE (GE:IERAL ADHERENCE TO RaNDOM)a,b SUN., ,;aN. 8, 1984 BAP + SMOKE 9AP + SP[.1M -------------- ----~----------- DAYS ON TEST PRESENT ABSEyT PRESENT ABSENT PROBaBILITY -------------------------------------------------------------- 1-28 0 2 0 3 1.0 29-56 0 18 0 3 1.0 57-84 0 33 0 0 1.0 85-112 0 11 0 1 1.0 113-140 0 3 0 1 1.0 141-168 0 4 2 3 .56 (.181 169-196 1 7 0 5 .99 (.54) 197-224 1 5 1 4 .93 (.58) 225-252 1 1 1 1 .94 (.62) 253-280 2 4 1 4 1.0 (.88) 281-308 3 2 1 4 .88 (.65) 309-336 2 1 5 3 .85 (.64) 337-364 3 1 6 3 .77 (.58) 365-392 1 1 2 3 .73 (.56) 393-420 4 6 8 4 1.0 (.93) 421-448 1 3 10 2 .57 (.44) 449-476 3 1 10 1 .46 (.35) 477-504 4 4 12 2 .19 (.14) 505-532 7 1 6 2 .27 (.20) 533-560 10 5 12 4 .22 (.17) 561-588 17 3 9 4 .44 (.36) 589-616 6 2 8 2 .41 (.34) 617-644 7 0 10 3 .61 (.52) 645-672 8 3 5 0 .46 (.38) 673-700 6 2 5 2 .49 (.41) 701-728 4 1 4 3 .61 (.53) 729-756 5 1 3 2 .74 (.65) 757-784 2 0 0 2 .94 (.84) 785-812 0 0 5 0 .94 (.84) 813-840 0 0 1 1 .94 (.34) 841-868 0 1 2 0 .80 (.71) 869-896 0 1 0 0 .80 (.71) 897-924 0 0 1 0 925-952 0 0 0 • 0 953-980 0 0 0 0 981-1008 0 0 0 0 1009-1036 0 0 0 0 1037-1064 0 0 0 0 1065-1092 0 0 0 0 1093-1120 0 0 0 0 1121-1148 0 0 0 0 98 127 130 72 --------------------------------------------------------------- Detined as the examinationm total mmber of animals with histopatholoq ic a b The•total aumb.r of animals in a given time intetval is the sun of the number of animals in the Ptes.nt and Absent col~=ns. CZ'R coNTRACTS 029358 11249108 Cf R f ff`''f 0'"f•'-•' f• 1''°•' f• f""-"

TABLE 84 INCIDENCE OF ALVEOLAR NON-COMPRESSING N000LES OR A.LVEO:.AR COMPRESSING NODDLES IN 8C3F1/CQM MICE AFTER TREAT:S=:1T WITH SAP AND LONG-TER!{ EXPOSURE TO 2R1 CIGARETTE SMOKE (STRICT ADHERENCE TO RANDOM)a,b SDN., JAN 8, 1984 3AP + SMOKE SAP + SHAM -------------- ---------------- DAYS ON TEST ?RESENT ASSENT PRESENT ASSENT ?R03ASILI:Y -------------------------------------------------------------- 1-28 0 2 0 3 '.0 29-56 0 18 0 3 1.0 57-84 0 33 0 0 1 .0 85-112 0 10 0 1 1.0 113-140 0 3 0 1 1.0 141-168 0 4 1 3 1.0 (.32) 169-196 1 7 0 5 1.0 (.88) 197-224 1 4 1 4 1.0 (.91) 225-252 1 0 0 0 1.0 (1.0) 253-280 2 4 1 4 1.0 (.85) 281-308 3 2 1 4 .62 (.40) 309-336 2 0 5 0 .62 (.40) 337-364 0 1 2 3 .79 (.56) 365-392 1 0 2 2 .62 (.42) 393-420 3 5 6 2 1.0 •( .93) 421-448 1 2 9 2 .69 (.52) 449-476 1 1 5 1 .54 (.39) 477-504 1 2 8 1 .25 (.18) 505-532 6 0 3 1 .39 (.28) 533-560 4 1 2 1 .47 (.35) 561-588 5 1 4 1 .51 (.39) 589-616 2 0 6 0 .51 (.39) 617-644 2 0 2 1 .63 (.49) 645-672 1 1 3 0 .48 (.37) 673-700 0 0 0 0 .48 (.37) 701-728 0 0 0 1 .48 (.37) 729-756 0 1 0 0 .48 (.37) 757-784 0 0 0 0 .48 (.37) 785-812 0 0 4 0 .19 (.13) 913-840 0 0 0 0 841-868 0 0 0 0 869-896 0 0 0 0 897-924 0 0 0 0 925-952 0 0 0 0 953-980 0 0 0 0 981-1008 0 0 0 0 1009-1036 0 0 0 0 L037-1064 0 ..0 0 0 1065-1092 0 0 0 0 1093-1120 0 0 0 0 1121-1148 0 0 0 0 37 102 65 44 --------- ----------------------------------------------------- a b Detined as those animals taken otf test randomly. The total nuaber of animals in a qiven tiae interval is the aum of the number of animals in the Present and Absent colunns. 180 CTf2 CONTRRCTS 029359 11249109 CTR i-14,4 044146

TA.BLE 8 5 ACTDARIAL aNALYSIS OF 9C3F1/CUN MICE OYING OF LU;tG CaNCER AFTER TREATMENT WITH SAP AND L0:1G-TERlt EXPOSURE TO ' 2R1 CIGARETTE SMOKE (GENERAL ADHERENCE TO DIED OR ri0RI3UND, aLL)a'h SDN., JAN 8, 1984 DAYS ON TEST. 3AP + SMOKE SAP + SEt3lM ------------------ ------------------ D/W TCMOR AT RISK D W/TII:SOR AT RISK 7ROHa8ILITY ----------------------------------------------------------------- 1-28 0 225 0 202 1.0 29-56 0 223 0 199 1.0 57-84 0 205 0 196 1.0 85-112 0 172 0 196 1.0 113-140 0 161 0 195 1.0 141-168 0 158 0 194 1.0 169-196 0 154 0 189 1.0 197-224 1 146 0 184 .91 (.271 225-252 1 140 1 179 .84 (.43) 253-280 0 138 1 177 1.0 (.81) 281-308 0 132 0 172 1.0 (.81) 309-336 1 127 3 167 1.0 (.74) 337-364 2 124 1 159 1.0 (.901 365-392 1 120 3 150 .97 (.77) 393-420 3 118 2 145 1.0 (.94) 421-448 2 108 5 133 .88 ( . 72,) 449-476 4 104 6 121 .74 (.61) 477-504 7 100 7 110 .85 (..73) 505-532. 3 . 92 5 96 .68 (.58) 533-560 12 84 13 88 .69 (.61) 561-588- 17 69 9 72 .87 (.79) 589-616 6 49 8 59 .93 (.86) 617-644 5 41 10 49 .99 (.93) 645-672 7 34 4 36 .95 (.88) 673-700 7 23 5 31 .7S (.69) 701-728 4 15 4 24 .67 (.60) 729-756 4 10 4 17 .58 (.53) 757-784 2 4 2 12 .50 (.45) 785-812 0 2 3 10 .54 (.49) 813-840 0 2 2 5 .59 (.54) 841-868 1 2 1 3 .58 (.53) 869-896 1 1 0 1 .55 (.49) 897-924 0 0 1 1 .55 (.49) 925-952 0 0 0 0 953-980 0 0 0 0 981-1008 0 0 0 0 1009-1036 0 0 0 0 1037-1064 0 0 0 0 1065-1092 0 0 0 0 1093-1120 0 0 0 0 1121-1148 0 0 0 0 91 100 a Detined as the total numb.= of aaimals that had histopathologic examination. b Th• numbsr of aniaals that died of the lesion or tumor. 181 C.TR CCNTRRCTS 42936Q 11249110 C~ TR HN 044149

TABLE 86 ACTOARIAL ANALYSIS OF 9C3F1/CIIH MICE DYING OF L7NC CANCER AFTER TREATMENT WITH BAP AND LONC-TERN EXPOSURE TO 2R1 CIGaRETTE SMOKE (STRICT ADHERENCE TO DIED OR HORIBQND)a,b SON., ,TAN 8, 1984 BAP + SMOKE BAP + SHAM ------------------ ------------------ DAYS ON TEST 0/W TOHOR AT RIS1C D W/TDHOR AT BISR PROBaBILITY ----------------------------------------------------------------- 1-28 0 • 86 0 93 1.0 29-56 0 86 0 93 1.0 57-84 0 86 0 93 1.0 85-112 - 0 86 0 . 93 1.0 113-140 0 85 0 93 1.0 141-168 0 85 0 93 1.0 169-196 0 85 0 92 1.0 197-224 L 85 0 92 .98 (.30) 225-252 1 84 1 92 .95 (.52) 253-280 0 83 0 90 .95 (.52) 281-308 0 83 0 90 .95 (.52) 309-336 1 83 3 90 1.0 (.80) 337-364 2 82 1 87 1.0 (.90) 365-392 1 79 1 83 1.0 (.90) 393-420 2 78 2 82 1.0 (.89) 421-448 0 76 1 78 1.0 (.92) 449-476 2 75 4 77 .79 . (.63) 477-504 4 73 3 72 .95 (.80) 505-532 1 68 3 67 .69 (.57) 533-560 7 66 11 63 .38 (.30) 561-588 13 56 7 50 .79 (.70) 589-616 4 42 4 42 .80 (.72) 617-644 4 36 9 38 .51 (.44) 645-672 6 31 2 28• .77 (.66) 673-700 7 22 5 26' .96 (.89) 701-728 4 14 3 19 1.0 (.99) 729-756 4 9 4 13 .98 (.91) 757-784 2 4 2 8 .91 (.84) 785-812 0 2 1 6 .94 (.88) 813-840 0 2 2 5 1.0 (.94) 341-868 1 2 1 3 .99 (.92) 869-896 1 1 0 1 .94 (.87) 897-924 0 0 1 1 .94 (.37) 925-952 0 0 0 0 953-980 0 0 0 0 981-1008 0 0 0 0 1009-1036 0 0 0 0 1037-1064 0 0 0 0 1065-1092 0 0 0 0 1093-1120 0 0 0 0 1121-1148 0 0 0 0 68 71 ------------ -------------------------------------------------- a Defined as those animals that died or were killed when cnoribund. The number of animals that died of lesions or tumor. 182 ' CTR CQNTRRCTS 42936I 11249111 CTR ~IN 0441~:~0

TA3LE 87 INCIDENCE 0? NALIGNANT TDHORS IN THE RES?IRATORY TRACT OF 3C3F1/CIIH MICE AFTER TREATMENT WITH SAP AND LONC-TERM EXPOSDRE TO 2R1 CIGARETTE SMOKE (GENERAL ADHERENCE TO RANDOH, aLL)a,b SQN., JAN. 8r'Z984 SAP + SMOKE BAP + SIiAH -------------- ---------------- DAYS ON TEST PRESENT A3SENT PRESENT ABSENT 2QOBABILITY -------------------------------------------------------------- 1-28 c) 2 0 3 1.0 29-56 0 18 0 3 . 1.0 57-84 0 33 0 0 1.0 85-112 0 11 0 1 1.0 113-140 0 3 0 1 1.0 141-168 - 0 4 0 5 1.0 169-196 0 8 0 5 1.0 197-224 1 5 0 5 1.0 (.37) 225-252 1 1 1 1 1.0 (.56) 253-280 0 6 1 4 1.0 (.92) 281-308 0 5 0 5 1.0 (.92) 309-336 1 2 3 5 1.0 (.88) 337-364 2 2 1 8 .78 (.52) 365-392 1 1 3 2 .88 (.62) 393-420 3 7 2 10 .59 (.42) 421-448 2 2 S 7 .55 (.39) 449-476 4 0 6 5 .2S (.17) 477-504 7 1 7 7 .07 (.05)* 505-532 3 5 5 3 .20 (.15) 533-S60 12 3 13 3 .25 (.19) 561-588 17 3 9 4 .15 (.10) 589-616 6 2 8 2 .18 (.14) 617-644 5 2 10 3 .22 (.18) 645-672 7 4 4 1 .31 (.25) 673-700 7 1 S 2 .25 (.19) 701-728 4 1 4 3 .19 (.16) 729-756 4 2 4 1 .24 (.19) 757-784 2 0 2 0 .24 (.19) 785-812 0 0 3 2 .24 (.19) 813-840 0 0 2 0 .24 (.19) 841-868 1 0 1 1 .20 (.17) 869-896 1 0 0 0 .20 (.17) 897-924 0 0 1 0 .20 (.17) 925-952 0 0 0 0 953-980 0 0 0 0 981-1008 0 0 0 0 1009-1036 0 0 0 0 1037-1064 0 0 0 0 1069-1092 0 0 0 0 1093-1120 0 0 0 0 1121-1148 0 0 0 0 91 134 100 102 --------------- ---------- ------------------------------------- a Defined as the total number of animals with histopatholoqic ezamination. b The total number of animals in a qiven tiae intarval is the sum of the number of animals in the prasent and Absent col=ns. 183 CTR CaNTRRCTS 029362 11249112 CTR f IN 04,41=51

TABLE 8 8 INCIDENCE OF HALIGNANT TUMORS IN THE RESPIRATORY TRACT OF BC3P1/CIIH MICE AFTER TREATMENT WITH BAP AND LONG-T?e't.4 EXPOSURE TO 2R1 CICARETTE SMOKE (STRICT ADHERENCE TO RANDOH)a,b SUN., JAN 8, 1984 SAP + SMOKE SAP + SdAH -------------- •--------------- DAYS ON TEST ?RESENT ABSENT PRESE;IT ABSENT ?ROBa8IL1TY -------------------------------------------------------------- 1-28 0 2 0 3 1.0 29-56 0 18 0 3 1.0 57-84 0 33 0 0 1.0 85-112 0 10 0 1 1.0 113-140 0 3 0 1 1.0 141-168 0 4 0 4 1.0 169-196 0 8 0 5 1.0 197-224 0 5 0 5 1.0 225-252 0 1 0 0 1.0 253-280 0 6 1 4 .93 (.28) 281-308 0 S 0 5 .93 (.28) 309-336 0 2 0 5 .93 (.28) 337-364 0 1 0 5 .93 (.28) 365-392 0 1 2 2 .53 (.18) 393-420. 1 7 0 8 1.0 (.60) 421-448 2 1 4 7 1.0 (.83) 449-476 2 0 2 4 .57 (.35) 477-504 3 0 4 5 .1,9 (.10) 505-532 2 4 2 2 .36 (.23) 533-560 5 0 2 1 .22 (.14) 561-588 4 2 2 3 .15 (.09) 589-616 2 0 4 2 .10 (.09) 617-644 1 1 1 2 .09 (.06) 645-672 1 1 2 1 .13 (.08) 673-700 0 0 0 0 .13 (.08) 701-728 0 0 1 0 .13 (.08) 729-756 0 1 0 0 .13 (.08) 757-784 0 0 0 0 .13 (.08) 785-812 0 0 2 2 .31 (.22) 913-840 0 0 0 0 841-868 0 0 0 0 869-896 0 0 0 0 897-924 0 0 0 0 925-952 0 0 0 0 953-980 0 0 0 0 981-1008 0 0 0 0 1009-1036 0 0 0 0 1037-1064 0 0 0 0 1065-1092 0 0 0 0 1093-1120 0 0 0 0 1121-1148 0 0 0 0 23 29 -------------------------------------------- ------------------ a b Defined as thoce animals taken off test randomly. The total ntmber of animals in a qiven tine interval is the sum of the numb.r of animals in the PLesent and Abs.nt columns. 184 c.TI2 coHTRacTS 029363 11249113 ~~ ~ CTR NN 0441

TABLE 89 aCTUARIAL aNALYSIS 0F BC3F1/CUM MICE DYING OF LONC CaNC 'zR CZ ALVEOLAR CCi•iPRESSINC NODOLES AFTER TREATMENT WITH 9aP AYD LONC-TERM EXPOSURE TO 2R1 CIGARETTE SMOKE (CE:7ER.1L ADHERENCE TO DIED OR MORIBUND, aLL)a'b CtON., JAN 9, 1984 BAP + SMOKE SAP + SH.1M - -------------- ------------------ DAYS ON TEST D/W TUMOR AT RISK D W/TUMOR AT RISK PROBABILITY ------------------------------------------------ ----------------- 1-28 0 225 0 202 1.0 29-56 0 223 0 199 1.0 57-84 0 205 0 196 1.0 85-112 0 172 0 196 1.0 113-140 0 161 0 195 1.0 141-168 0 158 1 194 1.0 (.37) 169-196 0 154 0 189 1.0 (.37) 197-224 1 146 0 184 1.0 (.88) 225-252 1 140 1 179 1.0 (.82) 253-280 1 138 1 177 1.0 (.78) 281-308 1 132 1 172 1.0 (.73) 309-336 2 127 5 167 ..gg (.79) 337-364 2 124 2 159 1.0 (.90) 365-392 2 120 4 1S0 .87 (.71) 393-420 4 118 ' 7 145 .66 (.54)' 421-448 2 108 11 133 .16 (.12). 449-476 4 104 7 121 .12 (.09) 477-504 7 100 11 110 .09 (.07) 505-532 6 92 6 . 96 .12 (.09) 533-560 13 84 15 88 .14 (.11) 561-588 19 69 12 72 .42 (.37) 589-616 7 49 10 59 .38 (.34) 617-644 7 41 12 49 .30 (.27) 645-672 10 34 4 36 .54 (.49) 673-700 7 23 6 31 .68 (.62) 701-728 5 15 6 24 .75 (.69) 729-756 5 10 5 17 .86 (.80) 757-784 2 4 2 12 .94 (.89) 785-812 0 2 5 10 .88 (.83) 813-840 0 2 2 5 .83 (.78) 841-868 1 2 1 3 .85 (.79) 869-896 1 1 0 1 .89 (.83) 897-924 0 0 1 1 .89 (.83) 925-952 0 0 0 0 953-980 0 ' 0 0 0 981-1008 0 ' 0 0 0 1009-1036 0 0 0 0 1037-1064 0 0 0 0 1065-1092 0 0 0 0 1093-1120 0 0 0 0 1121-1148 0 0 0 0 111) 138 ---------------------------------------------------------------- 3 Detined as th• total numb.r of animals that had histopathologic examination. The nu.zber of animals that died of the lesion or tumor. ~ 185 b CTR caHTRacT5 029364 11249114 CTR 11N 044157-3

TASLE 90 ACTUARLAL aNALYSIS OP BC3F1/CUM MICE OYI?JC 0£ LUNG CANCER OR ALVEOLAR COMPRESSING NOCQLES AFTER TREATMENT WITH 8AP AND LONC-TERM EXPOSURE TO 2R1 CIGARETTE SMOKE .(STRICT ADSERENCE TO RANDOM)a,b .SON., JaN., 9, 1984 BAP + SMOKE BAP + SHX%t ------------------ ------------------ DAYS ON T:ST 0/W TUMOR AT RISK D W/TU:SOR AT RISK PROBABILITY ----------------------------------------------------------------- 1-28 0 86 0 93 1.0 29-56 ' 0 86 0 93 1.0 57-84 0 86 0 93 1.0 85-112 0 86 0 93 1.0 113-140 0 85 0 93 1.0 141-168 0 85 1 93 1.0 (.35) 169-196 0 85 0 92 1.0 (.35) 197-224 1 85 0 92 1.0 (.951 225-252 1 84 1 92 1.0 (.93) 253-280 0 83 0 90 1.0 (.93) 281-308 0 83 0 90 1.0 (.93) 309-336 1 83 3 90 .83 (.57) 337-364 2 82 2 87 .89 (.67) 365-392 1 79 1 83 .90 (.70) 393-420 2 78 2 82 .94 (.75) 421-448 0 76 1 78 .76 (.59) 449-476 2 75 5 77 .42 (.31) 477-504 4 73 S 72 .38 (.30) 505-532 1 68 3 67 .26 (.19)• 533-560 8 66 1: 63 .15 (.11) 561-588 14 56 7 50 .48 (.41) 589-616 5 42 4 42 .57 (.49) 617-644 5 36 9 38 .39 (.34) 645-672 8 31 2 28 .73 (.66) 673-700 7 22 6 26 .86 (.79) 701-728 5 14 5 19 .95 (.88) 729-756 5 9 5 13 1.0 (.97) 757-784 2 4 2 8 1.0 (.97) 785-812 0 2 1 6 1.0 (.99) 813-840 0 2 2 S 1.0 (.94) 841-868 1 2 1 3 1.0 (.96) 869-896 1 1 0 1 1.0 (.99) 897-924 0 0 1 1 1.0 (.99) 925-952 0 0 0 0 953-980 0 0 0 0 981-1008 0 0 0 0 1009-1036 0 0 0 0 1037-1064 0 0 0 0 1063-1092 0 0 0 0 1093-1120 0 0 0 0 1121-1148 0 0 0 0 76 81 a ----------------------------------------------------------- Dafinad as those animals taken off test randomly. 5 The total numb.r of animals in a qiv.n tim• interval is the sun of the number of animals in the Present and aba.nt eoluaas. 186 CTR COHTRRCTS 029365 11249115 CT~.'~' f f N 044 1 =54

TABLE 91 INCIDENCE 0P ALVEOLaR COMPRESSING NODQLES OR L.. G C:..`JCEiLS. I:J 9C3P1/CIIM MICE AFTER TREATMENT WITH SAP AND LONC-TER.`! EXPOSQRE TO 2R1 CIGARETTE SMOKE (STRICT ADHERENCE TO RANDOM)a,b MON. JAN. 9, 1984 BAP + •SMORE 8AP + SPiAM -------------- ------------ ---- DAYS ON TEST PRESENT aBSENT PRESENT ABSENT PROBABILITY ------------------------------------------------- ------------- 1-28 0 2 0 3 1.0 29-56 0 18 0 3 1.0 57-84 0 33 0 0 1.0 85-112 0 10• 0 1 1.0 113-140 - 0 3 0 1 1.0 141-168 0 4 0 4 1.0 169-196 0 8 0 5 1.0 197-224 0 5 0 5 1.0 225-252 0 1 0 0 1.0 253-280 1 S 1 4 1.0 (.89) 281-308 1 4 1 4 1.0 (.93) 309-336 1 1 2 3 1.0 (.97) 337-364 0 1 0 5 1.0 (.97) 365-392 1 0 3 1 1.0 (.84) 393-420 2 6 5 3 .64 (.431 421-448 2 1 - 10 1 .44 (.28) 449-476 2 0 2 4 .87 (.65) 477-504 3 0 6 3 1.0 (.97} S05-532 5 1 3 1 1.0 (.95) 533-560 5 0 9 0 1.0 (.95) S61-588 5 1 S 0 1.0 (.88) 589-616 2 0 6 0 1.0 (.88) 617-644 2 0 3 0 1.0 (.88) 645-672 2 0 2 1 1.0 (.97) 673-700 0 0 0 0 1.0 (.97) 701-728 0 0 1 0 • 1.0 (.97) 729-756 0 1 0 0 1.0 (.97) 757-784 0 0 0 0 1.0 (.97) 785-812 0 0 4 0 .64 (.48) 813-840 0 0 0 0 841-868 0 0 0 0 869-896 0 0 0 0 997-924 0 0 0 0 925-952 0 0 0 0 953-980 0 0 0 0 981-1008 0 0 0 0 1009-1036 0 0 0 0 1037-1064 0 0 0 0 1065-1092 0 0 0 0 1093-1120 0 0 0 0. 1121-1148 0 0* 0 0 34 57 --------------------------------------------------------------- a Datined as those animals taken off test tandomly. The total nnmber of animals in a qivan time inte=val is th• sum of the number of animals in the Present and b Absent columna. 187 CTR CON1'RRCTS 029366 11249116 CT~' t-IN 04- 41I.-E-55'

TABLE 92 INCIDE:7CE OF ALVEOLait COMPRESSING NODULES OR LUNG C?..vC=RS IN HC3F1/CUlS MICE AFTER TREATMENT WITH SAP AND LONG-TERti EXPOSURE TO 2R1 CICARZTTE SMOKE (CENERAL ADHERENCE :'C'RaNDOM, aLL)a•b MON., .:aN. 9, 1984 SAP + SMOKE BAP + SHAt4 -------------- ---------------- DAYS ON TEST PRESENT A3SENT PRESENT A.BSENT ?ROBA3ILITY ----------------- -------------------------------------------- 1-28' 0 2 0 3 1.0 29-56 0 18 0 3 1.0 57-84 0 33 0 0 1.0 85-112 0 11 0 1 1.0 113-140 0 3 0 1 1.0 141-168 0 4 1 4 1.0 (.37) 169-196 0 8 0 5 1.0 (.37) 197-224 1 5 0 5 1.0 (.99) 225-252 1 1 1 1 1.0 (.99) 253-280 1 5 1 4 1.0 (.95) 281-308 1 4 1 4 1.0 (.95) 309-336 2 1 5 3 1.0 (.991 337-364 2 2 2 7 .88 (.65) 365-392 2 0 4 1 .75 (.55) 393-420 4 6 7 5 1.0 (.98) 421-448 2 2 11 1 .77 (.60) 449-476 4 0 7 4 1.0 (.96) 477-504 7 1 11 3 1.0 (.90) S0S-532 6 2 6 2 1.0 (.90) 533-S60 13 2 15 ' 1 1.0 (.94) 561-588 19 1 12 1 1.0 (.99) 589-616 7 1 10 0 .99 (.86) 617-644 7 0 12 1. 1.0 (.95) 645-672 10 1 4 1 1.0 (.96) 673-700 7 1 6 1 1.0 (.94) 701-728 5 0 6 1 .96 (.84) 729-756 5 1 5 0 1.0 (.95) 757-784 2 0 2 0 1.0 (.95) 785-812 0 0 5 0 L.0 (.95) 813-840 0 0 2 0 1.0 (.95) 841-868 1 0 1 1 .99 (.88) 869-896 1 0 0 0 .99 (.88) 897-924 0 0 1 0 .99 (.98) 925-952 0 0 0 0 953-980 0 0 0 0 981-1008 0 0 0 0 1009-1036 0 0 0 0 1037-1064 0 0 0 0 ` 1065-1092 0 0 0 0 1093-1120 0 0 0 0 1121-1148 0 0 0 0 110 138 --------------------------------------------------------------- a Defined as the total number of animals with histopatholoqic ezamination. b The total number of animals in a qiven time interval is the auon of the number of animals in the Present and Absent col=na. iRR C.TR CONTRRCTS 029367 11249117 CTR 11IN 0441=56

TABLE 9 3 ACTUARIaL ANALYSIS OF BC3F1/CQM MICE DYING OF E::'HER ANCN, ACN, AAC, 204;, .QR SCC A.FTER T?_.AT:SEZ)T WITY 3AP AND LONG-TERM EXPOSURE TO 2R1 CIGARETTE SMOKE , (GFNERAL ADHERENCE TO DIED OR MORIBUNO)a'o MON., JAN. 9, 1984 BAP + SMOKE BAP + SEiAM. -------------=---- ------------------ DAYS ON TEST D/W TUMOR AT RISK D W/TUMOR AT RISK PROBABILITY ------------------------------------------------=---------------- 1-28 0 225 0 202 1.0 29-56 0 223 0 199 1.0 57-84 0 205 0 196 1.0 85-112 0 172 0 196 1.0 113-140 0 161 0 195 1.0 141-168 0 158 2 194 .58 (.20) 169-196 1 154 0 189 1.0 (.69) 197-224 2 146 1 184 1.0 (.79) 225-252 2 140 1 179 .74 (.50) 253-280 2 138 2 177 .68 (.49) 281-308 3 132 1 172 .33 (.23) 309-336 3 127 8 167 .91 (.76) 337-364 . 3 124 6 159 1.0 (.97) 365-392 2 120 4 150 .94 (.82) 393-420 5 118 9 145 .69 (.59) 421-448 2 108 12 133 .19 (.13) 449-476 4 104 10 121 .09 (.071 477-504 8 100 13 110 ...~ *(.04)• 505-532 8 92 8 96 .08 (.07) 533-560 15 84 15 88 .13 (.10) 561-588 19 69 12 72 .35 (.31) 589-616 8 49 10 59 .36 (.32) 617-644 7 41 13 49 .26 (.24) 645-672 10 34 5 36 .43 (.39) 673-700 7 23 7 31 .51 (.47) 701-728 , 5 15 7 24 .55 (.50) 729-756 5 10 5 17 .64 (.59) 757-784 2 4 2 12 .72 (.67) 785-812 0 2 5 10 .66 (.62) 813-840 0 2 2 5 .62 (.5a) 841-868 1 2 2 3 .61 (.57) 869-896 1 1 0 1 .64 (.60) 897-924 0 0 1 1 .64 (.60) 925-952 0 0 1 1 953-980 0 0 0 0 981-1008 0 0 0 0 1009-1036 0 0 0 0 1037-1064 0 0 0 0 1065-1092 0 0 0 0 1093-1120 0 0 0 0 1121-1148 0 0 0 0 125 163 ------------------------ --------------------------------------- a Defined as the total number of animals that had b histopatholoqic examination. The number of animals that died of the lesion or tcsmor. 189 ' CTR COh{TRRCTS 029368 11249118 C~ 7"R I-IN 0441=z5t~"

TABLE 94 ACTOARIaL ANAYLSIS OF HC3l1/COM MICE DYING Ot•EIT3ER A.YCY, ACN, ACC, PDC, OR SCC AFTER TREATI".ENT PiITH BAP AND LONG-TER!•( EXPOSORE TO 2R1 CIGARETTE SMOKE (STRICT ADHERENCE TO DIED OR MORI3UND)a,b MON., JAN. 9, 1984 BAP + SMOKE BAP + SRAP3 --------- --------- ------------------ DAYS ON TEST D/W TOMOR AT RIS1C 0 W/TOMOR aT RISK PROBABILITY ------------------------------------------------ ---------------- 1-28 0 86 0 93 1.0 29-56 0 86 0 93 1.0 57-84 0 86 0 93 1.0 85-112 0 86 0 93 1.0 113-140 0 85 0 93 1.0 141-168 a 8S 1 93 1.0 (.35) 169-196 0 es 0 92 L.0 (.35) 197-224 1 85 0 92 1.0 (.95) 225-252 1 84 1 92 1.0 (.93) 253-280 0 83 0 90 1.0 (.93) 281-308 0 83 0 90 1.0 (.93) 309-336 1 83 3 90 .83 (.57) 337-364 3 82 4 87 ,72 (.54) 365-392 1 79 1 83 .75 (.57) 393-420 2 78 3 82 .64 (.49) 421-448 0 76 1 78 .51 (.38)- 449-476 2 75 5 77 .28 (.20) 477-504 5 73 5 72 .34 (.27). 505-532 . 2 68 4 67 .24 (.18) 533-560 10 66 12 63 .21 (.17) 561-588 14 56 7 50 .56 (.49) 589-616 6 42 4 42 .72 (.64) 617-644 5 36 10 38 .47 (.41) 645-672 8 31 2 28- .81 (.74) 673-700 7 22 7 26 .88 (.81) 701-728 5 14 6 19 .92 (.86) 729-756 5 9 5 13 1.0 (.94) 757-784 2 4 2 8 1.0 (.99) 785-812 0 2 1 6 1.0 (.97) 813-840 0 2 2 5 .98 (.92) 841-868 1 2 2 3 .97 (.90) 869-896 1 1 0 1 1.0 (.94) 897-924 0 0 L 1 1.0 (.94) 925-952 0 0 0 0 953-980 0 0 0 0 981-1008 0 0 0 0 1009-1036 . 0 - 0 0 0 1037-1064 0 0 0 0 1065-1092 0 0 0 0 1093-1120 0 0 0 0 1121-1148 0 0 0 0 82 89 ---------------------------------------------------------------- a Defined as those animals that died or were killed when moribund. b The number ot animals that died of lesions or tumor. 190 C.TR CONTRRCTS 029369 11249119 CTR HN 044158

TABLE 95 I.`1CID"c..*ICF. OF EITI'. ER ANCN : AC:1, AAC, PDC, OR SCC 'r:I BC3F1/CQM MICE AFTER TREATMENT WITH BAP AND L0:1G-TER*( EXPOSIIRE TO 2R1 CIGARETTE SMOKE (GENERAL ADHERENCE TO RANDOM, ALL) a,b MON., JAN. 9, 1984 BAP + SMOKE BAP + SFil1M -------------- ---- ------- ----- DAYS ON TEST PRESENT ABSENT PRESENT ABSENT PROBABILITY 1-28------------o-------2-------0--------3--------1.0------- ' 29-56 0 18 0 3 1.0 57-84 0 33 0 0 1.0 85-112 0 11 0 1 1.0 113-140 0 3 0 1 1.0 141-168 0 4 2 3 .S6 (.18) 169-196 1 7 0 5 .99 (.54) 197-224 2 4 1 4 1.0 (.90) 225-252 2 0 1 1 1.0 (.77) 253-280 2 4 2 3 1.0 (.91) 281-308 3 2 1 4 .69 (.49) 309-336 3 0 8 0 .69 (.49) 337-364 3 1 6 3 .63 (.461 365-392 2 0 4 1 .54 (.38) 393-420. 5 5 9 3 1.0 (.89) 421-448 2 2 12 0 .78 (.62) 449-476 4 0 10 1 .87 (.70) 477-504 8 0 13 1 .99 (.83) 505-532 8 0 8 0 .99 (.83) 533-560 15 0 15 1 1.0. (.98) 561-588 19 1 12 1 1.0 (.95) 589-616 8 0 10 0 1.0 (.95) 617-644 7 0 13 0 1.0 (.95) 645-672 10 1 5 0 1.0 (.95) 673-700 7 1 7 0 .97 (.82) 701-728 5 0 7• 0 .97 (.82) 729-756 5 1 5 0 .84 (.69) 757-784 2 0 2 0 .84 (.69) 785-812 0 0 5 0 .84 (.69) 813-840 0 0 2 0 .84 (.69) 841-868 1 0 2 0 .84 (.69) 869-896 1 0 0 0 .84 (.69) 897-924 0 0 1 0 .84 (.69) 925-952 0 0 0 0 953-980 0 0 0 0 981-1008 0 0 0 0 1009-1036 0 0 0 0 1037-1064 0 0 0 0 1065-1092 0 0 0 0 1093-1120 0 0 0 0 1121-1148 0 0 0 0 125 163 --------------------------------------------------------------- a Detined as th• total nuzaber of animals with histopathologic examination. b The total number ot animals in a qiven time interval is the svm of the numb.r of animals in the Prssent and Absent columns. 191 C.TR COhtTRRCTS 029370 11249120 C T R V I t - I C-) -1' 4, 1 '15; 9

;ABLE 96 ZNCIDE:ICE OF EZ.rs-.R ANCtt, ACN, AAC, ?DC, OR SCC I:J 8C7F1/CCti MICE aPTER TREATMENT WITH BAP AND LONG-TERM EXPOSURE TO 2R1 CIGARETTE SMOKE .(STRICT ADHERENCE TO RANDOH)a.b MON., JAN. 9, 1984 SAP + SMOKE BAP + SiiAM -------------- ---------------- DAYS ON TEST PRESENT aBSENT PRESENT ABSENT ?R08ABILITY -------------------------------------------------------------- 1-28 0 2 0 3 1.0 29-56 0 18 0 3 1.0 57-84 0 33 0 0 1.0 85-112 0 10 0 1 1.0 113-140 0 3 0 1 1.0 141-168 0 4 1 3 1.0 (.32) 169-196 1 7 0 5 1.0 (.87) 197-224 1 4 1 4 1.0 (.91) 225-252 1 0 0 0 1.0 (.82) 253-280 2 4 2 3 1.0 (.82) 281-308 3 . 2 1 4 .90 (.65) 309-336 2 0 5 0 .90 (.65) 337-364 0 1 2 3 1.0 (.82) 365-392 1 0 3 1 .97 (.73) 393-420 3 5 6 2 .83 (.63) 421-448 2 1 11 0 .54 (.39) 449-476 2 0 5 1 .64 (.47) 477-504 3 0 8 1 .73 (.56) S0S-532 6 0 4 0 .73 (.56) 533-560 5 0 3 0 .73 (.56) 561-588 5 1 5 0 .58 (.44) 589-616 2 0 6 0 .58 (.44) 617-644 2 0 3 0 .58 (.44) 6.s-672 2 0 3 0 .58 (.44) 673-700 0 0 0 0 .58 (.44) 701-728 0 0 1 0 .58 (.44) 729-756 0 1 0 0 .58 (.44) 757-784 0 0 0 0 .58 (.44) 785-812 0 0 4 0 .15 (.09) 813-840 0 0 0 0 841-868 0 0 0 0 869-896 0 0 0 0 897-924 , 0 0 0 0 925-952 0 0 0 0 953-980 0 0 0 0 981-1008 0 0 0 0 1009-1036 0 0 0 0 1037-1064 0 0 0 0 1065-1092 0 0 0 0 1093-1120 0 0 0 0 1121-1148 0 0 0 0 43 74 --------------------------------------------------------------- a b D.lined as those animals takan off test randomly. The totsl number ot animals in a the svm of the numb.r of animals Absect columns. 192 given time interval is 1n the Present and CTR CONTRRCTS 029371 11249121 f al I R ! / N yI' 44' 1 .U/. 'IYlr'

?inal Report Contzaet: C:R-0030 CTR-lO1A FIGURES 31-37 193 CTR COHTRaCTS 029372 11249122 CTR llh1I 044 161

I I e P I 380 223 1$0 %S So 23 I ftwl Rcp"t Co.tr.ct: CTt-oo]0 I fIGIIRE 31 i SMOKE TPM GENERATtON I1ND OEPOSITION ~ ....... ....• . ,.. •...• . . S 1• IS 20 . • %$ $• 23 30 3S tio MEEKS Of ExPOSURE ~ . 65 70 iS •o i , 95 9• 93 1o0 105 i

Fina1 Report Contract: CTR-0030 LEGEND. FIGURE 32. A MEAN PERCENT CARBOXYHEMOGLOBIN FOR 3-5 ANIMALS BLED IMMEDIATELY AFTER EXPOSURE TO 2R1 CIGARETTE SMOKE. SHAM EXPOSED AND UNEXPOSED ANIMALS EXPRESSED MEAN VALUES OF 1.43 AND 1.79, RESPECTIVELY, OVER THIS 31'MOt1TH PERIOD. B SAMPLES NOT COLLECTED. . 195 C.TR COh{TF2ACTS 029374 11249124 Wf I R I I 1 41I 044 163

t Final Geport Contract: CTR-0030 FIGURE 32 O tND w 1! Ul e oi 25 20 15 10 5 SONDJFMAMJJASONBDJFMAMJJASBONBUJFH l Jt I I 1 u 1978 1979 1980 1981 MONTNS OF TEST

Final Report FIGURE-33 Contract: CIR-003U BODY WEIGHT OF DC3F1/CUM FEMALE MICE AFTER EXPOSURE TO 2111 C I GARETTE SPIOKE, SIIAhI-EXPOSURE, OR UNTREATED 50 40 .. ~' 30 ~ .!T: 0." ~ ~ ~ 0 m 20 N 10 GJ -~! ~ . I I I 20 q0 60 80' 100 110 WEEKS ON TEST q0 SHELF SHAM SMOKE

Final.Report Contract: CTR-0030 LEGEND. FIGURE 34. SURVIVING FRACTION OF ANIMALS FOR 2R1 CIGARETTE SMOKE-EXPOSED, SHAM-EXPOSED, OR SHELF CONTRL 8C3F1/CUM MICE. t4ICE WERE 8-16 WEEKS OLD WHEN EXPOSURE WAS INITIATED. 198 CTR CQNTRACTS 029377 11249127 CTR NN 04,41 GG

I I i .,I ...I ~ I I Final Report Contract: CTR-0030 FIGURE 34 1.0 to_o-o-o_o_o-o-o-o- o-o-o-o_o,s_o SHAM ~8'g=8=8=8=6=8=8=6;$~ ° ~ ~- SHELF `°0.o-O ~ • 0 o 0 0~ SMOKE ~~ 0.5 e 20 40 60 80 WEEKS ON TEST I I I I 100 ; 120 1110 160 SMOKE EXPOSURE STOPPED i

I I I I I I I I` I I' :I I.~ 1 I~i Fiual Report Contract: CTR-C030 FIGURE 35 50 • • BAP SHELF . . N u 40 . • . .~~., .,..{ Ul. 10 • • BAP1+ SNAH , . . . . . ... •. .  . ' ~ ~ . • • + w • ~ •  , t t - • BAP + SMOKE 20 40 • a 60 80 100 120 TIfE Oq TEST

I 7Cf ~ m Final Report ' ~ Contract: CiK-0030 FIGURE 36 q0 30 20 ,t 10 1 2 3 \ I 111111 \ \ i ~ \ ~ \ \ 4 5 6 7 8 9 10 11 12 TIME ON TEST (MONTiis)

ii - iI I I I Final Rcpurl Contract: CTR-0030 z 0 ~. I-- u tl.. 1.0 0.5 20 40 I I;: I I,•'I 1 11 I I i I i I I FIGURE 37 60 80 WEEKS ON TEST 100 120 I

TA.BLE 91 INCIDENCE OF ALVEOLAR COHPRESSING NODQLES OR LUNG CANCERS.I;t BC3F1/CQ`i MICE AFTER TREATMENT WITH BAP AND LONG-TERti EXPOSIIRE TO 2R1 CIGARETTE SMOKE (STRICT ADHERENCE TO RANDOH)a.b HON. J.1N. 9, 1984 SAP + SMOKE BAP + SHAM --------- ----- ---------------- DAYS ON TEST PRESENT ABSENT PRESENT ABSENT PROBABILITY ------------------------------------------------- ------------- 1-29 0 2 0 3 1.0 29-56 0 18 0 3 1.0 57-84 0 33 0 0 1.0 85-112 0 10. 0 1 1.0 113-140 0 3 0 1 1.0 141-168 0 4 0 4 1.0 169-196 0 8 0 5 1.0 197-224 0 5 0 5 1.0 225-252 0 1 0 0 1.0 253-280 1 5 1 4 1.0 (.89) 281-308 1 4 i 4 1.0 (.93) 309-336 1 1 2 3 1.0 (.97) 337-364 0 1 0 5 1.0 (.97) 365-392 1 0 3 1 1.0 (.84) 393-420 2 6 5 3 .64 (.43) 421-448 2 1 10 1 .44 (.28) 449-476 2 0 2 • 4 .87 (.65) 477-504 3 0 6 3 1.0 (.97) 505-532 5 1 3 1 1.0 (.95) 533-560 5 0 3 0 1.0 (.95) 561-588 5 1 5 0 1.0 (.88) 589-616 2 0 6 0 1.0 (.88) 617-644 2 0 3 0 1.0 (.88) 645-672 2 0 2 1 1.0 (.97) 673-700 0 0 0 0 1.0 (.97) 701-728 0 0 1 0 1.0 (.97) 729-756 0 1 0 0 1.0 (.97) 757-784 0 0 0 0 1.0 (.97) 785-812 0 0 4 0 .64 (.48) 813-840 0 0 0 0 841-868 0 0 0 0 869-896 0 0 0 C 897-924 0 0 0 0 925-952 0 0 0 0 953-980 0 0 0 0 981-1008 0 0 0 0 1009-1036 0 0 (1 0 1037-1064 0 0 0 0 1065-1092 0 0 0 0 1093-1120 0 0 0 0 1121-1+48 0 0 0 0 3.4 5 7 ---------------------------------------- ------------------------ a Oetined as those animals taker off test randomly. b The total number ot animals in a given time interval is the sum of the number of animals in the P=.sent and absent eol=ns. 187 CTR CCNTRRCTS 429382 11249132 CTR NN 044.171

TABLE 92 INCIDENCE OF ALVEOLAR COMPRESSING N000LES OR LtTNC Ca,NCERS IN BC3F1/CIIH MICE AFTER TREATMENT WITH BAP AND LONC-TERlt EXPOSURE TO 2R1 CIGARETTE SMOKE (GENERAL ADHERENCE TO RANDOM, ALL)a.b MON., jAN. 9, 1984 3AP + SMOKE 8AP + SHAl4 DAYS ON TEST PRESENT ABSENT PRESENT ASSENT PROBABILITY -------------------------------------------------------------- 1-28' 0 2 0 3 • 1.0 29-56 0 18 0 3 1.0 57-84 0 33 0 0 1.0 85-112 0 11 0 1 1.0 113-140 0 3 0 1 1.0 141-168 0 4 1 4 1.0 (.37) 169-196 0 8 0 5 1.0 (.37) 197-224 1 5 0 5 1.0 (.99) 225-252 1 1 1 1 1.0 (.99) 253-280 1 5 1 4 1.0 (.95) 281-308 1 4 1 4 1.0 (.95) 309-336 2 1 5 3 1.0 (.99) 337-364 2 2 2 7 .88 (.65) 365-392 2 0 4 1 .75 (.55) 393-420' 4 6 7 5 1.0 (.98) 421-448 2 2 11 1 .77 (.60) 449-476 4 0 7 4 1.0 (.96) 477-504 7 1 11 3 1.0' (.90) 505-532 6 2 6 2 1.0 (.90) 533-560 13 2 1S ' 1 1.0 (.94) 561-588 19 1 12 1 1.0 (.99) 589-616 7 1 10 0 .99 (.86) 617-644 7 0 12 1 1.0 (.95) 645-672 10 1 4 1 1.0 (.96) 673-700 7 1 6 1 1.0 (.94) 701-728 5 0 6 1 .96 (.84) 729-756 5 1 5 0 1.0 (.95) 757-784 2 0 2 0 1.0 (.95) 785-812 0 0 5 0 1.0 (.95) 813-840 0 0 2 0 1.0 (.95) 841-862 1 0 1 1 .99 (.88) 869-896 1 0 0 0 .99 (.88) 697-924 0 0 1 0 .99 (.88) 925-952 0 0 0 0 953-980 0 0 0 0 981-1008 0 0 0 0 1009-1036 0 0 0 0 1037-1064 0 c 0 0 1065-1092 0 0 0 0 1093-1120 0 0 0 0 1121-1148 0 0 0 0 110 :38 ------------ --------------------------------------------------- a Detined as the total number of animals with histopatholoqic ezamination. b The total number of animals in a given time int.rval is the sum of the nuaber of aniaals in the present t AFi and Abaent col,=ns. CTR CoNTRAcTS 029383 11249133 CTI 1 NI 1

TABLE 93 ACTUARIAL ANALYSIS OF BC3F1/CUM MICE DYING OP EITHER aNCN, ;,CN, r1AC. PDC„ OR SCC AFTER TREATltENT WITB BAP ;~.YD LONG-TERM EXPOSURE TO 2R1 CIGARETTE SMOKE (GE:7ERAL ADHERENCE TO DIED OR MORIBQND)a.b MON., JAN. 9, 1984 BAP + SMOKE BAP + SFiAM ------------------ DAYS ON TEST D/W TUMOR AT RISK D W/TUMOR AT RISK PROBABILITY ------------------------------------------------=---------------- 1-28 0 225 0 202 1.0 29-56 0 223 0 199 1.0 57-84 0 205 0 196 1.0 85-112 0 172 0 196 1.0 113-140 0 161 0 195 1.0 141-168 0 158 2 194 .58 (.20) 169-196 1 154 0 189 1.0 (.69) 197-224 2 146 1 184 1.0 (.79) 225-252 2 140 1 179 .74 (.50) 253-280 2 138 2 177 .68 (.49) 281-308 3 132 1 172 .33 (.231 309-336 3 127 8 167• .91 (.76) 337-364 3 124 6 159 1.0 (.97) 365-392 2 120 4 150 .94 (.821 393-420 5 118 . 9 145 .69 (.59) 421-448 2 108 12 133 .19 (.15) 449-476 4 104 10 121 .09 ( .07j 477-504 8 100 13 110 • .05'(.04)~ 505-532 8 92 8 96 .08 (.07) 533-560 15 84 15 88 .13 G10) 561-588 19' 69 12 72 .35 (.31) 589-616 8 49 10 59 .36 (.32) 617-644 7 41 13 49 .26 (.24) 645-672 10 34 5 36 .43 (.39) 673-700 7 23 7 31 .51 (.47) 701-728 5 15 7 24 .55 (.50) 729-756 S 10 5 17 .64 (.59) 757-784 2 4 2 12 .72 (.67) 785-812 0 2 5 10 .66 (.62) 813-840 0 2 2 5 .62 (.58) 841-868 1 2 2 3 .61 (.57) 869-896 1 1 0 1 .64 (.60) 897-924 0 0 1 1 .64 (.60) 925-952 0 0 1 1 953-980 0 0 0 0 981-1008 0 0 0 0 1009-1036 0 0 0 0 1037-1764 0 0 0 0 1065-1092 0 0 0 0 1093-1120 0 0 0 0 1121-1148 0 0 0 0 125 163 -------------------------------- --------------------------------- a Detir.ed as the total numb.r of animals that had b histopathologic examination. The number of aaimals that died of the lesion or tcmzor. 189 .'CTR COHTRRCTS 029384 11249134 C/ / 1 / 1N 0!• 4Ya 7`1wP

TABLE 94 ACTUARIAL ANAYLSIS OF BC3F1/COH HICE DYING OF•EIT3EP. ANCN. AC:I, ACC, PDC, OR SCC AFTER TREATN.ENT wITH BAP A:JD LONG-TERM EXPOSURE TO 2R1 CIGaRETTE SMOKE (STRICT ADHERENCE TO DIED OR HORIBUND)a.b MON., JAN. 9, 1984 . BAP + SMOKE BAP + SHAH ------------------ ------------------ DAYS ON TEST D/W TUMOR AT RISK 0 W/TUMOR AT RISK PROSABILITY ------------------------------------------------ ---------------- 1-28 0 86 0 93 1.0 29-56 0 86 0 93 1.0 57-84 0 86 0 93 1.0 85-112 0 86 0 93 1.0 113-140 0 85 0 93 1.0 141-168 0. 85 1 93 1.0 (.35) 169-196 0 85 0 92 1.0 (.35) 197-224 1 85 0 92 1.0 (.95) 225-252 1 84 1 92 1.0 (.93) 253=280 0 83 0 90 1.0 (.93) 281-308 0 83 0 90 1.0 (.93) 309-336 1 83 3 90 .83 (.57) 337-364 3 82 4 87 .72 (.54) 365-392 1 79 1 83 .75 (.57) 393-420 2 78 3 82 .64 (.49) 421-448 0 76 1 78 .51 (.38) 449-476 2 75 5 77 .28. (.20) 477-504 5 73 5 72 .34 (.27) 505-532 2 68 4 67 .24 (.18) 533-560 10 66 12 63 .21 (.17) 561-588 14 56 7 50 .56 (.49) 589-616 6 42 4 42 .72 (.64) 617-644 5 36 10 38 .47 (.41) 645-672 8 31 2 28 .81 (.74) 673-700 7 22 7 26 .88 (.81) 701-728 5 14 6 19 .92 (.86) 729-756 5 9 5 13 1.0' (.94) 757-784 2 4 2 8 1.0 (.99) 785-812 0 2 1 6 1.0 (.97) 813-840 0 2 2 5 .98 (.92) 841-868 1 2 2 3 .97 (.90) 869-896 1 1 0 1 1.0 (.94i 897-924 0 0 1 1 1.0 (.94) 925-952 - 0 0 0 0 953-980 0 0 0 0 981-1008 0 0 0 0 1009-1036 0 0 0 0 1037-1064 0 0 0 0 1065-1092 0 0 0 0 1093-1120 0 0 0 0 1121-1148 0 0 0 0 82 89 ----------------------•-----------------------•-------------------- a D.fined as those animals that died or were killed wh.n moribund. b The number ot animals that died of lsaiona or t:unor. 190 CTR coHTRACTS 029365 11249135 CTR VIN 044 1774

TABLE 95 INCIDENCE OF EIT3ER ANCBi, ACN, AAC, PDC, OR SCC I:I 3C3F1/CQH MICE AFTER TREATMENT WITH 811P AND LONC-TZeL4 EXPOSQRE TO 2R1 CIGARETTE SMOKE (CENERAL ADHERENCE TO RANDOM, ALL) a,b !!ON., JAN. 9, 1984 3AP * SMOKE SAP + SH31Ii -------------- ---------------- DAYS ON TEST ?RESENT aBSENT 2RESENT ABSENT ?ROBABILITY -------------------------------------------------------------- 1-28 0 2 0 3 1.0 29-56 0 18 0 3 1.0 57-84 0 33 0 0 1.0 85-112 0 11 0 1 1.0 113-140 0 3 0 1 1.0 141-168 0 4 2 3 .55 (.18) 169-196 1 7 0 5 .99 (.54) 197-224 2 4 1 4 1.0 (.90) 225-252 2 0 1 1 1.0 (.77) 253-280 2 4 2 3 1.0 (.91) 281-308 3 2 1 4 .69 (.49) 309-336 3 0 8 0 .69 (.49) 337-364 3 1 6 3 .63 (.46) 365-392 2 0 . 4 1 .54 (.38) 393-420 5 5 9 3 1.0 (.89) 421-448 2 2 12 0 .78 (.62) 449-476 4 0 10 1 .87 (.70) 477-504 8 0 13 1 .99 (.83) .~ 505-532 8 0 8 0 .99 (.83) 533-560 15 0 15 1 1.0• (.98) 561-588 19 1 12 1 1.0 (.95) 589-616 8 0 10 0 1.0 '(.95) 617-644 7 0 13 0 1.0 (.95) 645-672 10 1 5 0 1.0 (.95) 673-700 7 1 7 0 .97 (.82) 701-728 5 0 7 0 .97 (.92) 729-756 5 1 5 0 .84 (.69) 757-784 2 0 2 0 .84 (.69) 795-812 0 0 5 0 .84 (.69) 313-840 0 0 2 0 .84 (.69) 841-86a 1 0 2 0 .84 (.69) 369-896 1 0 0 0 .84 (.69) 897-924 0 0 1 0 .84 (.69) 925-952 0 0 0 0 953-980 0 0 0 0 981-1008 0 0 0 0 1009-1036 0 0 0 0 1037-1064 0 0 0 0 1065-1092 0 0 0 0 1093-1120 0 0 0 0 1121-1148 0 0 0 0 a b 125 :63 ------------------ ----------------------------------------------- Defined as tl:e total numcer ef animals vith histopatholoqie examination. '"he total auaber o: animals ia a qiven t:=e interval is the sum of the number of animals in the Present and Absent coluzas. i91 C.TR CaNTRRCTS Q23386 11249136 CTIR NN ~"'.441'?`55

TABLE 96 INCIDENCE OF EiTR-;R ANC:7, AC.I, AAC, PDC, OR SCC I:1 8C3F1/CIIH MICE AFTER TREATMENT WITFi 8AP AND L0:1C-TERH EXPOSURE TO 2R1 CIGARETTE SMOKE (STRICT AOHERENCE TO RANDON)a,b MON., JaN. 9. 1984 BAP + SMOKE BAP + SR3AM -------------- ----------- ----- DAYS ON T:ST PRESENT ABSENT PRESENT ABSENT ?ROBASILITY -------------------------------------------------------------- 1-28 0 2 0 3• 1.0 29-56 0 18 0 3 1.0 57-84 0 33 0 0 1.0 85-112 0 10 0 1 1.0 113-140 0 3 0 1 1.0 141-168 0 4 1 3 1.0 (.32) 169-196 - 1 7 0 5 1.0 (.87) 197-224 1 4 1 4 1.0 (.91) 225-252 1 0 0 0 1.0 (.82) 253-280 2 4 2 3 1.0 (.82) 281-308 3 2 1 4 .90 (.65) 309-336 2 0 5 0 .90 (.65) 337-364 0 1 2 3 1.0 (.82) 365-392 1 0 3 1 .97'(.73) 393-420 3 5 6 2 .83 (.63) 421-448 2 1 11 0 .54 (.39) 449-476 2 0 5 1 .64 (.47) 477-504 3 0 8 1 .73 (.56) 505-532 6 0 4 0 .73 (.56) 533-560 5 0 3 0 .73 (.56) 56-1-588 5 1 5 0 .58 (.44). 589-616 2 0 6 0 .58 (.44) 617-644 2' 0 3 0 .58 (.44) 645-672 2 0 3 0 .58 (.44) 673-700 0 0 0 0 .58 (.44) 701-728 0 0 1 0 .58 (.44) 729-756 0 1 0 0 .58 (.44) 757-784 0 0 0 0 .58 (.44) 785-812 0 0 4 0 .15 (.09) 813-840 0 0 0 0 841-868 0 0 0 0 669-896 0 0 0 0 897-924 0 0 0 0 925-952 0 0 0 0 953-980 0 0 0 0 981-1008 0 0 0 0 1009-1036 0 0 0 0 L037-1061 0 . -0 0 0 1055-1092 0 C 0 0 1093-1120 0 0 0 0 1121-1148 0 0 0 0 43 74 ---------------------------------------------------------------- 3 J Oetined as those animals taken off test randomly. The total number oE animals in a given time interval is the sum of the number of animals in the Present and Absent coluans. 192 CTR CONTRRCTS 029387 11249137 C TR HN 044176

Final Report Concraet: CTR-0030 CTR-lOlA FICURES 31-37 L93 CTR CaNTf2RCT5 029388 11249138 CTR HN 044177`

I i fln.l Report Contr.ct: CfR-0010 f1GtM1E 31 SMOKE 1PM 6ENERATION ANO DEPOSIf10N 380 22! 150 \J•. /f ,.. ..• W "_ rTl ~O . ~ . -0 y 10 tS 20 2$ 30 15 ti0 4S SO SS SO SS 70 )S 00 OS 90 95 100 105 ..$, ~ MEEKS OF fxPOSURE Ln N (0 _ ~ f ` _-. . ' . .

Final Report Contract: CTR-0030 LEGEND. FIGURE 32. A MEAN PERCENT CARBOXYHEMOGLOBIN FOR 3-5 ANIMALS BLED Ih4'EDIATELY AFTER EXPOSURE TO 2R1 CIGARETTE SMOKE. SHAM EXPOSED AND UNEXPOSED ANIMALS EXPRESSED MEAN VALUES OF 1.43 AND 1.79, RESPECTIVELY, OVER THIS 31 MONTH PERIOD. a SAMPLES NOT COLLECTED. 195 C.TR CQNTRRCTS 029390 11249140 CTR NN 044179

I 1 .1 1 1. 1 1 1 i I I Final (:cport Contract: CiR-0030 I 25 . 20 a 15 N A tp A ~ ~ 0 10 ~ 5 m ..r.a .J.~J ..:k ~ Ln I FIGURE 32 SONOJFMAMJJASONBUJFMAMJJASsONBDJFM I_ 1 l-- 1 1 i U 1978 1979 1980 1981 MONTHS OF TEST w w 0.

I ~ I I I I I I i ::I : I .I . 1 I I I I I FinaP Report Contract: Cflt-0030 FIGURE 33 BODY WEIGIIT OF 8C3F1/CUM FEMALE MICE AFTER EXPOSURE TO 2111 CIGARETTE SP10KE, SIIAM-EXPOSURE, OR UNTREATED 50 N A A N l J ?U 110 ~ 30 n ~ ~ "r''~ ~ .-. X ~ ~ ~ Jou 33 ~ un r 0 CA I I I ~ 0 l0,- .~ tD ~ .~ w M i,.,, 20 60 80 ' 100 120 WEEKS ON TEST 0 .96 SIIELF SHAM SMOKE I .

Final Report Contract: CTR-0030 LEGEND. FIGURE 34• SURVIVING FRACTIOti OF ANIMALS FOR 2R1 CIGARETTE SMOKE-EXPOSED, SM4-EXPOSED, OR SHELF CCNTRL BC3F1/CUM MICE. MICE WERE 8-16 WEEKS OLD WHEN EXPOSURE WAS INITIATED. 198 CTR CONTRACTS 029 393 11249143 CI I"1 / I I 7 044 7 .6 6u.F l.a.. •

Final Repurt Contract: CTIt-0030 FIGURE 34 1.0 w-o-o-°..o-o_a°-o-o-o-o-o_ °,o_° SHAM . -B~=B=8=8=6=8~~$~ ° ~+-- SHELF % 0. -°~` 0 ~ o-o-o..o o q., '~ SMOKE W1 N WEEKS'ON TEST i SMOKE EXPOSURE STOPPED

; i i Fiual Iteporl Contract: CtR-UL']U FIGURE 35 50 • • • BnP SHELF . .- N O .. E u 40 30 • • BaP + SNAtI • ~.    . . .. & • ~• ~. i snP + SHOKE  •_ . 10 A  20 110 60 80 100 120 T I ME Otl TEST

tt , Final Report Contract: CTR-0030 NO 30 20 ]0 1 2 F Icur,E 36 ~ ~ ` %x \ ~ ~ ` \ ~ ` \ ~ ~ ~ ~ ~ ~ \kN \ ~ \ S& 3 tl 5 6 7 8 TIME ON TEST (MOnTiis) 9 10 11 . 12 I

i ~ T_ O N _to v n 7U i oC :l. ~ 1 l ~ N ~ N ~ V.JLI ~~~' aJ~ Final Report CollifdCL: CIR-0010 1.0 0.5 20 40 FIGURE 37 60 80 WEEKS ON TEST 100 120 1q0 i

CTR CaNTRRCTS 429396 11249148 CTR HN 044187

Final Report Contract: CTR-0030 CTR-1013 V. CTR-1019. EXPOSDRE OF BC3e^1/CQa MICE TO 2R1 AND 3A1 CICaRETTE SMOKE A. Introduction . During 1979-1980, an exposure regimen was developed that resulted in low toxicity for mice exposed to either 2R1 or 3A1 cigarette smoke (see discussion of CTR-1'17 below). These exposure conditions allowed the scheduling of a long term study whereby the potential biological effects of smoke from both cigarette types could be simultaneously evaluated in both female and male 8C3P1 mice. Such a long term study employing this smoke exposure regimen was proposed, approved and initiated during the 1980 contract year. In July 1980, however, it was decided that this study should not be completed and that smoke exposure under these newly defined conditions would be limited to the length of time during which animals in CTR-101A were to be exposed to smoke. The experiments conducted ie the CTR-1018, therefore, were redesigned to support three specific studies in order to obtain the most' information possible. Several •short-term assays were evaluated that have the potential to be early indicators of possible toxicologic consequences of exposure to cigarette smoke. The three specific studies are as lollowst o CTR-117 - A preliminary study to determine the toxicity of 2R1 smoksr in mice and rats using the continuous exposure regimen. o CTR-118 - A study where only female BC3F1/Cum 3ice were used, and the analysis of the assays were performed after daily exposure to 3a1 azd 2R3. cigarette smoke for 3, 6, 9 and 12 mcnths. o CTR-131 - A study where age-matched male and female mice were used, and the assays analyzed after 1 day, 1 week, 3 weeks, 3 months, and 6 months of daily exposure to 3A1 and 2R1 cigarette smoke usinc- this continuous regimen. 203 CTR CaNTRACTS 029399 11249149 CTR HN 044,168

8ina1 Raport Contract: CTR-0030 CTR-1013 B. Smoke Exposure Regimen, Toxicity and Histapatholoqy The smoke exposure regimen applied in the CTR-101B study was different from other exposure regimens used previously. I: contained the following features: o there were no rest periods betaeen successive cigarettes or "runs." o the exposure period for smoke was IS seconds per minute, o each "run" resulted in exposure to 120-150 seconds of smoka from 2R1 or 3A1 cigarettes (1 puff/minute, 15 seconds smoke/minute. 8 puffs/ciqarette), and, o smoke exposures were repeated "continuously," resultinq in 17 "runs" in 140 minutes. Because this regimen did not..provide rest periods between cigarettes, a preliminary study was performed to a~sess both the relative tozicity and the types of pulmonary lesions in• both male and female BC3Y1/Cum mice following this exposure (i.e. CTR-117). Circumstances of death for these animals are presented in Table 97. No sham exposed females were used in this study since sufficient data were available regarding the'response of female mice to chronic restraint. Sham exposed male mice were used. Data in Table 97 indicated that toxicity of 2R1-ciqarette smoke using this "continuous" regimen was approximately 0.4% per week for female. mice an 1.11 per week for male mice. In com parison, CTR-IOlA shoved that 2R1 smoke using a discontinuous regimen and many more animals, resulted in a toxicity of "1.6t per week. Microscopic diagnosis of the lunqs of these animals suggested that exposure to the continuot;s regimen resulted in occurrence of PAMA much earlier and with higher incidence than was observed in CTR-101A. Microscopic evaluation of the occurrence of PAH11 and any other pulmonary lesions are presented in Tables 98, 99, and 100. PAMA was the most prevalent lesion observed in both male and female smoke exposed mice, with an overall incidence of 97% for male mice and 951 for female mice that died after 16 weeks of daily smoke exposure (Tables 98 and 99, respectively). Thus, the continuous exposure regimen resulted in higher PAM71 incidence and shorter latency than occurred in CTR-101A. No other smoke associated lesions aere observed. 204 C.TR EOh{TRRCTS 029400 11249150 CTR I-IN 044169.-

Final Report Contract: CTR-0030 CTR-1013 C. Dosimetry 1. Particulate phase A dosimetry experiment using 14C-dotriacontane (OTC) labelled 2R1 cigarette smoke was carried out to determine the actual TPM deposition and distribution under this continuous exposure regimen. The tissues for this dosimetry experiment were analyzed for radioactive content at MA using the Packard 306 TRI-CARB Tissue Oxidizer. A brief description of the dosimetry experiment follows. BC3F1/Cum female mice, 12-14 weeks old, were exposed to the continuous exposure regimen for 4 weeks prior to the dosimetry experiment. On the day of the experiment, 64 of these mice were exposed to radiolabelled 1 4 C-DTC-2R1 cigarette smoke, along with an additional 10 mice that had never been exposed to cigarette smoke ("unadapted"). The experiment was designed so that 8 mice would be removed from smoke exposure after each run and the tissueslt nalyzed for radioactivity. The deposition and distribution of C-OTC disintegrations pe,r minute (dpm) are presented in Table 101. Deposition in the lung increased linearly with increasing smoke exposure time (correlation coefficient (=)"0.97), with a slope of -260 dpm/120' second exposure time or "run." ~e~ average of 74% (coefficient of variation (c.v.) = 0.041 of the DTC dpm was found in the lung for all eight "runs." Between-animal variation in deposition in the lung ranged from 0.21 (c.v.) to 0.39 (c.v.) for the individual "runs." A substantially highef4c.v. of 0.50 was observed in "snadapted" mice. The average OTC dpm in the lung was also suestantially higher in these animals, suggesting cnnl- unadapted mice exposed to smoke altered their breathing pattern in a manner that resulted in large intecindividual variation and in some instances, resulted in rather high levels of smoke deposition. In order to convert the 14C-DTC dpm to micrograms TPM, the specific activity of the TPM must be determined. A brief review of the mechanics of the procedures will be given here. The optical sensor was calibrated to detect the total amount of TPM generated from the cigarettes. A sampling pump withdrew smoke at a known rate through a"conditioned" Cambridge Filter. The amount of particulates in this filter could not be weighed reproducibly (<5 mg), however, sufficient radioactivity could be detected after elutinq the pacticulates and analyzing by scintillation counting. Yicotine could also be analyzed :rom tnis sample. Thus, the amount of TPM was determined from the optical sensor data and the pump sampling rate. The amount oE radioactivity was determined after elutinq from the filter and ccunting. These. measurements and the calculated specitic act:v:-y are presented in Table 102. 205 CTF2 COHTRACTS 029401 11249151 y.r / R / M( • f 0• . 1M' 0

Final Report Contract: CTR-0030 CTR-1018 Qsing the specific activity calculated in Table 102, lung deposition was calcalated to be 4, 9, 11, 19, 24, 29, 31 and 34 ug TPH for 120, 240, 360, 480, 600, 720, 840, and 960 seconds total smoke exposure time, respectively. These data were plotted in Figure 38. If these data are extrapolated to 2040 seconds or 17 "runs," an estimated deposition of 80 ug TPH/per day per mouse was made (Piqure 38). These values are much lower than expected based upon the high incidence and time of occurrence of PaKA (see previous Section V.B.). It must be speculated that removing the rest period between exposures and providing smoke every 15 seconds for -140 minutes modulated the macrophage response. .2. Gas Phase The mice in CTIt-131 r+ere monitored throughout the study for COHb levels after exposure to 2R1 cigarette smoke. •rhe overall mean COHb values for female mice were 35.5+9.1t. The values for male mice were 32.5 + 3.4%. These leveli are almost twice the levels observed in CTR=101A. D. Short-Term Assays to Monitor the Effects of Smoke The following assays were selected for analysis in age-matched male and female BC3r1/Cum mice exposed to 3A1 and 2R1 cigarette smok. for up to 6 months (CTR-131), and in female BC3ri/Cum mice for up to 12 months (CTR-118). These assays wero selected for their potential to predict or monitor lonq-term effects of cigarette smoke. Por some of these assays, previous experiments that used other smoke exposure regimens will be discuased and compared to the present studies. 1. Inhibition of pulmonary DNA repair in vitro (in collaboration with Dr. Rasmussen). 2. Stimulation of pulmonary DNA synthesis both in vitro and in vivo. 3. Induction o:'pulmonary, hepatic and renal aryl hydrocarboa•hydroxylase (XHH) activit;•. 4. Induction of pulmonary ornithine decarboxylase (0DC) activity. 5. Augmentation of the DNA damaging effects of a particular lung carcinogens. 6. Induction of sister c.hromatid exchanqe2 (SCE) in bone marrow (in collaboration with Dr. W. Benedict). 206 CTR CONTRRCTS 029402 11249152 CTR f f N 04'41'_041.

Pinal Report Contract: CTR-0030 CTR-101B 7. Alterations in physical and biochemical properties of the lung, such as lung weight (aet and drl) , and DNA, protein and hydroxyproline levels in lung. 8. Alteration in the immune response. 9. Alteration in rate of atresia (i.e oocyte toxicity) (in collaboration with Dr. 0. Hattison). E. Disposition of Animals The disposition of female'BC3rl/Cum mice that were to be evaluated after exposure to 3A1 or 2R1 cigarette smoke is given in Table 103. The disposition of aq e-matched male and female mice following daily exposure to 3A1 or 2R1 smoke is given in Table 104. As originally designed, the animals in these studies were to be chronically exposed ta smoke over their lifetime. Thus, over 1200 animals were scheduled for initiation on test when the chronic study was cancelled. The redesigned studies (CTR-118 and CTR-131) were an attempt to utilize these animals technically and economically to.obtain the most information during either a 6 or 12 month ezposure period. No microscopic diaqnoses were scheduled since it had been demonstrated previously that very few smoke-associated changes took plac• during the first year'of smoke exposure. The smoke esposure equipment at Microbiological Associates was shipped at the Council's direction to the Qniversity of Kentucky in July 1981. Thus, a:ter the final exposures, approximstely 400 animals were alive when the study was terminated as of August 1, 1981. F. Summary of Results 1. Inhibition of Pulmonary DNA Repair (in collaboration with Dr. R. Rasmussen) There is evidence that the initiating event for ^eoplasia may be a direct alteration of cellular DNA, i.e. somatic mutation. There is also evidence that if the alteration can be repaired, the otherwise exFected neoplasia will not occur. Thus, if cellular DE1A repair capacity is somewhow inhibited or reduced, the chance for the retention in cellular DNA of potentially oncoyenic alterations may be increased. The results presentM here were concerned with the effects of cigarette smoke expoe;.sre on enzymatic activities in 207 ' C.TR CCINTRRCTS 029403 11249153 i..r f R f f f"f 0441 :.~"~ 2.

Final Report contract: CTR-0030 CTR-101B lunq tissue associated with DNA reelication and r)vA repair. Repair was measured by 3eter:nini:.q• the Leve1 of 3:i-thyr..id:; e incorporated into DNA in the presence of hydroxyurea. This :s called unscheduled DNA s,vnt!~esis (UDS). Our previously published results (14) demonstrated the inhibition of in vivo DNA repair after only 11 weeks exposure to 2A1 cigarette smoke (Table 105). Experiments also demonstrated that the inhibition persisted for at least 6 months after exposure was terminated (Table 106) (14). A series of experiments Were.performed to investigate further the capacity of cigarette smoke to inhibit DNA repair. Data from all of these experiments and CTR-1013 .+ill be presented here so that conclusions can be drawn from the entire data base. a. Cigarette Type Three cigarette typea (2A1, 3A1, and 2R1) were compared for their capacity to inhibit DNA repair using a relatively high dose of smoke, i.e. approximately that used in CTR-100. Groups of BC3F1/Cum mice 8-10 weeks old were adapted to smoke from each cigarette type using the SEM II B. The exposure conditions utilized 10% v/v smoke concentration and standard exposure conditions of 2 second puff duration, and 35 ml average puff volume. -------------------------------------------------------------- EXPOSURE TOTAL SMOKE ESTIliATED CIGARETTE CYCLE EXPOSURE/DAY TPM TYPE SHOKE/AIR (SEC) DOSE ---------------------------------------------------------------- 2A1 30/30 3A1 30/30 2R1 20/40 Sham 0 Shelf 0 2400 •1 mg/day 2400 1 mg/day 1600 0.8 mg/day . 0 - 0 - --------------------------------------------------------------- Eiqht puffs per cigarette were used to reduce the toxicity of the high nicotine containing 2R1 cigarettes, yet maximize the dose. The exposure regimen was given in two sessions per day (5 cigarettes in the morning, 5 cigarettes in the afternoon). Ten minutes of air were given between each ciqarette. Mice were shipped to Dr. Rasmussen after 9, 13, 24, and 41 weeks of exposure for analysis of pulmonary DNA repair capacity. !iice -were analyzed for UD3 and replicative DNA synthesis kROS), as described previously (14). Results of RDS Will be discussed in the next section. 208 CTR CaNTRRCTS 029404 11249154 CTR HN 044193

Pin.l R.pott Contract: CTR-0030 CTR-101B The disposition of the animals for this study is shown in Table 107. The toxicity observed using this exposure regimen was 1.81, 2.4%, 2.3%, and 0.9% per week for 2AL, 3A1, 2R1 smoke and sham exposed mice, respectively. As a point of comparison, the toxicity observed in CTR-117 was 0.4% per week (see Section v.e.). The cumulative TPM deposition was estimated from the daily smoke exposure documentation, resulting in approximately 1 mg TPH/day/mouse for 2A1 and 3AI cigarettes and 0.8 mq TPM/day/mouse for 2R1 cigarettes. Animals were monitored in this study for the occurrence of PAltA and as shown in Table 108, a high incidence occurred in response to exposure to all thrae cigarette types. The lowest iacidence of '73t was observed with the 2R1 :igarette. Data are presented in Table 109 for the cos levels observed in vitro in lung tissue from BC3F1/Cum mice exposed to smoke from 2A1, 3A1, or 2R1 cigarettes. Results for vOS in tissues from smoke exposed mice were given relative to the common sham exposed controls. QDS was depressed after 13 weeks exposure to 2A1 and 3A1 ciqarette smoke, but not after exposure to 2R1 cigarette smoke. The lack of effect with the 2RL cigarette may be a consequence of the lower dose requ.ired to maintain equivalent toxicity with the 2711 and 3A1 cigarettes, or may reflect a real difference in the effect of the 2R1 ciqarette. These data were in marked.contrast to those obtained usinq the continuous exposure regimen. No effect on DDS ("DNA Repair") was observed for mice exposed to the continuous reqimen with either 3J11 or 2R1 ciqarettes for up to 12 months (Tables 110, 111, and 112). No differences betWeen male and female mice were seen at any of the time points. Thus, the inhibition of DNA repair by ciqarette smoke would seem to be d ependent upon both the cigarette type (2A1'3A1»2R1) and/or the exposure regimen (30 seconds smoke/min » 15-20 seconds smoke/minute): b. Chemical Additional studies were carried out in an attempt to find other agents that induced inhibition of pulmonary DNA repair capacity. The effect of intratracheally inoculated 3aP and Bap-7,8-diol was examined. BC3FI/Cum mice, 8-12 weeks old were inoculated intratracheally with 1.2 mq BaP in 0.02 ml qelatin-saline or with vehicle alone, once per weak for a total of three weeks. yice were shipped to or. Rasmussen one week after the last treatment. Opon arrival, sample animals from each group were assayed for oulmonary OttA repair and replication. The remaining animals were housed for approximately 9 weeks, at which time they were assayed 209 CTR CONTRRCTS 029405 11249155 CTR HN "11441 R.- 4

Final Report Contract: CTR-0030 CTR-101S ._i in a similar manner. Results are shown in Table 113. There was no effect of the BaP treatment on either DNA repair or replication as measured by this assay. The effect of BaP-7,8-diol on DNA synthesis rates in mice was evaluated in a different manner. :n this case. :+e analyzed the effect of intratracheally-instilled BaP-7,8-diol in animals that were previously exoosed to 3A1 or 2R1 ciqa3rette smoke _°or 58 weeks. The percent lunc cells labelled 'r:-Tdq is presented in Table 114. The number of cells labelled (or labelling index (LI)) was determined 4 hours after BaP-7,8-diol treatment and either immediately after smoke exposure, or 7 days after smoke exposure ceased. As expected, the smoke exposed mice demonstrated a 3-fold increase in lung LI relative to the sham or shelf controls. This LI was high 7 days later in the 241 smoke exposed mice, but had dropped to background level in the 3A1 smoke exposed mice. Treatment with BaP-7,8-diol resulted in a 2 to 4-fold increase in LI at both test times, however, this increase was not additive to that of the smoke exposed animals. Treatment of smoke exposed mice with BaP-7,8-diol yielded an LI that was quite similar to that observed in mice that were smoke exposed only. An exception may be that the group that was exposed to 3A1 smoke and held 7 days after termination of smoke exposure before BaP-7,8-diol treatment (see Table 143). For th.is• group, the BaP-7,8-diol animals demonstrated a high LI while the smoke exposed controls returned to background level. It would seem that both BaP-7,8-diol and cigarette smoke were capable of causing an._increase in rates of DNA synthesis and/or repair in pulmonary tissue of mice, but that this increase was not interactive when both agents were given simultaneously. C. Age In an attempt to find other conditions that induced inhibition of DNa repair capacity, we investigated the effect of two different ages of mice in four different strains: 9C3F1/Cu:n, C3H/Anf Cum. D9A/2J, and C57BL/6 Cum. riice of two different ages from each strain were shipped to Dr. Rasmussen for evaluation. Results are shown in Table 115. In each case, the older animals appeared to have less replicative activity in the lung. The only scrain showing any indication of reduced DNA repair activity in older animals was the 9C3P1/Cum. This strain also had the oldest animals and the greatest separation in ages. These results should be considered preliminary since only one set of animals was available for study. 210 . ~. L CTR CONTRRCTS 029406 11249156 CTR HN 044195"

Final Report Contract: CTR-0030 CTR-101B 2. Stimulation o: Pil:nonary DNA Synthesis a. In Vitro DNA Synthesis in Pulmonary Tissue Lunq slices from smoke exposed, or she1= control r3tice were incubated, as described for ODS, in the presence of H-TdR, but in the absence of inethyl methane sultonate (MMS) and hydroxyurea (HU). 3H-TdR uptake under these conditions reflected only replicative DNA synthesis (RDS) . DNA replication was increased -2-fold in mice exposed to 2R1, 3A1, or 2A1 cigarette smoke compared to sham exposed mice (see Tables 109-112). The increase could be seen as early as one day after 3711 or 2R1 smoke exposure and remained at this -2-fold differeace for tt:e entire time of smoke exposure. Exposure of 8C3Pl/Cum mice using the continuous or discontinuous exposure reqimern both causec: the same 2-fold increase in RDS. b. Ln Vivo DNA Synthesis in Pulmonary Tissue Rates of DNA replication, as measured by estimation of Labellinq Indez (LI) were determined in BC3F1/Cum mice after daily exposure to whole cigarette smoke using both the discontinuous exposure regimen as described in Table 109 and the continuous regimen. Imsediately following the list smoke exposure, lung DNA was labelled with 1.0 hr pulse of H-TdR and tissues processed for autoradioqraphy. The LI was evaluated in animals after 9 and 13 weeks of 2A1, 3A1, or 2R1 smoke exposure (Table 109). The LI from 2A1 and 3A1 smoke exposed mice was increased over controls by 2-fold, while the GI from 2R1 smoke exposed mice remained at control levels. Us;nq the continuous regimen, the LI in both 3A1 and 2R1 smoke expoted mice was increased over controls after 3 weeks of exposure and this increase persisted over the 58 week exposure period (Table 116). This apparent dependence upon the exposure regimen for LI from 2R1 smoke exposed mice needs to be investigated further. Of the 4000-6000 cells counted in both sham exposed and shelf control 3ice, not more than 3 cells had any significant number of grains. The percent of total cells labelled was 3-to-6-fold qreater in mice exposed to smoke •from 3711 and 2R1 cigarettes (see Table 116). In the lung tissue from smoke exposed mice, the distribution of cells with grains was reqular. No labelling of cells o~ the bronchial epithelium was found, rather•, the uptake of the H-TdR was confined to alveolar cells. Neither necrosis nor epithelial hyperplasia was observed in nny area of the lunq that would account 'for the increaso in LI. 211 CTR CONTRRCTS 029407 11249157 CT• R HN 04,4196

rinal Report Contract: CTR-0030 CTR-101H * The grain number per nucleus was estimated for those mice exposed for 13 weeks (3 months) to 3AL or 2v.1 cigarette smoke (see Table 117). The number of labelled cells with a countable (up to approx. 100) grain number was about 25-fold higher in smoke exposed mice than in sham or shelf controls. The number of labelled cells with a grain nuaber too numerous to count were only 4-to-7-fold higher in the smoke exposed groups relative to control (see Table 1•17). A similar distinction was also evident in mice exposed to cigarette saoke for 26 or 39 weeks (Table 118) , but the magnitude of the difference was not as high as obtained in the 13-week smoke exposed mice. The presence In smoke exposed lung of a large number of cells with a low grain count is of importance. A low grain number of cells is generally indicative of DNA repair synthesis. Thus, our data seem to suggest that whole cigarette amoke, while stimulating normal DNA replication may induce unscheduled or Dt77i repair synthesis as well. The degree of LI was also studied in other tissues of•BC3P1/Cum mice following exposure to cigarette smoke (Table 119). The LI was high in spleen and intestinal crypt cells, but smoke exposure only' seamed to enhance LI in tracheal cells. Whatever factors in smoke that induce DNA synthesis, thesi factors only seem to effect cells in close proximity to the point •ot smoke deposition. 3. Znduction of 71HH Activity Pulmonary AHH activity was determined 6 hours post final smoke exposure to 3A1 and 2R1 cigarette smoke. This enzyme activity was found to be stimulated (relative to sham- controls) approximately 4-fold after 3 months, and 5-fold after 6 and 9 months of daily smoke exposure (Table 120). Daily smoke exposure for 9 months also induced AHH activity in the kidneys (Table .121). Renal aHH activity was 4 to 6-fold higher in 3a1 or 241 smoke exposed mice. The increase was approximately the same whether the assay was performed 3, 6 or 9 hours after the last smoke exposure. No increases in hepaeic AHH activities were observed. e.xposure to cigarette smoke from either cigarette type resulted in induction of AHH activity in both pulmonary and renal tissue. This induced AHH activity likely remained at this 4 to 6-fold higher level during the enti.:e length of time of smoke exposure. 212 ' CTR CONTRRCTS 029408 11249158 C, T R M N 0 -1? 4 1 c.-f' "

rinal Report Contract: CTR-0030 CTR-lO1B 4. Induction of Pulmonary ODC activity 0DC is the rate limiting step in polyamine synthesis and has been used as a marker for the promoting capacity of unknown caemicals. Pulmonary ODC activity is stimulated -2-fold within 3 hours after daily exposure to 2R1 or 3A1 cigarette smoke for 3 or 6 months (Table 122). ODC levels decreased to background levels by 6 to 9 hours post smoke exposure. Daily exposure to cigarette smoke likely causes a small, but transient induction of pulmonary ODC. This level of ODC induction is much smaller than those normally associated with treatment with known promoters, such as 12-0-tetradeconylphorbol-13-acetate (TPA). Previous studies in our laboratories have shown that aerosolized TPA is capable of inducing pulmonary 0DC activity -10-50-fold (1S) . 5. Augmentation of DNA Damaging Effects of Lung Carcinogens The alkaline DNA elution assay measures the rate of elution of DNA from a polyvinylchloride filter in the presence o'f an alkaline buffer system. The rate at which DNA elutes from such a filter is dependent upon the molecular weight and strandedness of the DNA. DNA from chemically or physically damaged cells elutes from the filter at a faster rate than DNA from untreated control cells because single.strand breaks have been introduced into the DNA. This assay was used. in order to determine if cigarette smoke was capable of inducing single strand DNA breaks or if smoke was capable of modifying the ability of other chemicals in causing these single strand breaks. The results to date employing the alkaline elution to measure the effects of smoke exposure have been equivocal. Reproducibility of the assay has been a problem, particularly in the amov.nt of DNA recovered when using diaminobenzoic acid to determine the DNA spectrofluorometrically. The extent of damagu induced in the positive controls has also been variable and, in general, lower than expected. The results of daily smoke exposure for 3 or 6 3onths on the rate of DNA elution from cells derived :roz pulmonary tissue is shown in Table 123. Data from 3 months smoke exposure suggested that the rate of DNA elution was slightly higher in cells from smoke exposed animals, but no differences vere seen after 6 months of smoke exposure. The variability of response to the negative and positive control (i.e. w.MS) was evident. 213 C.TR Cah4TRRCTS 029409 11249159 CTR HN 044198

rinal Raport Contract: CTR-0030 CTR-lOlB The effects of smoke exposure combined with intratracheal t:eataent with BaP or BaP-7,8-diol are presented in Table 124. Chemical tzeat.3ent was given 24 hours prior to assay. ror this exposure regimen, no increase was observed in the amount of DNA eluted for 9aP + smoke or BaP-7,8-diol + smoke treatment, compared to the sham exposed groups. These results need to be repeated because the methods used to quantitate DNA were not very efficient. The poor reproducibility of the positive and negative controls could likely aask any effect that might have been caused by exposure to cigarette smoke (or exposure to any unknown chemical). 6. Lnduction of Sister Chromatid Exchange in Bone Marrow Cells (collaborative studies with Dr. '. Benedict) Sister Ct:romatid. Exchange (SCE) induction, postulated as a precarcinoqenic event, measures the number of times the genetic material of the chromosome is exchanged ot recombined per metaphase chromosome. We examined the effects of whole cigarette smoke in modifying SCEs in bone marrow cells o,f BC3F1/Cum female mice, with the view to studying possible clastogenic effects of smoke in vivo. Methodology for these studies was presented elsewhere (16). Shelt control as well as sham exposed mice consistently yielded approximately 4 SCS per metaphase chromosome. After only 1 week of exposure to 3Al. or 2R1 ciqaret.te smoke, the number of SCEs in•bone marrow-derived cells increased two-fold (Table 125 and 126). Continued daily exposure to cigarette smoke for 4, 12. 'and 46 additional weeks showed no further increase in the number of SCEs observed. These significant increases in SCL persisted even after cessation of smoke exposure for at least one week, whethe: the mice were exposed initially for 1 week or 46 weeks (Table 127). Data suggested that smoke contains specific chemicals that were distributed to bone marrow and these chemicals caused the formation of SCEs in these cells. Since these SCEs persisted for at least one week after smoke exposure was terminated, it would seem that these SCEs were either extremely long-lived, or that there were fairly long-lived smoke-associated chemicals that were :e-initiating these effects. A more complete description of this .study is given in (16). 214 CTR CQNTRACTS 029410 11249160 C.x f R f f f'f 04"T' 1991

?inal Repo=t Contract: CTR-0030 CTR-101B 7. Alterations in Physical and Biochemical Properties of the Lung a. Lung Weight Exposure to cigarette smoke using the continuous regimen resulted in an increase in the wet weight of lungs. The dry weights of the lungs were also increased, with the dry weight consistently representing 22-251 of the wet weight. The ratios of wet aeight-of lung to body weight for female and male mice are given in Tables 128 and 129. The ratios of dry weight to body weight for male and female mice are given in Tables 130 and 131. Increases in weight of the lung was observed within 3 months, and this increase became more apparent as the length of smoke exposure was continued over 12 months. b. ONA/Protein content Two other measures of the increase in pulmonar•y- lung weight associated with long-term exposure to cigarette smoke are total DNA and/or protein content. Pulmonary DNA and protein' content determined in the female mice exposed- to'3A1 and 2R1 cigarette smoke over 1 day through 52 weeks ..are -summarized in Tables 132 and 133. Pulmonary DNA content increased slightiy during the observation period. Protein content appeared to almost double during this same time for the smoke exposed groupa, but remained the same (or decreased) in the sham-or shelf control groups. Total protein content seemed to parallel•the increase in dry and wet weight observed in Tables 129 to 131. c. Pulmonsry Hydroxyproline Content It is known that a major consequence of inhalation of many aerosolized chemicals :s the alteration in collagen synthesis in the lung. We have attempted to study this phenomenon by determining the hydroxyproline (HyP) content in lung tissue. Collagen is unique in that it alone among proteins has a high concentration of HyP. Hence, the amount of HyP in lunq would reflect the extent of collagen synthesis in the tissue. Ho:aogenates of lung were prepared, acid hydrolized and the levels of HyP determined by a spectrophotometric assay adapted from Ptoessner at al., (17). The levels of HyP in smoke exposed and control lungs are compared in Table 134 and 135. U;: to 13 weeks, ir. both male and female mice, 215 CTR CaNTRACTS 429411 11249161 CTR I-IN 04420 ~"0

Final Report Contract: CTR-0030 CTR-1018 HyP levels were unchanged in the smoke exposed and control mice. By 26, 39 and 52 weeks a=ter smoke exposure, the levels of HvP had increased (Tables 134 and 135) in the smoke exposed animals compared to sham or shelf controls. Evidently, prolonged exposure to cigarette smoke induced collagen synthesis in the lung. Exposure to 3A1 or 2R1 cigarette smoke specifically increased the cellularity of pulmonary tissue and the rate of this inerease was proportional to the length of time that daily smoke exposure continued. This increase in tissue mass was likely the result of increased cellular proliferation. Moreover, this proliferation was probably a final manifestatio:: of the increased DNA synthesis rates observed in these smoke exposed animals (descrSbed at length in Section V.F.2). e. Alteration in the Immune Response After Exposure to Cigarette Smoke (Collaborative Studies with Dr. Herscowitz- Study ociginally Termed "CTR-109") Collaborative studies with Dr. H. Herscowitz (Georgetown University, Washington, D.C.) have investigated the immunosuppressive effects of cigarette smoke in BC3F1/Cum female mice. The following criteria were used to determine the immune response: 1) antibody production at the cellular level as reflected by the spleen plaque assay (P!C assay), 2) antibody production in vivo as determined by circulatinghemagglutinating antibody (HA), and 3) mitogen-induced proliferation of spleen cells. Previously, Dr. Herscowitz demonstrated that all three responses were substantially suppressed in BALB/c mice after exposure to 1R1 cigarette smoke, but not to lal cigarette smoke. Using the Walton Horizontal Smoking Machine (WHSM) (Progress Report, 1979), previous studies from this laboratory showed that exposure of BC3F1/Cum mice to 2R1 cigarette smoke did not result in a consistent depression of the antibody production (PFC assay) when r.ompared to sham exposed controls. Ln order to determine whether the lack of depression of the PFC•response in BC3F1/Cum mics was due to differences in strain, experiments were designed to expose both BC3rl/Cum and 9ALa/c •mice. The BALB/c mice would serve as positive controls for inhibition and the results from the PFC assay for the two strains could be compared directly. 216 I CTR CaNTRRCTS 429412 11249162 \...r )/ 1 1 aI I I 7.h 44/f~ 0 1

Final Report Contzact: CTR-0030 CTR-101B a. 9ackqround Experiments to Optimize tae System The system employed to measure systemic i=unoloqic suppression was the Jerne plaque technique. Animals were injected IP with 0.2 ml of a 101 suspension of Sheep Red 31ood Calls (SRBC). Three to seven days later, animals were killed, their spleens were removed and teased apart, and the resulting spleen cells were assayed for their ability to produce antibody against SRBC (PFC assay). The kinetics of the PFC response are given in Table 136 and show that both eC3F1/Cum and 3ALB/c Cum mice demonstrated a maximal response at 5 days post SZBC injection. Cyclophosphamide, a widely used chemotherapeutic agent and bifunctional alkylatinq agent with well characterized immunosuppressive qualities, was used as a positive control for the PFC assay. Results in Table 137 show that cyclophosphamide caused a dose-dependent decrease in the PFC response both in old (59 weeks) and younger (22 weeks) BC3F1/Cum mice. Cyclophosphamide treatment was used as a positive control for the PFC assay in the following experiments. . b. Effects of Acute Cigarette Exposure on the Systemic Immune Response of Mice Out initial attempt was to repeat. the previous observation that acute exposures to high nicotine containing cigarette smoke (1R1 cigarettes) for 1 to 13 days could substantially suppress systemic immunity. BALB/c Cum or BC3F1/Cum mice were exposed to 101 (v/v) smoke from 2R1 cigarettes (similar to 1R1 cigarettes) on the SEM Ii. Exposure conditions were 20/40 seconds smoke/air per minute over 8 minutes (8 puffs/ cigarette). Ten minutes smoke/air followed each smoke .exposure. The exposures were repeated for a total of 4 sessions per day (equivalent to 4 cigarettes/day). Animals were exposed for 5 days post SRBC injection for a total of 10 days. Results are shown in Table 138. No immunosuppression resulted from exposure to 2R1 cigarette smoke under these conditions. A repeat of these experiments using a 30/30 seconds of smoke/air per :ainute over 8-10 minutes similarly failed to cause any decrease in the splenic PFC response. Thus, it would appear that -ipcuta exposure to smoke from 2R1 ciqarettis did not cause any i:nmuaosuppression. 217 ' CTR CoNTRACTS 029413 11249163 CTR HN 044202

rinal Report Contract: CTR-0030 CTR-1018 ,.. Effects of Chronic Cigarette Smoke Exposure on Systemic *_rusunity In a further attempt to confirm the previous observation by Or. Herscowitz that substantial is,munosuppression was caused by chronic exposure to high nicpt:ae containing cigarette smoke, the following experiment was performed. BC3F1/Cum mice were exposed to 2R1 cigarette smoke for 52 weeks under the exposure regimen used in CTR-lOlA (10t v/v) smoke, 20/40 seconds_smoke/air per minute over 8 minutes, followed by 8 minutes of air, 5 repeated exposures/day. Five days prior to the termination of the experiment, the micc were immunized with sasc. Smoke exposure was continued for 5 days following iclmunization. The results in Table 139 show that no immunosuppression occurred after chronic exposure to 2R1 cigarette smoke. in conclusion, neither acute nor chronic exposure to 2R1 cigarette smoke caused immunosuppression after exposure to 2R1 cigarette smoke in either BALB/c Cum or BC3F1/Cum mice under the conditions employed in these experiments. The reasons for these discrepancies with previous observations arr not clear. Literature reports regarding the immunosuppcessive effects of cigarette smoke are conflicting. While many investigators have shown that immunotoxic effects are mediated by cigarette smoke exposure, the magnitude of these effects has varied greatly. The studies presented here were defined in terms of the health status of the animal and the smoke exposure regimen used. whether these variables play a role in these resuits remains under investigation. 9. Alteration in Rates of Atresia. (collaborative studies with Dr. 0. Mattison - study originally termed "CTR-126") Collaborative studies with Dr. Mattison (National Institute of Child Health and Human Development, Bethesda, MD.) have examined the effects of various treatments, including exposure to cigarette smoke, on the rate of depletion of oocytes in the mouse. A relationship has been suggested to exist between smokinq and onset of menopause in humans, indicating that women between the ages of 44 and 53 years, who smoke one or more packs of ciqarattes per day are more likely to be poatmenopausal than non-smokers (18). menopause likely occurs when the ovaries are depleted of oocytes. The number of oocytes decreases from a maximum at birth to zero at about 50 years of age in humans and at about one. year in mice. Since ovvilaticn accounts for only about 0.06% of the oocytes in the ovarl-, factors other than 218 CTR CaHTRRCTS 429414 11249164 CTR MN 04'~`~0~

Pinal Report Contract: CTR-0030 CTR-1018 ovulation are clearly important in determining the age of menopause (19). The major factor is thought to be a poorly understood process called atresia, whereby oocytes are destroyed in the ovary (20). The question raised by these observations was whether oocytes were destroyed at an accelerated rate in mice exposed to whole cigarette smoke. Since the smoke exposure studies utilized lifetime exposure of animals to smoke, the oocyte depletion was determined as a function of age for BC3F1/Cum, C57B1/6 Cum (86 C;:m), C3H/An! Cum (C3 Cum), and DBA/2J (02J) mice. The oocyte depletion was also determined as a function of exposure to 2R1 cigarette smoke. a. Genetics o: Murine Atresia For the studies of atresia as a function of age, 3 mice per strain were sacrificed by cervical dislocation every 2 months for a total of 18 animals per strain. Ovarectomies were performed izumediately after sacrifice. Both ovaries were removed with the fat pad intact and fixed for 24 hours in Bouin's solution. Ovaries were then •::ansferred to 70% ethanol for storage until delivered to .3r. Mattison fot sectioning and analyses. The entire ovary was sectioned and the oocytes in every 20th section determined. Data are presented as a mean number •of oocytes per ovary as a function of the age of the mouse. Data are presented in Figure 39 for the four strains held at MA (C3 Cum, BC3F1/Cum, B6 Cum and D2J) and two strains held at azCHD (DaA/2N (D2H) and C5781/6 (86N) ). The C3 strain appeared to have the most rapid rate of atreeia. There were no differences between the B6 Cum and 86N strains. The D2J and D2N also appeared to have similar rates of atresia. The rate of atresia from BC3r1/Cum mice appeared to be intermediate between the C3 Cum and B6 Cum strains. b. Effects of Cigarette Smoke Exposure on Atresia The ovaries from BC3F1/Cum mice on test for s=oice and sham exposure uader CTR-lOlA were utilized to determine t::e rate of oocyte depletion. Ovaries were taken from age-matched smoke and sham exposed mice at selected intervals, fixed, sectioned and the number ot oocytes counted as described above. Data are presented in Figure 40 for the number of oocytes/ovary from BC3F1/Cus mice from 6 to 39 weeks of age. Mice were first exposed to 2Ri cigarette smoke or sham treated at 3-10 weeksL of age. Smoke exposure conditions were described previously (see CTR-101A). The data indicate that the rate of 219 CTR CaHTRRCTS 029415 11249165 CTR I-IN ~'44 22C.,14

Final Report Contract: CTR-0030 CTR-1018 oocyte depletion was not influenced by exposure to 2R1 cigarette smoke compared to sham exposure or untreated controls (compare Fiqures 30 and 40). c. Effect of BaP Given IT on Atresia Or. Mattison has previously demonstrated a dose response for oocyte destruction by BaP given IP. These results were compared to BaP given IT to determine whether chemicals given via the lung could exert any effect on the ovary and be toxic to oocytes. Pour weekly doses of 1.2 mq (BaP) were given IT to groups of BC3F1/Cum mice (1.2, 2.4, 3.6, or 4.8 mq/mouse. Nica were sacrificed at weekly intervals, one week post BaP treatment. No effect of BsP.was detected on the number of oocytes in any of the treated mice. These data supported data from previous expnriments in that the BC3F1 heterozygote :nouse may be more resistant to oocyte destruction than the inbred parental strains (CS781/Cum or C3H/Anf Cum) (21). 220 CTR cONTRRCTS 029416 11249166 CTR MN 04-4,220~'~

F:nal Rapor_ Contract: CTR-0030 CTR-1018 TABLES 97-139 221 CTR coNTRACTS 029417 11249167 CTR HN 044206

TABLE 97 flnd) Report MUNOERS Of OClf1/CtM fENAIE 11M0 WttE MI[E 011NG Of 1ARIOl/S C~1uSES Contract: CTR-003U DWIIRC IIIE ftRSt 1tA0 of tfpoSl/RE 10 211 cIGREnt StA[Ea.,.c ~nw ~ wr- Irt(RS A11tR fIRSI ftYOSWIEd Yescr/vtlon of Oeeth SEM 11 f.t•,hosur. Sea 4 • 12 16 20 21 is It 161f10 44 10 S2 56 lot.is Iblder r+lated d.atbsO 2R1 S.ot. Q 0 0 2 0• 0 0 0 0 1 0 0 0 0 0 l 2R1 S.ots ~' 2 0 0 2 1 0 / 0 0 0 6 SMa. 1' I 0 • 0 • 0 0 0 0 Ibdol.t .pened too 2111 Sowte v 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 ~.pldll/4.pp+d/ 20) S.ot. I' 0 0 • 0 • 0 0 0 0 0 0 SAa. - 1' • 0 f/ •• 0 0 0 0 • 0 Docweented l.pruper 2R1 Sw1. Q 0 0 0 16 0 0 0 0 0 0 0 0 0 0 16 rr.t./.Ir fl.w 201 SarA. 1' 0 0 fl • 0 • 0 0 0 0 0 Slu. j' 0 0 • • 0 0 1 0 • 0 4 E.pwtwre r.l.t.d 2R1 S.ots ~ 0 0 1 1 4 1 2 1 1 0 ] 0 0 2 16 OealR.! 221 ~s l' ! 2 1 6 1 1 6 l 6 9 10 N S ha. ~ 2 1 • • 0 0 0 l 0 • 6 A t a [ase rel.ted deaths 221 Srota ( 1 0 0 0 0 0 0 0 0 1 0 0 0 0 2 _ 211 S.ot. ; 1 0 fl fl • f/ f/ 0 0 0' 1 O) 01) Shes J • 0 fl 0• • 0• 0 0 0 fompd dead 1n ca0. 221 S.ok. 1 0 0 1 0 0 r 1 0 0 0 0 0 1 1 2111 h ts 2 fi • 1 0 1 f/ 1 0 0 II ~ +. Sha. ~ fl 0 0 0 0• 0 0 0 • 0 0 /6urltwod, tlll.d 2R1 S.uk. 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 2R1 Sn.l. 0 0 0 • 0 0 0 ~ c.ll.t+or.tlo. Oot/.etry 211 S.A. y 0 0 0 0 1S 0 10 2 6 0 22 35 0 0 9S , 2R1 Swds ~ ' 0 0 0 0 0 0 0 0 0 0 0 Sh.. j • 0 • 1 0 0 0 0 0 0 1 ` l 2R1 Saot. 2 0 3 10 19 1 12 4 6 1 30 h ]S 0 lh 176 20) Sewi. ~ 13 1 o 2 0 6 4 6 S h 6 6 U1 - - -- -- Sh.. ? L .~.r--1 , aS.u1. .apetwre c.nflslf of IOi 241 clS.rett. s.ote: IS tecendt of s.ole alternat/ny w/th IS teconsis of alr for a perl.d of 0-IO .leotet (0-10 plfft/ehar.lt.). Se.er/eeA such e.pot.res wen $/e.e each d.y rltlrout a reet period Mlw.on e.p.e.ra. 1'1el.l nw.6.r .f .al.alf: 291 s.o1e ~- IIO; 2R1 frole a~ 160; SI..r d-]0. cS.ele eapted /c..lef pet no test 1-1-00; trqk• .apted sales put .l test S-S-bO; sh.s e.poted put on test S-S-Ou. dilec;c .fter first e.posrr. are represeoted 4r the laft week (n th. Inter..i I.e.. 4 represent weeks 0-1; 0 re- pretents weeks S-0 etc. eC..fse .f iea16 /aclod. '1.praper loadlaf' t.swlt/o0 /. .on-a1111wert of ta+ .oie wllh tla wud.le (fwffecatlon) aod .la. etrotyllel (a the hold.rf or Iwlttl.! (. tlt. Uold.re reawlilny /o brete. .ecta. fDal.pel wll.nctlo.. K.riet of death (nclu.le rlte fornd dead .o tMe .ndwle (pefore. dwrla9. or .fter .eposwre), or found de.J In tLe 4olsler or c.ee wlthln ai. hawr aftur ..Iewre. hI

i Finjl Report ~:ntract: CTR-0030 TABLE 98 111ST0PAT1/OLOGY SUMMARY IN 8C3F1/CUy MALE MICE AFTER CHRONIC CONTINUOUS EXPOSURE TO 2P.1 CIGARETTE SMOKEa WEEKS AFTER FIRST EXPOSUREC NUHBER OF ANIMALSb 4 8 12 16 20 24 28 32 36 40 44 48 52 56 Totals Number with necropsy 14 13 3 8 2 5 6 4 6 5 d 66 Number diagnosed gnoscd 14 13' 3 8 2 5 6 4 6 5 66 DIAGNOSIS -~ N Negative 6 5 1 1 0 0 0 1 0 0 14 A tD ~ J rn ---~ PANA 0 0 1 7 2 5 6 3 6 5 35 All o 0 1 0 0 0 0 0 0 0 1 ANCM 0 0 0 0 0 0 0 0 0 0 0 ACN 0 0 0 0 0 0 0 0 0 0 0 n n MC 0 0 0 0 0 0 0 0 0 0 0 "4 0 N N W ASC 0.0 0 0 0 0 0 0 0 0 0 SM 0 0 0 0 0 0 0 0 0 0 0 rY .~ SN 0 0 0 0 0 0 0 0 0 0 0 . ~ SCC 0 0 0 0 0 0 0 0 0 0 0 33 Congestion 7 7 0 0 0 0 0 0 0 0 14 ~. •-~ Peribronchtolitls 1 0 0 0 0 0 0 0 0 0 1 eost mortem autolysis • 0 1 0 0 0 0 0 0 0 0 1 U1 Interstitial pneumonta 0 1 0 0 0 0 0 0 0 0 1 ~ 0 Peribronchial lymphold infiltratlon 0 0 1 0 0 0 0 0 0 0 1 W d Ilo further histopathology was performed. aSmuke exposure consists of 10% 2R1 cigarette smoke; 15 seconds of smoke alternating with 45 seconds of air for a period of 8-10 minutes (8-10 puffs/cigarette). Seventeen such exposures were given each day without a rest period between exposures. 4The number of animals put on test was 150 o'on 5-5-80. cRepresents weeks 0-4; S-6; 9-12, etc.

I I I I final Report Contrdct: CTR-0030 I I I TABLE 99 HiSTOPATHOLOGY SUMMARY IN 8C3F1/CUM MALE MICE AfTER CHRONIC CONTINUOUS SHAM EXPOSUREa i YEEKS AFTER FIRST EXPOSURE NUMBER OF RNIMALSb 4 8 12 16 20 24 28 32 36 40 44 48 52, 56 Totals Number with necropsy 2 1 0 0 0 0 4 3 0 0 10 Number diagnosed 2 1 0 0 0 0 4c - - - 7 DIAGNOSIS J ...a N Negatire 2 0 0 0 0 0 1 3 A u7 J V n  PAMA AH 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 7D ANCN 0 0 0 0 0 0 0 0 ACN 0 0 0 0 0 0 0 0 l 1 N N AAC 0 0 0 0 0 0' 0 0 CJ P ASC 0 0 0 0 0 0 0 0 ~ SM 0 0 0 0 0 0 0 0 SN 0 0 0 0 0 0 0 0 ."~ SCC 0 0 0 0 0 0 0 0 70 ~ Congestion 0 1 0 0 0 0 3 .4 ~ ~ aSham exposure consists of 136 minutes on the sham exposure machine. U1 hThe number of animals put on test was 30 d'on 5-5-80. O N .A h) O cNo further histopathology was performed.

I 1! I.: ~ I Final Report Contract: CTR-0030 TABLE 100 I I I ! I ! ! ; ' !-^ r ~ IIISTOPATIIOLOGY SUMMARY IN 8C3F1/CUM FEMALE MICE AFTER CHRONIC CONTINUOUS EXPOSURE TO 2R1 CIGARETTE SMOKEa WEEKS AFTER FIRST EXPOSUREC NUMBER OF ANIMALSb 4 8 12 16 20 24 28 32 36 40 44 48 52 56 Totals Number with necr6psy 2 0 3 18 4 1 2 2 2 1 3 0 0 2 40 Number diagnosed 2 0 3 18 4 1 2 2 2 1 3 0 0 2 40 ~ DIAGNOSIS ...~ N Negative 0 0 0 1 0 0 0 0 0 0 0 0 0 0 1 A n PAMA 0 0 3 17 4 1 2 2 2 1 3 0 0 2 37 V -I AN 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 ANCN 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 ACN 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 i I t A ~'.1 ~ ~ N N MC 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 5..+/ f'-, N ASC 0 0 0• 0 0 0 0 0 0 0 0 0 0 0 0 i SM 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 ~ -- J SN 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 SCC 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 ~ ?U M Congestion 2 0 0 0 0 0 1 0 1 0 0 0 0 0 4 ~ 0 . Peribronchial lymph. inflltration 0 0 0 0 0 0 1 1 0 0 0 0 0 0 2 Perivascular lymph. Infiltration 0 0' 0 0 0 0 1 0 0 0 0 0 0 0 1 ln Lyophoid Infiltration 0 0 0 0 0 0 0 0 0 1 3 0 0 0 4 aSaoke exposure consists of 10% 2R1 cigarette smoke: 15 seconds of smoke alternating with 45 seconds of air for a period of B-10 ainutes (8-10 puffs/cigarette). Seventeen such exposures were given each day without a rest period between exposures. ' bThe number of animals put on test was 180 g on 1-7-80. cRepresents weeks 0-4; 5-8. 9-12. etc.

fln.l Rryurt Contr.ct: CIR-0030 lABIE 101 DEfOSIIIUN ANU DISIkIbUIIOM Uf (UTAt I/C•UIC (W91) IN 0C3f1/CUM fltWt NICE AfiER ExP0Su11E 10 2R1 CIGWT1l N101cE GENENAIlO IN SIN, tK11k: IS/IS SECOND E11P05uRE CYCEE, 8 fuFFS/CIGAREflE a IUTAt SNOKl EtYOSUkE N0. Of 1601 i1NE (SECONDS) CIGAREITES txxrN[ 120 1 a• 9.18 (cv 0.8]) 240 2 a- 21.59 (cv 0.69) 360 . 7 a• 21.89 (cv 0.75) 400 4 - 26.85 (cv 0.37) 600 S a- 32.99 (cv 0.41) 120 6 a- 35.23 (cv 0.65) 840 1 a- ]9.56 (cv 0.49) 960 IS a- S1.58 (cv 0.46) 9G0 8 ttnaaoytea - 16.24 (cv 0.93) /ACKGROU100c 4 CUtD C16S. fREAWIlEO NICE a- 11.52 (cv 0.11) UNAOAr1E0 NICE a- 51.9 (cv 0.09) iMC11EA a- 9.91 (cv 0.52) x- 14.18 (cr 0.58) x- 30.56 (cv 0.59) x- 41.20 (cv 0.62) a- 51.11 (ct 0.59) x- SS.S1 (c. 0.17) a- 39.41 (cv 0.69) a- 11.83 (c. 0.6s) a-125.2 (cv 0.49) a• 255.9) (cv 0.39) a- 630.99 (cv 0.21) a• 900.35 (CV 0.39) a-11/1.41 (cv 0.21) a•1161.95 (cv 0.21) a-16t1.01 (cv 0.30) x-1111.99 (cv 0.26) a-2212.59 (cv 0.21) a-3263.19 (cv 0.50) a- 51.6 (cv 0.06) , a-'41.6 (cv, 0.14) a- S1.8 (cv 0.19) x- $0./ (cv 0.15) a- SL S(cv 0.06) a- 49.6 (cv 0.14) a- 54.5 (cv 0.06) a- Y1.21 (cv 0.09) IURe11ut1E a- 11.54 (cv 0.41) a• 61.30 (cv 0.14) a- 19.]1 (cv 0.61) a- 97.21 (cv 0.34) r-1S0.3S (cv 0.60) a-111.19 (cv 0.25) x•133.85 (cv 0.42) a-353.19 (cv 0.62) a-212.48 (cv 0.31) S101NCN a- 33.88 (cv 0.34) a-111.48 (cv 0.27) x-170.81 (cv 0.21) a-261.35 (cv 0.53) x-318.61 (cv 0.11) a•M.83 (cv 0.25) a-112.31 (cv 0.22) a-650.80 (cv 0.SS1 a-S36.36 (cv 0.30) t1ttG a E19At 1ce per tlre po/nt were e.poteJ to 101 221 clg.rette snu*e coatalning 14C-OlC for 120 seconds (8 a 16 tcconds)per clgarette, r/th no rest between. N1ce were Prcvlouslr e.poseJ to smoka eefore the day of tAe Jotlmetry eaperl.ent. ~ Coefflclent of varlatlun. C 8utyround valuet:

Final Report - Contract: CTR-0030 TAdIE 102 SUMMARY OF TPM AND 14C-7TC '!EASUREMENTS FOR THE CTR 119-5 uOSIaETRY EXPERIMENT OPTICAL SENSORa CAMBRIDGE FILTER SAMPLESb "RUN" `IU"i$ER DIGITAL VALUE ' (my) TOTAL.TPM GENERATED (mg) 14 C-DTC (dpm) c NICOTINE (mg) CALCULaTEDc SP CIF:C ACTI'/ITY (1~C-DTC-OP*i/Tg TPM) 0 3482 174.1 - 0.40 - 1 3138 159.9 92,108 (0.11)e 0.50 57.6 2 3335 166.8 112,205 (0.14) 0.50 57.2 3 3506 175.3 137,802 (0.02) 0.60 78.7 4 3667 183.4 138,574 (0.02) 0.52 75.7 5 3854 192.7 115,042 (0.03) 0.45 :9.6• 6 3798 189.9 106,446 (0.11) 0.45 56.0 7 3762 188.1 105,138 (0:02) 0.50 55.9 8 3707 185.4 123.060 (0.03) 0.40 66.5 1-8 32,249 1440.1 699,990 (0.01) 1.42 57.0 a Optical sensor placed directly In snioke stream and monitored the total Tr.4 generated. Conversion factor of 0.05 used to convert digital readout to mg/T74. yFilter samples taken at 100 cc/minute or 1% of the TPM was withdrawn on the Ciau„brid;e filter. c Calculated from the total 14C-DTC dpm eluted from the filter and 1/100 of the :otal T?M generated. d vic:.t'ne was determined by gas chromatography after elution of T?M frcm '!tter. - C.-efficient of variation. 227 .CTR CaNTRACTS 029423 11249173 CTR HVI 04' 421:2

Final Rsoort Cantract: C'x-=0 TABLE 103 DISiOSITiCt OF ANINAII FOLLCUINO C3lROKIC CCNTINUOUS EI70SURE TO 3A1 0R 2A1 CIGARtTft SnOUt (Cr!•ilfl a•0 NUMlQ CN TE3T 3A1 SIOrOt a60 2R1 S?QRt 360 SIWI 440 UNC(FOSED 1S6 EXPOSUAt AELATED ACCWT21. CfATNS' 30 3 12 • EXPOS1Dtt RELATED CF.1TNSd 49 SO 41 - OEJtD IN CAGE; !CRI8t910, KII:FO 6 10 13 0 0TW CAUSES 12 9 18 9 T0: OR. SQIEDICT • SISTEt C'StOlIATID 6 4 8 8 EXCWlAf+E T0: 0R. OINOYIIZ - 00C/1U01 ASSAT 60 60 60 18 T0: 0R. NSIANG - OOSIrFiRT 65 2 0 0 TO: 0R. c.tNAGALLVGAII • ulrJUt0I0GRNIR 40 40 43 32 TO: 0R. RABA6AL1NtJW - iI0C10:NiSTRY 24 Z4 14 20 TO: 0R. r.tNAGALIyGtM • El_-~Otl MICROSCOFT 12 12 12 8 T0: 0R. 6::8ET • 1UCALINE •C1A EIIRIGI 19 22 33 4 T0: 0R. l'.3ET • oLJIQUi FCxM2•'10 CELL ASSAT 11 11 tt 2 T0: OR. 'Ul17tsJ1 • ATtESIJ< 0 1S 1S 0 T7: 0R. ztStUSSCI •:NA tE7AIR 12 12 12 3 t07AL nWntR FUT :+t TLSTa a80 360 "0 156 TDTAL !ItMER RE+OYm FRCx T1iST 360 274 302 104 TOTAL ,vtalOl ALIVE CN 8/1/81 120 86 138 S2 a :andlttons for saocs uroosure: 101 3A1 cigarette a.ot• or 10% 2R1 ctqaracte uwte; 17 ctgar.cus oar aar; 1•10 ouffs oar ctgarettat IS seconds of sreaa altarn.ttna rtch 45 seconds of atr, contlnuous uroosure (no rest oarnods) vroqrsa 1 on cna SEJ, II. Shas a:oosurs consisted of 136 .tnuces on tne Sham ucoosun mcnlnM. Slnu Junt. 1;80. :+coari:,.nt CTt•11t. :wtns fnciud. 'laoreoar laading• ntulttnq In non-.I/gneent of the iose with the .oaula causing suftocatton. broken nea:s aua to .1c• scrupgl/ng against tNe holder er trlstlng in the nolder. dasnoots' malfunctlons. dacuaentad deatHs due to Loreoar s®ta or air f1ar. OaeNS Includ• .tce found dead an ti+a mdule (before. during or after ucposun), and ./ce fovnd dead In tlut holder or cage within one hour aootur.. Sar+as I on test 6/02/80 • 160 3A1: 180 iAt; 130 shaa; 56 unuoosed. Ser1n 2 an cast l/1t/80 • 120 3A1: 80 fhaw. Serlas 3 on te:t S/11/80 ! 180 3A1: 180 2R1; 180 sha.= 100 unasooted. b c 0 0 228 c.TR coNTRacTS 029424 11249174 C ~'i`~ f IN 0442 1~.."~

Flnel Raoart Cantrte:: CTR-0030 TAitl 104 OISiOSITTCUI Of AGE•MATCMED MAI.E A110 F[NAL[ OC7F1/C171 MIC[ FOLLOMINO E7VOStUIE TO Zll1 01 3A1 C:ZUttTE S110Rt (CTR-131) A. EJIiE1IINENTAL GxOUPS A1t0 tNMiETt OF 1Ull[ ANO FLMALt IC3f1/CtAI MICE SCNEDUIED F011_ CNAOMIC CONT1Nl10ltS EIPOSCiE TO OETEANINE SNOET-TEi3( t.rOrotMTS. ^..+~,g I r:po:un T/,. Oau For A11ar1 Ses _?At _ 2t1 Shiw She11 I 6 aoetns a/1E/el K so so So ts F so 54 6o 20 _ tt 3 eoetAS U0t/11 !I aS js tS IS F as 4s .s is III 3 weets 1/12/E1 4 iS ts tS 1S F 41 43 as is IY 7 dars 2JIf/11 4 I 4S aS aS tS 1 45 1s 45 is v I dar 3/17/!1 + aS as tS is F aS as 45 IS S. JISFOSIT:071 OF AMIMALS FER TIN[-iOINT F;ll0i1ING E1POStAI[ (5 TtNE POINTS) 'O.olect I Ses fA1 ?t1 Sht4 Shel1 C+u Ileoatr 4 3 3 3 0 F 3 3 3 0 ANM/00C M iS is is A f is 1S li t crfye. M s e a 0 s~ror.~e+o~ F s s e o lacellno !1 to 10 t0 6 Indas. F to to to S tw+o/Iiodr Yaqnt Ratlef. MydA=fOr'e11ne Content 229 cTR caNTRacTS 029425 11249175 CTR HN 0442214

ftnel Report Contract: C1R-0010 lAllE 105 ODS AAD ONA REPEICATTON IN LUNG TISSUE IN YITRO fROM 11C3f1/CUM IttCE EIU'OSEO 10 2A1 CIGARfTiE SNDRE a N w O ]1M dpo/e9 DKA CINA AT 1 YE EL'OSIME UuS IMIA REPtICAT1YE SYM/IIESIS MEEI: IUMO ~ SMOSf-EltPOSED SIUN-EIPOSEO SIIOr.E: SIWI SIIOKE-E1P0SED SIYIH-EIPOSED SMORF: SWW 1 0.5 2S1 s 6E 109 i 17 1.34 1ele a 61S 622 a 200 2.95 4 6.7 eA t 12 101 t 20 0.86 927 e 160 369 1 102 2.51 6 14.6 12] 1 28 138 1 IS 0.09 963 t 127 SS6 a 91 1.7) • 26.4 9/ a 4S US 1 30 0.75 1240 t 13 Sei a 116 2.13 11 45.2 101 t 22 176 1 32 0.57 13W s 112 731 1 202 1.16 Is 70.2 e9 a IS 1]1 t 72 0.6e M7 s 9z 10e2 1 226 1.14 11 $2.1 119 c 23 2S4 s 19 0.47 1512 1 46 699 e 76S 2.19 e Ass.ys were done .s described In the te.t. DNA da.age w.s induced by 2. 10 S. 4NQO. Y.lues .re the pooled .e.ns from triplicate weesure.eets •f ).Ice a Standard Devletto. (SO). (R.smussen et .1., 19tl1). b IPM dose estl.ated froi qu.ntlt.tlve doslwetry resulls. t ~ I

iIn.l R.port tMtr.ct: CiR-0030 TAGEE 106 uDS IN Sltlltf-E1P0SE0 OClfl/CtN lr]uSE IuNG IREATED IN 91TR0 MITN 10 S N 4NQO AfTER CESSATIOM 0F EtfOSURE 10 S110tE E1pERI1lCM1Al 6NO11p a N A t0 ~ ~ SMORE SIINf V '.~ V ~ N SIl1Kf W r S11N1 l J C1MJU111vE ElPOSuMle . 'M dl./uy LWA MEEK es)/1PN LtthG II/E S1NCE EAST StIDKE OR SIWI IAS 0NA REIIIGITION l5 195.2 S llonths 11~ 411b 7S - i lb.tAt 1]6~ 40/° 3S 195.2 5 IfootAs, 2 Mee1s 11) 1]1C SSI i 69c 35 - S Noethf. ! Yeett Ifel t lSc 6]7 1 9]c a TP/1 dose estleatcJ fros qu.ntlt.tlve doslmetry. (R.s.usten et .1., 1981). b E.cA v.lu. Eor u0S .ad uMA r.pllc.tlon Is the .v.r.ye frc. 2.Ic.. c[.c0 v.lw Is the e+e.a i St.nA.ra Devl.llon (SO) (ro. ].lu assayed. r. ~

Final Report Contract: CTR-0030 TABLE 107 DISPOSITION OF 8C3F1/CUM MICE AFTER EXPOSURE TO illGll LEVELS OF 2A1,','3A1, Olt 2R1 CIGARETTE SMOKEa z ORIGINAI. NUMOER OF MICE WEEKS 0N TEST CIGARETTE MUMBER OF ' TYPE MICE DISPOSITIOtI 4 8 12 16 20 24 28 32 36 40 42b 2A1 120 Scheduled Sacrifices 0 21 21 0 0 5 0 0 0 0 5 Other Deaths 7 5 6 3 2 3 3 1 5 1 0 Alive 113 87 60 57 55 47 44 43 38 37 32 3A1 120 Scheduled Sacrifices 0 21 21 0 0 5 0 0 0 0 5 Other Deaths 2 6 1 8 6 11 5 1 '2 1 0 Alive 118 91 69 61 55 39 34 33 31 30 25 W 2R1 120 Scheduled Sacrifices 0 21 21 0 0 5 0 0 0 0 5 Other Deaths• 4 S 1 4 12 7 3 3 3 0 0 Alive 116 90 68 64 52 40 37 34 31 31 26 Sham 120 Scheduled Sacrifices 0 21 21 0 0 0 5 0 0 0 5 Other Deaths 4 3 2 0 5 0 2 2 1 1 2 Alive 116 92 69 69 64 59 57 55 51 53 46 Shelf 50 Scheduled Sacrifices 0 8 8 0 0 2 0 0 0 0 5 Other Deaths 0 0 0 0 0 0 0 0 0 0 0 Alive 50 42 34 34 34 32 32 32 32 32 27 a L Exposure conditions on the SEM II were 10% smoke concentratlon, 8 puffs/cigarette, elther 30/30 seconds smoke/air for 8 uinutes (2A1 and 3A1 cigarettes or 20/40 seconds seioke/atr for 8 minutes. five such exposures with 10 minutes of air between exposures, were given In the morning and five in the afternoon. Sham exposed animals were treated the same, but without smoke. Siauke and sfiam exposure stopped due to the initlition of CTR101-U (CTR-118), which required all SEM 11 B and C nachtne time.

final Iteport Contract: CTR-0030 TABLE 108 CTR-96F. INCIDENCE AND DISTRIBUTION OF PIGMENTED ALVEOLAR MACROPIIAGE ACCUHULATION AFTER EXPOSURE TO 2R1, 2A1, or 3A1 CIGARETTE iMOKE a - ~ 0 Z. Weeks on Test Ci9urette Type 4 8 12 16 20 24 28 Total - - 2R1 - 0/1 2/2 4/5 9/14 4/5 3/3 22/30 2A1 - 0/2 6/6 2/2 2/2 1/1 3/3 14~16 .~ 3A1 - 1/2 4/4 6/7 13/14 3/3 28/30 OSmoke exposure consisted of 10% smoke and B puffs/clgarette for all three cigarette types.. for 2A1 and 3A1 !?~ n clgarettes, exposure times of 30 seconds per lnute for 8 consecutive minutes were used. For 2R1 clgafettes, "~ exposure tlmes of 20 seconds per minute for 8 consecutive alnutes were used. Flve such exposures were given ..~„ ln (n the morning and repeated ayaln In the afternoon. N O LD .P tn N .~ ~ r\.) 5-4 CD

I 1-4 flnal Report IAOCE 109 Coetracts CIR-00]0 UNSltd1AA1D (UUS) ANO MCPIICATIY[ (RIK) UNA SYNIII[SIS ININRIO III v118U IN ItUN: StIl.11 OOIAUtCD fM011 8Qf1/CUtl Nltf CIIPUSEO 10 Sf10RE 110" 3 RIfEREnCE CIGARCTfCSA CIWUtE11E 11PE 2A1 111 0 WS IMUS f • 'N-UPN/ g OMAc 3 11-01N/ 11 ONAd MttICS 0f EtPOSURf CUUTATIY[ iPN OEPOSItE06 (ay) SMUItF S1tAn- SItORE /SN/ly SMllltE- sIIAN- S.10R[/sNN1 t 1e t t) 9 47.8 425 t 65 412 a 47 1.03 1907 a 81t 171s a 114 4.58 0.80 13 64.4 236 a 22 430 a 42 0.55 3204 a 132 1121 a 241 1.91 0.82 24 105.2 460 a]2 620 /10) 0.14 876] a 482 243) a 259 7.60 41 214.4 438 / 69 539 a 59 0.01 8688 t 1218 2618 t]43 3.32 9 51.1 786 a 61 412 a 47 0.94 7216 a 550 1125 t 114 4.18 0.91 13 71.2 221 t 21 430 a 42 0.51 4141 t 134 1721 a 241 , 2.16 0.81 24 116.2 571 a 58 620 t10] 0.92 8650 t S00 2411 a 2S9 7.56 41 211.6 391 t 117 Sl9 a 59 0.14 1192 a t010 2618 t34] 2.75 9 25.8 $)1 a 62 412 a 47 1.29 3770 a 2l6 1125 1 114 1.95 0.29 a] 36.4 47S a3l 410 t 42 1.01 4112 a 707 1721 e 241 2.42 0.20 24 66.6 592 a1)S 620 e10] 0.95 4841 t32S 2433 a 2S9 1.99 11 174.2 Sl2 a 81 $19 a S9 0.99 6151 a 459 2618 a34) 1.91 Anl.rls were e.posed to 1N s.oke and 8 pufft/cl9arettes for all three clgarette types. for 2A1 and ]A1 clgarettes, eapsure tl.e of 30 secunds per .Inute for 8 consecutive ./wles were used. for 2A1 clgarette e.posure, tl.es of 20 seconds per .larte for 8 consecutive .Inutes were .sed. flve sYcA e.posures were glven /a the .orniny and repeated a9aln In the afternoon. UOS and ONA repllc.tloe atsayt were perforred by Or. R. Rasmussen. s Deposition deteiIned fro dosl.etry results and daily s.oke records. C UDS at determined after treatment of lung sllces with .ethyl.ethane suilonate /• the presence and absence of hydroarurea. DNA replication ras determined frum lung sllces incubated I 11-thy./dtne (3 11-1dR). Mlc: were Injected with 100 uCl 3N-fdR le.rcd/alely after treat.ent and sacrif/ced 30-60 .Inutes later. 1.1 /s perceet o/ a11-idR labelled cells dlvlded sf the total n+.oer of cells coaeled a 100i. Approl..tely 2500 cells from 18-20 randu.ly selected flelds were counled. SAa. and thelf controls had EI valres of 0.19 - 0.44. 4 e . ~~(

Final Report Controct: CIIi-UU311 TAULE 110 UNSCIIEUULEU DNA SYNTIIESIS (UDS) AND REPLICATIYE..ANA SYNTHESIS (RDS) IN VITRO IN LUNG TISSUE FROM BC3FI/CUM FEMALE MICE EXPOSED 10 3A1 and 2R1 CIGARETTE SMOKE a•b l J 0 r!+~ 3:) ..v. n (f) ~ 0 ~ rw. ., w , 311-DPH PER NICROGRAH DNA _ E XPOSURE 3A1 2R1 SHAM SHELF TIME LIDS RDS UDS ItUS UUS RDS UDS __ RDS 3 Munths 378 • 23 4151 t 184 309 # 36 3250 • 94 406 • 65 1959 * 130 499 & 42 20511 • 251 6 Months 375 • 29 3437 • 199 406 # 3U 3633 • 216 456 • 30 1744 ~ 112 N.U . C 9 Months 246 • 32 2651 • 267 272 • 26 2969 t 170 311 * 18 1543 * 228 N.D. N 12 Months 216 + 23 1896 • 247 228 • 17 1930 t 276 256 • 20 1095 t 237 N.D. W Ln a Uptake of 3H-TdR Into DNA was measured. Each value represents the mean of data from 3 mice, each of which was carried out in triplicate. UDS was Induced by methyl methane sulfonate (MMS) In the presence of hydroxyurea (11U). RDS was measured in the absence of HNS and IIU. b C118. Exposure conditions were 10% 3A1 or 2R1 cigarette smoking using the continuous reginen of 17 "runs", 8 puffs/cigarette. c Not determined.

Final Report Contract: CTR-0030 TABLE 111 UNSCHc']UL:O 1NA SYNTHESIS (UOS) AND RE?LIC.ITIYE V1A SYNTHESIS (ROS) :N YITRO IN LUNG TISSUE FROM SC3F1/CUN MALE 4ICE EXPOSED TO 3A1 AND 2R1 CIGARETTE SMOKEa EXPOSURE SMOKE 3H-0Py PER MICROGRAM OyAh TI4E _XPOSUR= DNA REPLICATICN CNA REPAIR 1 Day 3A1 1801 ± 775 373 t 18 2R1 2380 ± 883 359 ± 88 Sham 719 ± 31 326 ± 30 1'aeek 3a1 2544 ± 337 311 ± 57 211 3636 ± 585 301 ± 1.4 Sham 1057 ±.285 270 1 18 3'aeeks 3A1 4928 ± 325 368 t 32 2R1 4214 ! 921 402 ± 3 Sham 1486 ± 251 414 ! 31 3 Months 3A1 2384 ± 473 235 ± 10 2R1 2000 ± 187 234 ± 10 Sham 980 ± 1S2 263 ±. 21 6 Months 3A1 2842 i 578 316 = 28 2R1 1904 ±.303 237 ± 8 Sham 941 ± 415 215 ± 54 a b Exposure conditions were 10: 3A1 or 2R1 cigarette smoke using the continuous regim,en of 17 "runs", 8 puffs/cigarette. Uptake of 3i-TdR Into DNA was measured. Each value represents the cean of data from 3nice3 each of which was carried out in triolica:e. UOS was Induced by methyl methane sulfonate (?^iS) in the presence of hydroxyurea (HU). 20S was measured In the absence of `"HS and HU. 236 CTR CQNTRRCTS 029432 11249182 CTR VIN 04 4 2r2' 1

Final Report Contract: ETR-0030 TaBL: I12 UNSCHEOULEO DNA SY4THESIS (UOS) ::10 REPLICaTIVE ONA SYNTHESIS (?CS) IN VITRQ IN LUNG TISSUE FRC!i BC3F1/CUM FEMALE MICE EXPOSED TO 3A1 and ZR1 CIGARETTE SMOKE a EXPOSURE Ti4E SMOKE EXPOSUR E 3H-4PM PER MICROGRAy O4Ab CNA R:?!ICaTION DNA REPAIR 1 Day 3A1 2937 +_ 341 361 ± 24 ZR1 2633 _• 929 408 ± 59 Sham 960 •_ 230 339 ± 15 1'deek 3A1 1998 ± 946 336 ± 54 2R1 2712 1 821 297 ± 34 Sham 1611 = 535 271 ± 43 3 Weeks 3A1 3916 = 1053 323 ± 28 2R1 3523 = 1095 393 « S5 Sham 1384 ± 328 374 + 64 3 Months 3A1 2400 + 494 263 + 22 2R1 2393 + 322 262 + 25 Sham 1273 + 88 249 + 41 6 Months 3A1 2070 _ 281 132 + 70 2RI 990 + 21 167 + 52 Sham 1123 T 338 332 + 33 a Exoosure conditions were 10: 3A1 or 2R1 cigarette smoke using th_ continuous regimen of 17 "runs", 8 puffs/cigarette. Uptake of 3H-TdR into ONA was rreasured. Each value represents :ie -ean of data from 3 rice, each of whicA was carried out in trioli:a:e. UJS 4as Induced by methyl methane sulfcnate (MMS) in the presence .• hydroxyurea (HU). ROS was measured in the absence of MMS and 4U. 237 'CTR CQNTRACTS 029433 11249183 CTR HP-1 044~"2-*.2-~

Final Report Contract: CTR-0030 TAaIE 113.... EFFECT OF INTRATRACHEAL DOSE OF 8AP ON DNA REPAIR Ct,PACITY OF BC3F1/CUM MICEa ;REATMENT TINE OF ASSAY (WEEKS) 3 CNA REPAIR .X-DPM/MICROGRAM DNA 3 ON A SYNrr!E SIS H-OPM/MICROGR.IM CyA SAP 1 104 t 18b 798 t 61 Gelatin-Saline 1 33 t 17 879 t 32 Untreated 1 114 t 4S 769 =40 BAP 10 36 = 9 540 -:7 Gelatin-Saline 10 45 : 10 505 - 36 Untreated 10 54 t 9 591 : 21 a b Mice were treated with 1.2 rg 8aP/0.02 ml gelatin-saline by intratracheal inoculation once per week for a total of 3 weeks. One week after the last trea cr.ent, riice were shipped to Or. Rasmussen and assayed, as previously described. Standard Ceviation. 238 CTR CQNTf2RCTS 029434 11249184 CTR I-IN 0442,223

Final Report Contract: CTR=0030 TABLE I:4 L.`.SE : :.i`.iG INDEX (t L.13E:.LE:. C.:L L.S) CB SEe'tVrD i:f T8E 1--uG OF BC3F1/C4a FESSALE !iIC : EX?OSED TO eriiCL.E C:G:u^,,; :':Z S:SCSF::- cOR 58 h%tiCS FOLL04.i,.D BY TFZE.1TaE:IT NITS SE.*IZO (a) PYitE2IE 7, 8-DIOL 02i DAY 0 AND DAY 7a DayO Dav7 rxposure Grou: 3a?-7;8=dio1 Vehicle Vehicle Control__3aP-7,8-dioL Control 3a.1 0.93 0.83 1.03 0.10 (_0.10) (±0.31) (±0.37) (±0.003) 2R1 0.73 0.97 1.12 0.85 (±0.13 (±0.28) (+0.39) (+0.20) Shaas 0.56 0.36 0.41 0.25 (±0.15) (±0.12) (±0.23) (±0.09) Shel- 0.48 0.10 0.25 0.08 (+0.13) (+0.10) (+0.05) (+0.05) HaP-7,8-diol or vehicle (gel/saline) was instilled i._. i=adiately followinq final ssioka exposure, or 7 days after cessitioa of saoke exposure. :n !;oth exFeri3ents H-thj=idiae was administered i.a. within 20 min.of BaP-7,8-diol. The animals were sacrificed 4 T.ours altar eaP-7,8-diol adainistrat:on, ::e luag fixed, sectioned and processed auto- radiograpnically. (2) Tha ncmoer of calls lah.llad amanq a total of approx. 4000 total cells counted was determined, and ;.resanted as % of total calls labelled. 239 CTR CQNTRRCTS 029435 11249185 C~` T~' ~wI ~~ 044 :~ ~ ~~

Final Report Contract: CTR-0030 T/,aLE .:: EFFECT OF AGE ON REPAIR CA?AC:if I!{ VARIOUS STRAINS OF FENALE yICEa 5~1Iy AGE WEEKS MEAN +IEIGi{T (GRANS) :NA REPAIR 3r-C:~!/MICROGRAM ONA ONA RE?LICaT:CN 3H-OPM/MICROGRA~`4 C'1A 3C3FI/CL:m 5 21 = 1 96 t 29 934 : 70 109 37 = 12 56 s 10 337 : 25 C3H/Anf Cum 8 y•D•b 60 = 12 1017 = 51 33 - N-.D. 87 t 17 496 = 20 CBA/2J 8 18 ± 1 69 t 9 694 = 35 41 19 s = 84 = 13 358 = 31 C5731/6 Cum 11 22 t 1 42 s 8 593 = 93 22 21 - 1 33 t 13 456 s 48 a Untreated mice housed at MA were shi;ped to Or. Rasmussen for analysis as previously described. b Not deterTined. 240 C.TR CQNTRACTS 029436 11249186 CTR VIN 044

Final Report Contract: CTR-0030 TABLE 116 L:.Bc'LLI:1G I4CEX ('. LABELLED CELLS) DETEPMINED IN THE L'J.'1G OF BC3F1/CUM F:4AL= HICE EXPOSED TO CIGARETTE St10KE OVER 58 WEEKSa EXPOSURE PERIOD 3A1 2.11 SH/L4 SHELF 3 Weeks 1.15 1.15 13 Weeks 0.38 0.62 1 1 26 Weeks 1.13 0.53 0.23 0.1 6 39 Weeks 1.31 0.81 I ( 58 Weeks 0.83 0.97 ' a (1) For each exposure period 3H-thymidine was administered i.o. immediately foll:aing final smoke exposure, animals sacrificed 1 hour later, and lung fixed, sectioned and processed auto- radiograprtrcally. (2) The number of ce11s labelled among a total of 4000-6000 cells counted was deter.eined, and presented as S of total cells labelled. (3) Data from 4 mice per exposure group (3A1, 2R1 and sham) and 2 shelf control nice were analyzed. 241 CTR CONTRACTS 029437 11249187 C~``~' ~"~;~~ ~'.4-~-k: ~~

Final Report Contract: CTR-C030 TABLE :17 ANALYSIS OF THE DISTRIBUTION OF NUCLEAR GRAINS IN LUNG CELLS OF BC3F1/CUM FEMALE NICr _XPOSED TO CIGARETTE SMOKE FOR 3 yCNT1iSa EXPOSURE GROUP % CELLS WITH "CCUNTABLE" GRAINS/NUCLEUS 5 CELLS WITH TNTC G2AINS/NUCLEUS 3A1 0.67 0.22 (+0.15) (±0.15) 2R1 0.51 0.12 (=0.05) (;0.04) Sham 0.021 0.024 (i0.005) (_0.006) Shelf 0.016 0.033 (10.006) (t0.013) a(1) =or each exposure period 3H-thymidine was administered i.p. i.--edtately followinq last s-+oke exposure. animals sacrificed 1 hour later, and lung processed autoradlographically. (2) ,otal number of cells containing 120 labelled cells -.as selec:ed. The labelled cells were categorized into those with "countable" (usually, approx. 100) gratns,and those where the grain number was Too Numerous to Count (TNTC). These categories are presented as : total calls with the said grain distribution. 242 . CTR CONTRACTS 02943e 11249188 ~ T~` MN 0'•'• ~.~ ~

Final Report Contract: GTR•0030 TADLE 118 ANALYSIS OF TIIE DISTRIQUTION OF NUCLEAR GRAINS IN LUNG CELLS OF UC3F1/CUM FElU1LE MICE EXPOSED TO CIGARETTE SMOKE FOR 6 AflD 9 MONTHS a 6 M01iT11S 9 MONTHS X CELLS X Z CELLS 1 CELLS WITH "COUtIT- CELLS WITH WITH "COUNT- CELLS WITH EXPOSURE GROUP ABLE" GRAINS TNTC GRAINS ABLE" GRAINS TNTC GRAINS 3A1 0.89 0.33 0.88 0.89 (+0.32) (+0.09) (+0.33) (+0.28) 2R1 0.43 0.10 0.69 0.12 (+0.17) (+0.05) (±0.23) (40.08) ~ Ln .w. ~ O N N P w SIIMI 0.20 0.15 0.23 0.11 (10.07) (*0.03) (+0.08) (±0.06) SIIELF 0.11 0.14 0.05 0.22 a Total number of cells containing 120 labelled cells was selected. The labelled cells were categorized fnto those with "countable' (usually, approxiwately 100) grains, and those where the grain number was Too Numberous to Count (TUTC). These categories are presented as S total cells with the said grain distributions. I.

i'1nal Rcpurt Contrdct: C(R-0U3U TABLE 119 A QUALITATIVE ASSESSMENT Of TIIE DEGREE OF LI OOSERVED IN VARIOUS TISSUES OF NICE FOLLOWING A 13-WEEK EXPOSURE TO 2A1, 3A1 AND 2R1 CIGARETIE SIiOKE AND IN SIIAM- ANO SIIELF-CONiROLS.a N ~D l J O ~t N ~ V P ~ ~ 0 N ~ -LO A TISSUE Intestine Exposure Conditions lun9c Trachea Bladder Cry1t V1111 Lfver Kldneyl) Spleen 2A1 t+ t+ + ttt t t + ttt 3A1 *t tt + t+t t t t tt. . 2R1 tt tt t ttt t t t ttt Shelf t t t ttt t t t ttt Shdlp + t + itt +t t t ttt a. The assessment was made on the basis of examination of a Ainiaurm of 5 fields per section at a.w9nlflcatlon of 400m per field. b. Nust of the label was In the cortex. c. t` Lightly labelled - < S labelled cells/field it - Moderately labelled ~ 5-10 labelled cells/field +tt a Heavily labelled - > 10 labelled cells/field

Final Report Contract: CTR-0030 TABLE :20 P!'L`".aNARY ARYL HYOROCAR80N HYOROXYLASE (AHH) ACTIVITY IN 3C3F1/CM F::aALE MICE EXPOSED TO 3A1 OR 2R1 CIGARETTE SNOKE . i:R ::::1G TnE CCNTINUOUS EXPOSURE ZE;:MEY FOR 3, 5, AND 9 MONTHSa A H H U!1 I T Sy LElIG i rl OF SMOKE-EXPOSED EXPOSURE (MONTHS) 3A1 CIGaRETTES 2R1 CIGARETTES SHAM-EXPOSEO 3 146 (0.16)c 142 (0.17) 34 (0.38) 5 228 (0.15) 226 (C.15) 42 (Q-.24) 9 205 (0.20) 234 (0.33) 36 (0.36) a:xposur conditions were lOt 3A1 or 2R1 cigarette smoke, 17 "runs", 3 puffs/cigarette. b Data are from animals sacrificed 6 hours after smoke exposure with :HH uni:s In picomoles7 3-OH-Denzo(a)pyrene formed/mg mi crosa-•al protei n at 37°C. c Cceffic:ent of variation. 245 CTR CONTRRCTS 029441 11249191 • CTR I IN 0442 30

Final Report Contract: CTR-0030 T:+BI_ 121 REtlAL AHH* ACTI'JITY /4 MICE :vRC4ICALLY EXPOSED'TO 3A1 AND 2R1 C:u':.R i i c::".OIC? FOR 9 MONTHS TIh'E AFT"eR EXPOSURE TO LAST CICviRETT"c 3A1* 2R1** MACHINE** CONTROI !/ACHINE*' CONTRCL 3 Hours 15.7 = 3.0 11.9 t 0.8 4.1 : 0.7 3.2 : 1.2 6 Hours 15.7 t 5.5 23.2 = 6.7 4.0 t 1.2 9 Hours 15.5 t 1.3 18.8 t 2.5 4.0 t 0.5 * AHH - picomoles 3•OH-benzo(a)pyreme formed/mg mlcrosomal protein TM!!ean t Standard Deviation of ,'ive mice per time point 246 CTR CONTRRCTS 029442 11249192 CTIR I-Irl 04- 42-~~ 1

final Report Contract: CTR-0030 TABLE 122 PULMONARY ORNITIIINE OECARIlOXYLASt (OUC) ACTIVITY IN RC3F1/CUN FEMALE l11CE EXPUSLU TO 3A1 AND 2R1 CIGARETTE SMOKE USING TIIE "CONTINUOUS' REGIMEN FOR 3 SNO 6 MONTNSa 3 MONTHS 6 MONTHS TIME AFTER SHELF LAST EXPOSURE (i1 RS.) 3A1 2111 SIWM 3A1 2R1 SIIAM CONTROL n 15.4 (.10) b 13.9 (.47) 8.0 (.38) 23.7 (0.30) 24.9 (0.22) 12.8 (0.24) 6 14.6 (.19) 16 8 ( 35) 2 9 ( 18) 3 13 (0 14) 16 1 (0 18) 14 1 (0 21) n 0 9 7.0 (.11) . 6.9 . (.10) . 4.5 . (.20) . 14.9 . (0.35) . 17.1 . (0.25) . 12.7 . (0.13) ~ r"( N IV 33 A V a xposure conditions were 10% 3A1 or 2R1 cigarette sawke, continuous regimen (17 "runs", 8 puffs/cigarette). n ~ b ODC-p moles C02/aig proteln/20 minutes at 37°C. Coefficient of variation Is given in parent(iesls.

F1na1 Report Contract: CTR-0030 TABLE :23 PULMONARY DNA OAMAGE :VOUCEO I;1 VIVO AFTER EXPOSURE TO 3C3F1/C;tM FwALE MICE TO 3A1 OR 2R1 C:Gr'.ZEVic SVOKE• PERCEEYT CVA ELUTEDb SMOKE EXPOSURE TR'ATME-W GXOUPS PERIOD 3A1 ZR1 SMAM MMSd 3 Mont.tis 15.7 16.1 9.7 I2.5 6 Months 3.3 3.1 5.0 30.0 a b c d C118. cxposure conditions were lOS 3A1 or 2R1 cigarette smoke using the continuous regimen of 17 'runs", 8 puffs/cigarette. CNA is determined spectrofluorometrically using diamfnobenzoic acid. ?ercent of ONA eluted is calcuiated from the amount of DNA in the eluted fractions minus :.he background, divided by the total amount of DNA placed on the filter. `!ethyl .-ethane sulfonate (.1:MS) 120 ig/kg body weight, given t.a.,.4 hours prior to sacrifice. Means of data from two different assay days. 248 CTR CaHTRaCTS 029444 11249194 CTR- I-IN 0444-.-.3 %3

Final Report Contract: CTR-0030 T~BL' 124 PULMONARY DNA DAMAGE INDUCED IN VIVO BY SAP OR BAP-7,3-ol0l IN 8C3F1/CUM FEMALI MICE EXPOSED TO 3A1 or 2R1 CIGARETTE SMOKE PERCENT ONA ELUTEOb SMOKE EXPOSURE TREATMENT GROUPS PERIOD 3A1 2R1 SHAM MMS c 3 Months 16.3 - OMSOd 13.2 18.00 SAP 11.6 13.6 - - 8AP-7,8-diol 21.5 12.8e - - 6 Months 30.0 OMSO - - - - - - - - SAP BAP-7,8-dioi 14.2 11.1 8.7 - 9 Months DMSO 6.4f 1.4f 1.19 SAP - - - 3AP-7,8-diol 9.8f 11.79 13.39 - a C118. Exposure conditions were 10% 3A1 or 2R1 cigarette smoke using the continuous regimen of 17 'runs", 8 puffs/cigarette. As described in Table 123. As described In Table 123. Dimethylsuifoxide, vehicle for SAP and BAP-7,8-diol, 60 ug/0•02 ni. Means of data frorn two different assay days. ONA recovery was 60-80%. DNA recovery was 80%. 249 CTR CONTRRCTS 029445 11249195 CTFZ HN 04 4 2 "34

Final Report Contract: CTR-0030 TABLE 125 SISTER-CIIROMATIU EXCHANGES (SCEs) IN BONE MARROW OF MICE FOLLOWING ONE-WCEK EXPOSURE TO WHOLE CIGARETTE SMOKEa,b t Treatment Number of Mice Total SCEs Range of Mean SCEs per Mouse Average SCEs per Metaphaae per Mousu Experiment 1 Shelf 2 422 3.86 - 4.58 4.22 t 0.51c Sham Control 2 408 3.86 - 4.30 4.08 + 0.31c ~ 2H1 Cigarette Smoke 2 646 6.38 - 6.54 6.46 t O.lld N A co 3A1 Cigarette Smoke 2 754 7.28 - 7.80 7.54 t 0.37d ~ ^'{ rn ~ o Experiment 2 Shelf 4 971 4.28 - 5.34 4.86 t 0.53e Sham Control 2 475 4 62 90 - 4 20e 76 + 0 4 -4 ~ 201 Cigarette . . . . Smoke 2 852 8.18 - 8.86 8.52 t 0. 48f 70 3A1 Cigarette Smoke 2 790 7.78 - 8.02 7.90 t 0.17f ~ M . .~ n °Oetails of exposure conditions are described !n Methods. Ln bA total of 50 cells per mouse were scored for SCEs. Data are given as mean number 0 0 ot SCL•'s per wetaNhass Ler wou:ce (t standard deviation). c . N Aoy two iaeans followed by the same letter are not signiEicantly different at 1) < 0.05. ~ ILD .R -A 01

Final Keport Contract: CTR-U030 TABLE 126 I SIS'rGR-CUItOMATID EXCUANGES (SCEs) IN 60NE MARROW OF MICE EXPOSED DAILY TO WIIOLE CIGARETTE SMOKE FOR VARIOUS PERIODS OI` TIMEd~b t Treatment Number of Mice Total SC Es Range of Hean SCEs per House Average SCEs per Metaphsae per House 4 Weeks Shelf 2 392 3.88 - 3.96 3.92 + 0.03' Shar Control 2111 Cigarette 3 625 3.62 - 3.84 3.72 t 0.11c N A to Smoke 3A1 Cigarette 3 1135 6.78 - u.88 7.57 + 1.14i1 i (D v N ?U ~ ~ Smoke 12 Weeks 3 1326 7.36 -10.62 •8.84 t 1.65` Shelf 3 670 4.10 - 4.96 4.47 t 0. 44cof ~ n Sham Control 2 396 3.92 - 4.00 3.96 + 0.06e ~ K 0 2R1 Cigarette Smoke 2 842 7.1C - 9.69 8.43 + 1.80f ~ 3A1 Cigarette F ?U Smoke 2 838 7.00 - 9.76 8.38 t 1.95 ..~~ 33 46 Weeks .~ --i Shelf 4 838 3.04 - 4.52 3.99 ~ 0.689 ` .. tn Sham Control 2R1 Cigarette 2 •418 ' 4.04 - 4.32 3 4.18 * 0.20 0 Smok e 2 8 50 8.00 - 9.00 0.50 ~ 0.7111 ~ 3A1 Cigarettc ~ N Sooke 2 924 8.54 - 9.34 0.94 ~ 0.571k . i -P ~ a DcLdils of exposure conditiona are described in Methods. ~ ~ 1'A total of 50 cclls per mouse were scored for SCEs. Data are givcn as mean number (,t SCEs t.4cr aw.:taph.ise p.:r onouse (t atandard devintion) . `'Aliy two acans followed I)y the aume letter are not significantly different p< 0.05. 1

Flnal Report Contract: CTR-0030 TABLE 127 SISTEH-CIIItOMATID EXCIIANGES (SCEs) IN BONE HARROW OF MICE ONL WEEK AFTER CESSATION OF EXPOSURE TO WUOLE CIGARETTE SMOKE40b Treatment Number Total SCEs Range of Mean SCEs of Mice per House Average SCEs per Metnphsae per Mouse 1 Week of Prior Smoke Exposure Shelf 4 971 4.28 - 5.34 4.86 0.53c•a Sham Control 2 408 3.86 - 4.30 4.08 + 0.31c 2111 Cigarette Smoke 2 508 4.84 - 5.32 5.08 t 0.34`l 3A1 Cigarette Smoke 2 681 5.74 - 7.88 6.81 ± 1.51d ~•~ ~•. 46 Weeks of Prior Smoke Exposure Shelf 3 675 4.83 - 4.69 4.50 t 0.16c Sham Control 2 439 4.28 - 4.50 4.39 * 0.16e 2111 Cigarette Smoke 2 713 6.6 4- 7.62 7.13 t 0.89f 3A1 Cigarette Smoke 2 750 7.20 - 7.80 7.50 t 0.42t aDetails of exposure conditions are described in Methods. bA total of 50 cells per mouse were scored for SCEs. Data are given as the mean numLer of the SCEs per metaphase per mouse (t standard deviatlon). cALy two means followed by the same letter are not significantly different p< C.05.

Fina1 Report Contract: CTR-0030 TABLE 128 COMPARISON OF T3E RATIO Lri:IG WET 'oFEZGFiT/BODY iIEZGiiT OF BC3F1/CQI4 F'E.ALE MICE EXPOSED T0 3A1 A,vD 2Q1 CIGARETTE SMOICE OVER A 12-MONT$ PERIOD Lung Wet Weight/Body Weight Excosure Per 3A1 iod Ciqarettes 2Rl Ciqarettes Sham Control Shelf Cont:ol 1 Day 0.0063 0.0062 0.0067 0.0060 (+0.0009) (+0.0008) (+0.0028) (+0.0004) 1 Week 0.0080 0.0082 0.0067 0.0067 ..(±0.0004) (+0.0008) (+0,0001) (+0.0013) 3 Weeks 0.0078 0.0074 0.0072 0.0069 (+0.0004) (+0.0007) (+0.0007) (+0.0001) 13 Weeks 0.0070 0.0069 0.0064 0.0051 (+0.0080) (+0.0010) (+0.0004) (+0.0006) 26 Weeks 0.0076 0.0062 0.0061 0.0059 (+0.0004) (+0.0007) (+0.0005) (+0.0012) 39 Weeks 0.0091 0.0068 0.0054 0.0066 (+0.0006) (±0.0002) (+0.0010) (+0.0003) 52 Weeks 0.0097 0.0084 0.0060 0.0046 (±0.0007) (±0.0006) (±0.0011) (y0.0007) 253 C.TR CONTRACTS 029449 11249199 C TR MN 0442,38

' Final Report Contract: CTR-0030 TABLE 129 COMPARISON OF TEE RATIO LII:IG DRY WEIG3T/80DY 'AEIGHT OF BC3F1/CIIa F'r.`iALE MICE EXPOSED TO 3A1 AND 2R1 CIGARET'::.' S::ORE OVER A 12-MONT3 PERZOD Lunq Dry Weight/Body Weight Exoosure Per 3al iod Ciqarettes 2R.1 Ciqarettes Sham Control Shelt Control 1 Day 0.0015 0.0015 0.0018 0.0016 (±0.0001) (+0.0001) (+0.0003) (±0.0002) 1 Weak 0.0018 0.0018 0.0015 0.0016 (+0.0001) (+0.0002) (+0.0001) (+0.0004) 3 Weeks 0.0019 0.0016 0.0017 0.0017 (+0.0002) (+0.0003) (+0.0002) (+0.0001) 13 Weeks 0.0016 0.0015 0.0014 0.0012 (+0.0002) (+0.0002) (+0.0001) (+0.0001) 26 Weeks 0.0018 0.0015 0.0014 0.0013 (+0.0001) (+0.0002) (+0.0001) (+0.0003) 39 Weeks 0.0021 0.0015 0.0012 0.0014 (+0.0002) (+0.0001) (+0.0002) (+0.0000) 52 Weeks 0.0024 0.0019 0.0014 0.0010 (+0.0002) (+0.0003) (+0.0003) (+0.0001) 254 CTR CaHTRACTS 029450 11249200 -, CTR HN 04423 q

Final Report Contract: CTR-0030 TABLE 133 COMPARISON 0? TZE RATIO LQNG WET itEIC3T/30DY WEICHT 0! BC3F1/C?a :'.1.Lc. MIC E72OSED TO 3A1 .L`7D 2R1 CIG;,'L!TT.~.' S.*i01CE OVER A 6-MONTB PEFL*.OD Luag Wet weiah_/3odv Weiaht 'xaosure Per 3A1 iod Cigarettes 2R1 Cigarettes Sham Contro'_ She1t Cor.trol 1 Day 0.0052 0.0055 0.0051 0.0053 (+0.0007) (+0.0027) (+0.0012) (+0.0008) 1Week 0.0068 0.0060 0.0066 0.0062 (+0.0026) (+0.0006) (+0.00::) (+0.0011) 3 Weeks 0.0075 0.0068 0.0057 '0.C052 (*0.0003) (+0.0004) (_0.0002) (_0.0008) 13 Weeks 0.0064 0.0072 0.0055 0.0043 (+0.0009) (+0.0001) (+0.0003) (•0.0004) 26 Weeks 0.0078 0.0076 0.0053 0.0044 (+0.0004) (+0.0003) (+0.0004) (+0.0001) 255 C.TR CONTRRCTS 029451 11249201 CTR HN 044.';40

Final ReDort Contract: CTR-0030 ?ABL= 131 C0.'4?.'lRISOii OF T3.. RATIO 7.v^.tG :E7C i1EIGiiT/BODY idEIGFiT OF BC3F1/CQM M.'ILE :lSCM S7CPOSZED ':0 3?,l AND 2Q1 CIGARETTE SMOlCS OVER A 6-AlGNT3 PERIOD LunQ 0 ^7 weiaht/9odv Weight ?xnosv.=a ?er 3A1 iod Ciaarattes 2R1. a:arettes Sham Control Shelt Con_ol 1 Oay 0.0013 0.0013 0.0012 0.0013 (+0.0001) (+0.0001) (+0.0003) (+0.0002)- 1 Week 0.0017 0.0013 0.0014 0.0017 (+0.0002) (+0.3001) (±0.0003j (+0.0006) 3 0.0017 0.1013 0.0014 0.0012 (+0.0003) (+0.7001) (+0.0001) (+0.0:.02) 13 Weeks 0.0015 0.3017 0.0013 0.0009 (+0.0003) (+0.3002) (+0.0001) (±0.C000) 25 Weeks 0.0017 0.0016 0.0011 0.0010 (+0.0001) (+0. 3001) (+0.0001) (+0.0C01) 256 CTR CQHTRACTS 029452 11249202 C T R N N 0 4 4L2'4' 1

Final Report • Contract: GTR-0030 TABLE 132 DNA CONTr'NT (=q) Ia :'8E Lu^NG CT BC3F1/CGZi FEMALE KL*CE EXPOSED TQ iiHOLE SMOICE FROM 3A1 A.`1D 2Rl CIG.1.eZ..'TTr'S OVER 12 :40;)T)iS. Exmosu.:e Period 3a1 Cicarettes 2R1 Ciqarettes Sham Cont_-ol Shell Control 1 Day 0.797 0.688 0.767 0.531 (+0.154) (+0.042) (+0.163) (+0.064) 1 Week '_.123 1.296 0.983 0.919 (+9.075) (+0.126) (+0.049) (+0.038) 3 Weeks 1.256 0.992 L.143 1.102 (+0.205) (+0.155) (+0.125) (+0.0701 13 Weeks 1.125 0.968 0.823 0.941 (+0.178) (+0.148) (+0.063) (+0.025) 26 :7eeks 1.261 0.841 0.993 1.137 (y0.324) (+0.190) (+0.:90) (+0.239) 39 Weeks 1.265 0.909 0.655 0.641 (+0.14) (+0.25) (+0.:3) (+0.11) 52 Weeks '_.159 1.243 0.919 0.841 (+0.403) (+0.153) (+0.::57) (+0.04v,) 3~ata :=om 3 3:ce pet exposure group per exposu.re peri--d -fere i::d:v:d- _ally deter=ined. :he aaan and staada:d daviat:oa rer grot:a are ;resented. 257 CTR COHTRRCTS 429453 11249203 CTR ~*"~~">~ 0'''~• 2'-• ~

Final Report Contract: CTR-0030 TABLE 2:3 2ROTSy`I Cp:ITL';tT (rq) T_Y T3;E LL*NG OF 3C3F1/Cr^•'I F r'MALE a2CE E7Q0SED TO WHOLE SMOKE F RC.`i 3Al .V`JD 2Rl REFERENCZ CZ^'....RETTy'S OVER 12 }SOI`ITSS. a Zxnosure Per+od 3A1 Cigarettes 2R.1 C.r,arettes__ Sham Control Sk:el: Cont=ol 1 Day 18.28 24.50 30.90 34.10 . (+1.86) (+5.7a) (+2.94) (+0.91) 1'rteek 23.26 24.35 19.03 16.97 (+5.77) (+3.92) (+1.51) (_2.52) 3'+teeks 25.84 20.93 23.10 23.36. (+2.76) (+1.12) (+2.06) (+0.98) 13 Weeks 23.01 21.72 21.97 21.81 (+4.46) (+1.T3) ' (+2.29) (+1.39) 26 'Reoks 33.12 26.70 23.51 23.23 (+6.44) (+3.01) (+1.95) (+1.2:) 39 Weeks 34.93 27.75 19.44 25.7i (+1.34) (+1.38) (+3.54) (±0.56) 52 Weeks 35.60 35.73 27.13 22.79 (+2.02) (_4.39) (±4.16) (+2.57) ~ata Oro= 3 z:ce per exposure group per exposure period were i,di::d- a, :al-y deter=;n ed. The mean and standa.r.ad deviat:oa per group are ^•esented. 258 C.TR CQNTRACTS 029454 11249204 L..Y T) ! NN 044O'aa. 4r..i'

Final Report Contract: CTR-0030 ?AaL? L34 3YDR0XY?ROL+:IE CONTr:IT (mq) IN TEE LUNG OF 8C3F1/CL:d Fr.'L~I.r. :SZG ~C1G527 O. YHCLG SMOe'CE FROM 3Al AND 2R1 PL.:FTE'cL82iCE CZ ;.4zETTES OVER 12 :tC:T:SS . a rxoosuse Period 3Al Cigarettes 2R1 Cigarettes Sham Control 8:elf Co::t.:o: 1:.ay 160.0 144.1 141.3 144.0 (+38.5) (+ 25.8) (+36.9) (+24.5) 1 rieek 142.0 134.6 134.3 34.4 (+50.7) (+ 87.5) (+16.8) (+21.7) 3 Weaks 123.6 121.9 105.3 35.6 (+45.2) 19.1) (±35.2) (} 3.:) 13 Waeks 172.3 188.2 137.6 :73.0 (+43.6) (+ 40.4) (+66.2) (-46.3) 26 Weeks 401.9 379.1 236.1 :52.9 (+87.3) (+111.3) (+ 4.4) (- 2.3) 39 ~ee;cs 470.6 358.8 221.9 234.: (+48.5) (+ 26.4) (+38.0) 2.3) B 2 Weeics 431.5 350.4 225.5 224.3 (+87.0) (+ 42.3) (+ 6.1) (- 1.3) 3':)a:a :=oa 3 a.ice per exposure group : ar axposure rariod were :ad:•r:t- •-allJ 3etey :ed. The meaa and staada.rd daviation rer grou? are -esaated. 259 CTR COHTRRCTS 029455 11249205 CTR HN 0442-14,

Final Report Contract: CTR-0030 TABLE 135 H-ZDROXYYROLZ:7E (34) 2=t T3E LUNG OF 8C3F1/GVII :1-1La: aZCE EXPOjED TO HHOLG S:SOs.: FRC.K ::.1 A..`iD 2R1 QEFERE•.ICE CIG.lRE:"TSS OVF.aZ 6 aO.1TFiS. ?x•:osure Periad 3a1 Ci:arettes 2' R]. Cigarettes Shaa Control 3he1! Control 1 Day 221.0 171.0 145.3 119.1 (+61.3) (+50.7) (+11.1) (+14.7) 1 neek 222.9 164.9 159.7 186.5• (+18.9) (+41.2) (+ 5.6) (+51.6) 3 heeks 158.4 71.0 113.9 135.5 (+27.8) (+ 6.9) (+26.7) (+37.2) 13 Weeks 190.8 160.5 153.6 157.1 (+15.6) (+10.1) (+43.3) (+31.5) 26 weeks 351.7 338.3 250.1 277.7 (+56.7) (+84.4) (+15.1) (+44.7) 'Jata :rcm 3 aice =er exrosure group per exposure rericd were i:div:d- ua11y deterzized. :he mean and standard deviation per group 3=e presented.. 260 CTR CONTRRCTS 429456 11249206 CTR MN 04•

F1na1 Report Contract: CTR-0030 TABLE 136 KINETICS OF TLtE PLAQUE FORHING CELL (PFC) RESPONSE STRAIN OF MICE 3AYS AFTER SRBC INJECTIC`1 NUMBER OF PFC/105 SPLEEN CELLS3 BC3FI/Cun 3 42 BC3F1/Cum 5 1787 3C3F1/Cum 7 13 BALB/c Cum 3 21 dALB/c Cuca 2 1553 BALB/c Cuca 7 12 a Average r,umber of PFC's frocn five tr.dividual animals per grouo. 251 CTR CONTRRCTS 029457 11249207 CTR HN 044246

Final Report Contract: CTR-0030 TABLE 137 EFF:CT CF CYCLOPHOSPHAMIOE ON THE PFC ?ESP045E OF BC3F1/CUM MICEa AGE OF MICE DOSE OF CYCLOPHOSPHAMIOE (mg/kg BODY WEIGHT) NUMBER6 OF PFC/10 SPLEEN CELLS 22 Weeks 0 1554 22 Weeks 15 mg/kg 360 22 Weeks 2:0 mg/kq 30 59 Weeks 0 1379 59 Weeks 75 mg/kg 307 59 Weeks 250 mg/kg 75 aAnimais were given cyclophosphamide (IP) I day prior to the auninistrat'cn of SRBC. PFC assay performed 5 days post SRBC i njecti on. 262 CTR CONTRRCTS 029458 11249208 C ~9/ ! I I I~I 044, iu.+ I• l~ `

Final Report Contract: CTR-0030 TABLE 123 EFFECTS OF ACUTE C:;AR TTSMOKE EXPOSURE ON THE PFC RESPONSE CF BC3F1/CUM MICEa STRAIN OF MICE TREATMENT GROUP PFC/106 EXPT I SPLEEN CELLS EXPT-II BALB/c Cun Shelf 1553 1211 Sham 1504 1275 2R1 Smoke 1848 1222 BC3F1/Cum Shelf 2156 1727 Sham 1933 2064 2R1 Smoke 2363 1810 a Animals were exposed to 2R1 c'.garettes for 5 days prior to SR3C injection and 5 days post SRBC injection. The exposure regimen was 4 ctgarettes/day; 8 puffs/clgarttte; 20 sec/puff. 253 CTR CDNTRRCTS 029459 11249209 CTR VIN 0442-41' 18-4

Final Report Contract: C'R-C030 TABLE 123 -"FECTS OF CHRONIC CIGARiTZ :wOKE :i(?OSI;RE ON T'rE PFC RESPONSE OF BC3F1/Cli4 MICEa T REA,'`"EVT PFC/106 EXPERIMENT I SPLEEN CELLS EXPERIMENT I I Shelf 1348 1840 Sham 2231 1980 2R1 Sr„oke 2199 2340 a;nirals were exposed to 2R1 cigarettes on the SEH II machine .`or :2 weeks prior to i~munization, and 5 days post ir.munization. 264 CTR CCNTRRCTS 029460 11249210 M.J' I R / 1) ! 0l b) F.•v , 7-SW* .

?1na1 ROpozt Contract: CTR-0030 CTR-1018 ?IGQR:S 33-40 265 CTR CCh4Tf2ACT5 029461 11249211 C TR HN 044250

Flnal i(cuurt Contract: CIR-0030 FIGUItE 38 UeNurlLluu ul Tutul 1'uI.Llculrtu NALtaC (TI'N) lo Lungs ot• 111ca lapuuwl tu 281 C11jureLte Swuke fur Various Perlod• of Tlae (CTk 119-5) • ---1----~---f 1-~1--t--1 1 120 240 360 4110 LUU 720 840 'l60 1080 1200 1320 1440 1560 1680 11100 1920 2040 (17 runs) Tuta1 5ccuudr of kYpuuurc

Final zeDort-. Contract: t-M-0030 FIGURE 39. 2.17-r. OF ATRESIA IN 5 INBRED Si?.1IYS A,VO CN HYBRID S ii AIN OF !'.CUSE. 257 "CTR* CONTRACTS Q23463 11249213 CTR I~It"I 0442!52

F{nal ReOort Contract: CTR-0030 0 ~ i 4 R x 3 - 2 1z, 1 .> 0 ~ ~ Uj ~. 4 ~- O 3 O u. 2 O 1 O. Z 0 4 3 2 1 0 4 3 2 0 i i ~ i ir --- 1 ,~ ~ ~ . ~ a - - , 10 20 30 • 40 AQ E (wo.ka( 268 CTR CONTRRCTS 029464 FIGURE 39 t 3 2 1 0 .. T .. r 4 3 z I .. . . . a s z {f E Strain C3H/Ant Cum I z Straln BC3F1 / Cum 3 ~ Stn/n C Straln 57BL/g Cum ~ 11 z ~~gg{ ~.~~ o S~ra~n - T Straln Strain ' L Straln Strain - ~ 4 11249214 8A /?J DBA/2N , CS7 BL/6N CTR HN 0,44253

Ftnal Report Contract: CTR-0030 F:Gi;RE 40. RATE OF ATRESIA IN BC3F1/C'.;M :!ICE EXPOS'c0 TO 2R1 CICy.RETi"c S`!OKfi (AS C=SCRI3E0 UNOER CTR-101A) OR SF:r`1 EXPOSED. 259 CTR CCNTRRCTS Q29465 11249215 C~'~'~' HN 04' 4254. .

I Ina I IIclKrrt Luntract: LII(-(JU]U 5 ~- 1 l..OC > N ~ n -,, 3 ~ ?j .. y C)) ~ N t.. o V 0 0 A 2 0 Q SMOKE EXPOSED • SNAM EXPOSED ~ ?U ~ 33 $ n i ~ Ln Q ~ ~ 0 10 20 .~. lT V ~ ~ . ~~ wq~ twaoksl t IGUItE 4U , 0 Ul 1

CTR cOHTRaCTS o2s46"(" 11249217 CTR HN 0,4425~'.;~

Final Report Contzact: CTR-0030 COROLLaRY STUDIES •)I, COROLL.AeZY STCDIES: =jAR:SACCKI:7ETICS OF I:IH LED :!ATER?ALo The ?harmacokine:ics and metabolic fate of inhaled materials provide a basis for =ae comparison of the bioloqical activity of compounds wita diverse chemical str::c:ares. :'.`.e procedures and tec::nii:es used in these st-sdies ;;ave been described in References 7, and 22-24. The deposition and distribution after niala::on in smoke will be desc::bed :or :ou: natural constituents of •+nole cigarette smoke: nicotine, 3aP, dotriacontane (DTC), and catechol. The pharmacokinetics of two chemicals that were of interest as single chemical aerosols will also be described: 12-0-tetradeconylphorbo1-13-acetate (:'?A) and catechol. A. yicotize, 3aP, and DTC The internal distribution of the particulate phase of whole cigarette smoke was compared in male C3H/Anf C-= :aice• at various times after saoke exposure (see Ref. 2`). Cigarette smoke was generated f:om reference 2A1 cigarettes containing either 14C-DTC, L4C-nicotine, 14C-BaP using a Walton Horizontal Smoking Machine under standard conditions of puff duration, puf: volume, puff f:g~uency, s~oke concent~Ition and total smoke exposure time. C-OTC. - C-nicotine, C-Ba P represent the stzaiqht chain hydrocarbons, the alkaloids, and the polycyclic aromatic hydrocarbons, :espectively, of the total particulate matter (TPM) of cigarette snoke. Tye yield of T?M and amount of :adiolabel per cigarette were quantified during exposure. After the 10 minute smoke exposure (the time required to smoke one ciqarette),4total i;zi:tal T?!1 ~ose as reflected by smoke containing • C-DTC, C-nicotine, 4C-BaP was estimated to be 229, 292, or 147 ug T?!t per mouse, respectively (Table 140). T::e internal distribution of DTC was determined to be as follows: 721 in the lung, <1t in the larynx, 5% in the head, 119% in the stosac::, and 5% in other tissues (':able 141). For nicotine, the distribution was 2% in the lunq, <1t in the larynx, :7i :z t`:e :zead, 31 in the stomach, and 78% in other tissues (Table 141). ?or 9aP, the distribution was 2{1 in the lunq, 4% in the Larynx, 131 in the head, 14% in the stomach and 45% in other tissues (Table 141). The half-life fo= internal retention in the :aouse :is greater than 24 hours :or 14C-DTC, less than 1S minutes for ' C-nicotine, and less than 1`:our for 14 C-BaP (Table 142) . The :ni•ial distribution of.':?!!, as measured by DTC deposition, was -sinLy in the lunq (721) and stomach (18t), however, dur:sq a:e :J minute exposure period, extensive redistribution of two o: the 271 CTR CONTRRCTS Q29466 11249218 CTR I-IN 044257`

Final Report Contract: GTR-0030 COROLL.Aa? STIIDIES tobacco cont:tuents occurred. '7:coti e and BaP were rapidl~ redistributed ;resu.tably via the b_ood stream to the head (L7t and 1'31, :es;ecti•iely) and other internal tissues (781 and 451. respectively). H. Cateclol in Cigarette Smoke Catecaol, 1,2-dihydroxybenzene, is present in tobacco '_eaf (25) and :s the most abundant phenol :n smoke ranging °:om 0.08 to 0.28 _q/ciqarette (26). '!an Duuren and co-workers (27) reported that catechol was a strong cocarcinoqen on mouse skin when applied together with SaP. T::ese cocarcinoqenic properties have been confirmed by Hecht at al., (29), who reported that catechol is ane of the major cocar:inoqens in the weakly acidic fraction of ciqarette smoke condensate. In addition, moriaoto and Wol:: (29) have reported that eatechol was a potent inducer of sister chromatid exchange and delayed cell division in cultured human lymphocytes. It has been suggested that reduction of catechol :n cigarette smoke miqht be expected to lead to a smoke condensate with lower tumoriqenicity (28). An investigation of the pharmacokinetics and metabolic fate o: inhaled catechol would allow pharmacological analysis of its action and could provide additional information f or carcinoqenicity experiments. Details of this study may be found in Hwanq et al., (24). The deposition and distribution of 3 K- c a t e c h o 1 i n 3C3F1/C- mice exposed to LOts(v/v) <entucky Reference 2R1 ciqarette srscke labelled with ei-catec:ol are shown i1 :able 143. The radiolabelled catechol for tkess experiments was synthesized :n our laboratories (30). It has been previously shown that 3H-catechol can be added to ciqarettes, the cigarettes burned and -401 of the added 3H-catechol can be recovered unchanged with the TPM of the smoke (24). No radioactivit f was transferred to the gas phase under these conditions. Major organs and tissues of mice were analyzed at 0, 5, 1], 30, zJ and 120 minutes after exposure to Ta-catechol-:R: :+ho1e ci;arette smoke. The amount of radioactivity was greatest between 0 to 10 minutes after exposure. The highest radioactivity levels (disintegrations per minute, dpm) were observed in the lunqs, blood, and total respiratory tract at zero time, whereas in the liver, kidneys, and stomach, the highest d;.m values were found 5 to 10 minutes after exposure. The blood contained the greatest amount of radioactivity at all tize points, (i.e. 40-50t). Thirty minutes after exposure, nearly 3-fold :ess radioactivity was present in the l::nqs, total respiratory tract and blood. Approximately 1.5-fold less radioactivity was found in the l:ver n^d stomach, and 2.6-fold less was found in the 272 CTR CCHTRRCTS 429469 11249219 ti..X T) "`! 1 7 N 0M / 4firn M58

Pinal Report Contract: CTR-0030 CDROLLaRY STUDIES kidneys. ':`-jo hours after exposure, only -111 of label found at zero time remained in t:e total c:dy. It appeared that 3K-catec.^.ol in smoke was not only rapidly redistributed within :he mouse, but also was rapidly re=oved from the mouse after exposure. Urine and feces were collected after exposure for :20 a:nutes. Approximately 91% of the t:itium label was exc:eted in :!:e urine within 2 hours of exposure. Less than it of tne label remair.ed in _-e l~ng, =ir5inates, liver or kiC::ey 2 hours after exposu:e (see Ref. 24 for details and Table 1:1). C. Catechol as a Chemical Aerosol 1. Aerosol -jeneration, Characterization and Honitoring Aerosols of catechol vece generated using a:odi:ied Collison Nebulizer described by May (31). The nebulizer was designed and fabricated by the Analytical Chemistry Division, Oak Ridge yational Laboratory, Oak Ridge, Tennessee. The design was such that an aerosol may be produced continuously for 10 minutes '-oa only 2 milliliters of starti::q solution. The concentration and particle size are a:snct:on of the starting solution concentration. The catechol solutions in ethanol were placed :a t=e small vol=e reservoir, which was cooled with an ice bat:^.. Ai: (15-20--PSI pressure) was forced through the inlet tubing and exited at a high velocity through the orifice. Solution was aspi:ed through the drip tube f::? the reservoir and the high velocity air flow dispersed it :ato droplets. These droplets impinged upon the inner surface of the reservoir tube. Large la:t:cles coagulated and returned to the reservoir, while saall partic:es resained dispersed and exited with the air flow through :':e aerosol outlet. The relatively large volume of air flowing :h:ough the system evaporated :;e alcohol from the drapLe:s leaving solid catechol suspended as particulates. Two solutions of catechol in ethanol were used to generate aerosols, 0.2 and 1.0% (w/v) (Table 144). A seven-stage Cascade Impactor (Sandia Resear::: and Development, Albuque:que, vev ae:ico),:+as used to compare the size distribution of two aerosols (Table 144). The particle size distribution for bocl aerosols were shown to be similar, with 78 and 901 of t::e particles less than 0.45 um aerodynamic diameter (Table 144). An on-line lig::t emitting diode optical sansor (desc:ibed in Section II.C.) was used to monitor the aecoso: cor.cent:stion. The optical sensor was interfaced with a st::a j :73 CTF2 CQNTRaCTS 029470 11249_2C Cf f'6 Vf N 0`°f' `422.v,.~ ~E .

Final Report Contractt CTR-0030 COROLLAR? STOD28S chart recorder :or permanent documentation of the exposure. The data demonstrated that the aerosol concentration must be measured and documented :or each exposure (Table 145). This was done for all subsequent aerosol experiments. 2. Deposition and Distribution of Catechol Aerosols The deposition and distribution of radioactivity in major organs and tissues from 8C3P1/Cum mice after a 10 minute lxposure to aerosols of 45 ug 3H - c a t e c h o I/ L a n d 6 6 u q H-catechol/L are shown in Tables 146 and 147, respectively, (32). The total amount of radioactivity found in the mice immediately after either exposure was very similar. Lmmediately after either exposure, '48% of the radioactivity was found in the blood; '24t in tae total respiratory tract, with 'St in the lung and '16% in the.turbinates= '12t in the kidneys; and '9t in the liver. The total amount of catechol recovered at zero time was 3 uq/mouse. Within 5 minutes of exposure, only 44-54% of the I initial amount of radioactivity was found in the tissues. Within 120 minutes, only 6-8% of the material was found (Tables 146 and 147). These data suggest not only that rapid redistribution of catechol and catechol-related material occurred within the mouse during and after exposure, but that catechol was rapidly removed from the mouse, either in urine and feces or in re-expired air. Urine and feces were measured for catechol and related material and the data are shown in the last column of Tables 146 and 147. Between 671 and 88% of the radiolabelled material found at zero time was accounted for in the urine and feces collected :or 120 minutes after exposure. Total radiolabelled material recovered 2 hours after exposure was 72% and 95% (Table 146 and 147, respectively) of the material found at zero time. 3. :.omparison of Tissue Distribution in lice Pollowinq Exposure to R-Catechol Civen as an Aerosol or in Cigarette Smoke The percent distribution of radioactivity in mice exposed to catechol as an aerosol (from Table 146) was compared to the distribution in mice exposed to catechol as a component of Kentucky Reference 2Rl cigarette smoke (Table 148). Distribution data are shown for 120 cninstes after aerosol or cigarette smoke exposure. The distribution of the radioactivity in the tissues, blood, urine and feces :s 274 CTR CaNTRRCTS 029471 11249221 CTFR I-IN 0,44-f ; 0

Final Report Contract: CTR-0030 COROLLARY'STODIES identical for eit::er the catechol aerosol or catec::ol in cigarette smoke. ~ 4. Conclusions a. Catechol can be generated from etaanol solutions as a particulate, respirable aerosol. b. The aerosol has a particle size of <0.45 ..m aerodynamic diameter. c. Generation of aerosols from ethanol containing either 0.21 or 1.0% catechol resulted in aerosol concentrations of 45 and 66 ug catechol/L, respectively. d. Deposition and distribution of catechol in mice exposed "nose-only" to these two aerosols were quite similar with a total dose of '3 ug catechol/mouse/10 min exposure. e. During the 10 minutes exposure period, extensive redistribution likely occurred, so that immediately :ollo:+inq this exposure period, the blood, respiratory tract, kidneys and liver contained 481, 24%, 12%, and 9%, respectively, of t::e total material found. f. Catechol was rapidly eliminated :rcm t;:e internal tissues so that only one-half of that found at zero time was found in internal tissues 5-10 min after exposure. After 5-10 min, the half-life of catechol in the internal tissues was 20 minutes. g. ?rine collected over the 120 rin period following exposure contained 67-881 of initial amount :abelled catechol (or 3etabolites). h. Lnhalation of aerosolized catechol likely =spid results in catechol deposition in the respiratory tract, transfer to the blood, redistribution via blood to the zajor organs, (especially the liver and kidneys), and `inally, excretion within 2 hours of exposure. i. The phar=acokinetics of aerosolized catechol is not altered in the presence of cigarette smoke. 275 CTR COh{TRRCTS) 029472 11249222 C TR PIN 04.426 1

Final Report Contract: CTR-0030 COROLLARZ STQDIES D. TPA as a Chemical Aerosol TPA is ti:e active constituent of croton oil and the most potent prcmotor in the two-stage (initiation and promotion) animal -,ode1 of chemical carcinogenesis on mouse skin. Data from our laboratories had shown that TPA caused a specific biochemical event (induction of pulmonary ODC) (15) and t1i's suggested that pulmonary tissue nay be sensitive to the processes of initiation and promotion, similar to skin epithelium. In order to mote carefully defi.^.e this potential two-stage carcinogenesis model system in lung tissue, the deposition and retention characteristics of TPA in mouse lungs were determined. Two experiments will be described. In the 3ii -:'?A aerosol was generated by the nebulization of a 0.2% ethanolic solution. 3C3!1/Cum mice were exposed to three different aerosol concentrations, 24 mice per exposure. The aerosol concentrations were 54, 75, and 83 ug/L, generated over a 6 minute period. The control group (12 mice) was exposed to aesosolized ethanol under similar conditions. The animals were sacrificed by carbon dioxide asphyxiation, 4 mice from each exposure for each of the following time points: 0-5 minutes, 30 minutes, and 1, 4, 8, and 24 hours. Seven tissue samples were removed from each mouse: lung, larynx, head, stomach, liver, hide and a composite sample of the remaining tissues. The hide sample included all of the skin and :ur. Thes.e samples were frozen in dry ice and sent to Oak Ridge mational [.aboratcry for analysis of radioactivity. Data are shown in Table 149 for all three aerosol concentrations immediately after exposure (0-5 minutes). The deposition in the lung increased linearly from 195 ng TPA per lung to 255 ng TPA per lung. 3etveen 48 and 651 of the TPA was found in the lungs i.=ediately after exposure, with 66-d4= in the respiratory tract. Results from this experiment indicated that TPA was rapidly cleared f:om the lung, with a half-life of less than 30 minutes. The clearance data for 54 ug/L aerosol concentration are shown in Table 150. Similar clearance data were obtained for the other two aerosol concentrations. This rapid clearance of T?> from the lung was associated with a concomitant redistribution to the stomach, 1ide, and composite remains. These data suggested that a more thorough examination be performed using more tissues over a longer period of•time. In the second experiment, 3H-TPA was again generated from a 0.2% ethanolic solution. The deposition and distribution of radioactivity in 21 organs and -issues, (including urine and feces) after exposure to a 70 ug/L 3H-TPA aerosol are presented :n Table 151. As shown in the first experiment, 451 of the TPA •.+as four.d in the Lung and 611 in the respiratory tract. These data showed tZat TPA was rapidly absorbed into the blood stream 276 CTR caNTRacTS 029473 11249223 CTR ~~~~ ~,~~4~-~1=021

rinal aeport Contract: CTR-0030 C0R0LIJ1aT STIIDIES s and gastrointestinal tract. Approximately 181 of the TPA was found in the stomach iatmediately af:er exposure, suggesting :hat •he mice may have regurgitated and saallowed TPA during the exposures. Within 30 minutes, 38t of the initial TPA was lost from the :aouse# either through re-expired air or metabolism. The =ota1 dose to the mouse was -2.5 ug T?A. xhile the TPA in the respisatory tract decreased to less than 21 of the initial amount withiz a hours, tha total amount of TPA recovered in the internal tiss;:es remained approximately the same duri.^.q this time. Between 8 and 24 hours -65t of the TPA was removed `-.-m the mouse via the .°eces and urine, with the =eces the major excretory route. Forty-eig a hours after exposure, Less than 8% of the initial dose remained within the mouse. Large increases iz dpm were observed in the urine and :eces at 4a hours. Thus, TPA and/or its metabolites were deposited in the respiratory tract, rapidly redistributed via the blood to other organs and tissues, especially the liver and gastrointestinal tract. After a rapid (<30 minutes) initial decrease in total TPA levels, no further decrease occ::rred until 24-48 hours. The distribution and clearance of T?A was quite different f:oa catechol or other smoke related chemicals. 277 . CTR CaNTRRCTS 029474 11249224 CTR M4 04• 42G3"-

FinaL Report Contraet: CTR-0030 COROGLIIRY STUDIES 'aBLES 140-151 278 CTR CQNTRACTS 029475 „?49225 CTR HN 044264,

Final Report Contract: CTR-0030 TABLE 140 OEPOSIT:C,1 CF TFM IN C3H/ANF CUM MICE AFTER EXPOSURE TO 10% . 14 2A1 CI3:.2cT;" SMOKE LABELLED WITH EITHER 14C-OTC. C-NIC, GR 1~C-aaPa DEPOSITION OF T%4 ug TISSUE 14C-OTC 14C-NIC 14C-3AP LUNG AND LOWER T;AACHEA 161 (0.26)b .6 (0.07 35 (0.27) LARYNX AND UPPER TRACHEA <1 (0.10) 4 (0.16) 6 (0.25) HEAD 13 (0.74) 44 (0.14) 20 (0.02) TOTAL RESPIRATORY TRACT 174 (0:29) 54 (0.13) 61 (0.14) STCGtitACH 43 (0.49) 9 (0.16) 20 (0.30) REMAINING TISSUES 11 (0.49) 219 (0.11) :6 (0.26) TOTAL BODY 229 (0.34) 282 (0.11) 147 (0.02) a b Total smoke exposures of 300 seconds resulted from 30 seconds of smke followed by 30 seconds of air each minute for 10 consecutive minutes. Mice were sacrificed icrrediately (<15 seconds) after the 10 cninute exposure oeriod. Oata are from 8-12 mice per experiment. Coefficients of variation are given in parenthesis. 279 CTR CONTRACTS 42g476 11249226 CTR PIN ~'.4426>5'

Final Reoort Contract: CTR-0030 TABLE 141 PERCrM' CF 14=-RAOIOACTIYITY IN C3H/ANF ::;;1 MICE AFTER EXPOSURE TO 10% 2A1 C:G:.RZ7= SraoKE L18ELL"co WITH :IT:?_R 14:-OTC, 14C-YIC OR 14C-aaaa =:1CENT OF TOTAL OPM TISSUE 14C-OTC 14C-HIC 14C-8AP LUNG ANO LOWER TRACHEA 72 (0.07)° 2 (0.72) 24 (0.24) LARYNX ANO UPPER TRACHEA <1 <1 4 (0.35) ~~ 5 0.36 :7 0.09 ?3 0.02 TOTAL RESPIRATCRY TRACT 77 (0.04) 19 (0.07) 41 (0.15) STNMACH 18 (0.18) 3 (0.27) 14 (0.3:) R:LAI4ItiG TISSUES S(0.16) 78 (0.02) 45 (0.25) 3 Values talculated frcm data in Table i=0. ~ Coeff°cfent ;,f variation. 290 CTR CaHTRaCTS 029477 11249227 CTR MN 04,4266

final Report Contract: CTR-0030 ;2L: .=2 :LEA~PICE T:ME FOR 50: OF RADIClr3:LL:: yATERIAL (Ti) IN C3H/A;1f `!:C: AFiER EXPOSURE TO 1C: ZA1 CI6:+RE7TE SMOKE LABELLED WITH EITHER 14C-OTC, 14:-YIC, OR 14C-SAPa HOURS TISSUE 14C-OTC 14C-YIC 14 LUNG :ViD LOWER TRACHEA > 24.0 0.25 0.2: ::,2YNX AND UPPER TRACHEA 3.0 0.25 < 0.2: iiEAD 1.0 < 0.25 0.75 TOTAL RESPIRATCRY TRACT >24.0 < 0.25 0.25 STGVCH 3.0 4.0 4.0 RE.uAINI!iG TISSUES 15.0 < 0.25 0.25 TOTAL 3CDY 24.0 < 0.25 0.75 i=xcerinental conditions as in Table-'.10. Yice were sacrificed after ;he 10 minute exposure period at zero (15 < seconds) , 0.25, 0.:, :.7, :.0, 16.0, and 24.0 hours post exposure. =91 CTR CONTRRCTS 02947e 1124s2-1s CTR ~'I~~ 0~~4."~`

Iinal keyurt Contrdct: CiR-UU3U n 0 ~ ~ 41 ~ TAULE 143 DEPOSITION AND DISTRIBUTION OF 311-CATECIIOL IN BC3F1/CUM MICE EXPOSED T0 10% (v/v) 2R1 CIGARETTE SMOKE LABELLED WITH 311-CATECIIOL a 0 P Mb TIME AFTER EXPOSURE (MINUTES) TISSUES 0 5 10 30 60 120 I.UNG 243 (0.30)c 196 (U.32) 183 (0.45) 75 (0.44) 46 (0.67) 30 (0.79) TOTAL kESP1RATURY TRAC T 346 2'16 25B 113 :,tf 46 BLUUUd 163U (0.40) 1300 (0.56) 1246 (0.52) 529 (0.20) 238 (0.16) 129 (0.77) LIVER 371 (0.22) 433 (0.39) 430 (0.48) 233 (0.48) 163 (0.34) 83 (0.75) KIDNEY 400 (0.38) 479 (0.57) 442 (0.48) 150 (0.69) 180 (0.56) 42 (0.49) STOMACIIe 71 (0.64) 133 (0.81) 79 (0.83) 42 (0.98) 17 (1.22) 21 (0.57) RLIS/1INItlG TISSUES 104 167 125 58 54 4 TOTAL BODY 2933 2808 2588 1125 638 325 m N Total of 48 BC3F1/Cum female mice were exposed to 10Y(v/v) smoke generated on a Walton from 3H-catechol-2R1 ~ cigarettes. Standard exposure conditions were used to generate 30•seconds of smoke alternating with 30 seconds ~-.. Ln of air per minute. A total of 300 seconds smoke exposure time was obtained. Eight mice per time were used. w:. (ibang et al., 1902). rc.~. N b Values are DPM of 311-catechol per tissue per mouse. ~ Q C Coefficient of variation. ~ ~ d Represents the whole blood volume of lhe mouse. e ~ ~ Includes contents. , bg 1 ~e9..=

Final Regort Contract: CTR-C030 TABLE .:4 ?ARTIC_E SIZE OISTRIBUTICN (3) OETER.MIYEO FOR 3H-CATECHOL AEROSOLS USING A SEYEN-STAuE ^.:+SCA:E I!"PACTCR ~:STRIBUTICN aERCCYN.I.MIC 0 LL4E7ER 10.9 ug CAT:CHOL 15.9 ug CATECHOI (~,y) AEROSOLIL AEROSOL/L ...29 0 0.1 :. :9 0 0.1 2.93 0 0.8 2.;8 0.2 0.9 1.35 3.8 6.5 0.38 6.4 13.6 < 0.:5 89.6 78.0 293 CTR CONTRACTS 029480 11249230 CTR HN 044,2~'~9

Final Report Contract: CTR-0030 TA.BL= 1.45 CATECHOL AEROSOL CCNCct'ZATION OETERMINED AT VARIOUS SOLU-,:CN ::,NCENTRATIONS SOLUTION CONCENTRATION (PERCENT w/v) CALC:~.TEO AEROSCL CCNCc.'YiRATION (u;/L)a RECOROER VALUES (mv) 0 0.2 20 : 4 0.2 .0.4 75 : 5 0.4 0.6 165 - 5 0.7 0.8 247 : 7 1.1 1.0 320 : 10 1.4 a b Aerosols were sampled at :CC .1-11/minute for 3 rinutes, collected onto Cambridge fi?:er pads, chemicals eluted from the pads and concentra:'.on determined by UV absorOance. Mean and standard deviation `roe three separate determinaticns. Millivolt (mV) values determined from direct response of optical sensor during generation of aerosol. 234 CTR CONTRACTS 029481 11249231 r `~"~'~' ~~~~ 0442 '~`#:~

~s= s-saF~:::~ isi ssi:s:_=: s~xs~z:sxx==o~ 7 ss?~ssssssdss z:sg«es::_:~s- w ~t R~~ S: 0~ 3 3~ 3 3 143a =lmi 3 1 a 35=: xt 3 ~ i. ~iss ts~sss~~st~:s sxs'~s~ix---i= i i a a ~~I?s$S i~IY ® C.TR COHTRRCTS 029482 11249232 C-TR NN 0-4-4,22"'1

f fln.l Report Coatrat• CIR-00]0 IAOtE 1U6* DEPOSIII(111 AMO OISTRIOUTION Of RAOIOJICTIYITT (0fN) IN TISSUE$ Of 8C]fl/CU1 NICE AT YNtIOUS 111ES Af1ER EIU'OSURE 10 46 ry AEOOS0l12E0 SN-CAilLllql/E f1MEkAfEO fkOM A 0.21 CAIEq10l SOEUEION (St. ACT+12  106 Apyy) IIME Af1ER E1FOStWE (MIf1UfES) Iliiu[S 0 L IU lU 60 120 UU1c 2.001 (0.22)' 1.025 (0.30) 916 (0.45) 492 (0.38) 268 (0.16) 199 (0.26) TIAVIIMAIE 6.624 (0.2]) 1,368 (O.SS) •14 (0.61) 1,009 (0.49) 381 (0.65), 190 (0.61) IkACIu 391 (0.3s) - 183 (0.u) i4e (0.10) . 61 (0.M) 32 (0.s1) 30 (0.43) tARllli 466 (0.26) 194 (0.s1) 160 (0.38) 91 (0.15) 51 (0.36) 50 (0./2) lOTAE RESflRAl00T ~ TtACT ~ N • 9.484 2,1)0 2.118 1.651 1/1 469 l'' c-1 > • OtOOD 19.211 (0.21) 10,1]2 (0.46) 9,21] (0.18) 955 (0.36) 3.213 (0.s1) 800 (0.42) w ,,i EsOPwlp15 IJl (0.32) 166 (0.39) 100 (0.34) 52 (0.31) 26 (0.53) 32 (0.40) SIOW1Ca 410 (0.9s) 1,0]0 (0.64) 1.899 (0.61) 1,240 (0.24) $40 (0.69) 199 (0.41) 4n YEA0IKR 92 (0.19) 69 (O.SS) 51 (0.69) 61 (0.62) 21 (0.12) a 0.1 ~ SP/EEM 203 (0.1s) 126 (0.96) 116 (0.51) 61 (0.)4) 29 (0.11) 26 (0.15) Q [IONET 9.858 (0.25) 7.T21 (0.47) 2.135 (0.45) 1,075 (0.S1) 210 (0.40 127 (0.20) ~ 1ANCREAS ]s9 (0.67) 219 (0.64) 220 (0.48) lu (0.48) a (0.40) 54 (0.26) ~ /[ARI )es (0.91) 3]) (0.56) 291 (0.45) 121 (0.v) 4S (0.3f) 39 (0.29) ORAIN s9z (0.19) 360 (0.s>t) 408 (0.3e) 212 (0.40) 110 (0.21) 152 (0.31) ~ ~ AOREMAIS 89 (0.51) 62 (0.41) 6o (0.19) 21 (0.42) fs (0.16) 16 (0.96) • Y (7 OYARIES 99 (0.]6) 61 (0.12) 54 (0.43) 54 (0.22) 11 (0.42) 11.(0.61) U1ERUS 303 (0.19) 231 (0.16) 224 (0./10) 139 (0.60) so (0.a6) u (1.06) ~ ~ ~ EIrER ).506 (0.28) 2.]91 (0.95) 2.469 (0.43) 1.131 (0.57) 316 (0.44) 289 (0.42) ~ N IUTAE 8U0T 40.226 21,912 20.584 6,861 5,901 2,2)] URI/IE 26.841 b ~ ~ fccES ~ a) _ .. _....._^.--- -- -- .rw w + l cll u Icel ul r+~ ~+1/•m. S.r'le (wciudes rs lea fn.c 0 tu !'LU rip. ./ter e.puaure ~~

Final Report Contract: CTR-0030 TABL_ 145 CATECHOL AEROSOL CCNCE4"j7ATION OETERHINED AT VARIOUS SOLU^ CN CONCENTRATIONS SOLUTION CONCENTRATION (PERCENT w/v) CALCUTATED AEROSOL CCNCE;'(TRATION (ug/L) a RECORDER VALUES (mV) b 0.2 20 : 4 0.2 0.4 75 : 5 0.4 0.6 :65 - 5 0.7 0.8 247 t 7 1.1 1.0 320 : 10 1.4 a b Aerosols were sampled at :CO a1/minute for 3 minutes, collected onto Cambridge filter pads, chemicals eluted from the pads and concentration determined by UV absortrance. Mean and standard deviation from three separate determinations. Millivolt (mV) values determined from direct response of optical sensor during generation of aerosol. 234 CTR COHTRRCTS 029481 11249231 C"'*7a R ITI`1 0-11`1-12 f "3

11n.1 Mrpuil Cenlrcct: CIM•OOIU IALI( 1// UlYU1111UN ANO DISIMIDUIION 0f NADIOACIIYIIT (IM71) IN f(SSUES 01 MC)fl/CIM NIC[ A/ VAAIOUS 11NES A111tt (11'01UN[ 10 66 mg A[NOSp(IlEO J11-CAIECMO(/l UM(IAI(0 f11u1 A i.01CA1([IIOI S0EU110N (Sr. ACI.% 23 .C1/.f) 111E Af1ER EIPOSINtE (IIIMUIES) /(SSUES 0 5 10 30 60 120 IUNG 1.120 (0.26) a 065 (0.20) 101 (0.7]) 300 (0.1)) 16] (0.24) 154 (0.22) 11MtlIMAI[ 6,0/) (0.]6) 026 (0.42) 459 (0.U) 320 (0.22) 220 (0.40) 2S6 (O.Su) 1 NACIfEA t(2 (0.36) AIS (0.32) )) (0.4)) 16 (0.)S) 15 (0.48) L1 (0.)4) IANTMI So1 (0.11) 251 (0.14) 1s1 (0.48) 99 (0.4e) 62 (0.314) 40 (0-.51) 101A1 Il(SrIWl10Nr 10AC1 •.300 1.991 1.554 110 460 461 MI OUO 10,S6/ (0.1u) /.D00 (0.21) 6.144 (o.ls) 2.142 (0.01) )07 (u.14) )01 (e.4u) [SWnUU.U1 210 (0.90) 19 (D.SS) )9 10.6/) 70 (0.))) Iz 10.60) 19 (11.69) N S1fMY1CM 904 (0.26) 1.291 (0.54) 1.005 (0.62) 2,590 (0.46) 664 (0.21) 9SS (0.44) c0 N >>• OLADD[N 1s] (0.34) 90 (0.20) se (0.Q) 26 (0.))) 12 (1.0s) 41 (1.21) W J ~ SE'l[[N 20I (0.20) 9S (0.11) 13 (0.3/) 11 (0.04) 20 (0.40) 29 (0.49) A OL ~ [IOM[Y 4.46e (0.20) 2,451 (0.26) 2,70A (0.65) S9S (0.20) 102 (0.21) 177 (0.20) t•AMCRf AS 439 (0.40) 21S (0.41s) 166 (0.2)) 120 (0.43) 6li (0.29) 16 (0.26j n IEEAiI 509 (0.24) 238 (0.1]) 102 (0.7!) 97 (0.01) S2 (0.10) SS (0.29) 0 tiMIN S2S (0.13) 2ss (0.12) 2S9 (0.21) llt (0.16) 142 (0.07) 141 (0.29) AON[NAl S )s (0.1]) 44 (0.1!) ]) (0.43) 14 (0.64) 5 (1.91) /2 (0.52) 0vAE11ES 6) (0.74) 26 (0.6s) Il (0.26) 2e (0.6y) 14 (0.61) 20 (0.66) ~ ~ YIEIWS 628 (0.56) 241 (0.31) 200 (0.51) 145 (0.22) 64 (0.52) 19 (0.e1) .J-J~ l ir[ti 7,)0/ (0.10) 2.11? (0.12) 1.617 (0.24) IeC (0.12) ]79 (0.20) 424 (0.]S) IOIA( ti00r 36,828 16.915 1].OSI l.)S1 t,)tl6 ),151 ..~a4 Ul MOIN( - »,s~lp •"17 (Q 1[CES 9. N a Coe/flcleel uf e.rl.l/un. ~ OD b $r/Ie Iac1vGeb arlne end fece/ /rw 0 to 120 .In. efter eeyos.re +s! J' -L

F1na1 Report , Contract: CTR-0020 TABLE 143 . ~ COMPARISCN OF TISSUE t~ISTRIBUTIC;i . 'hiiEN -}t-C.ITECHOL IS GIVEN EITFiER AS AN AEROSOL OR AS A COMPONENT i;l 10: 2R1 CIGARETTE SMOKE a PERCENT DISTRIBUTION TISSUE OR SITE AEROSOLIZEO 3H-CAT=CHOL 3H-CATECHOL IN CIGARETTE SMOKE LUNG 0.7 0.5 TURBINATES 0.7 0.2 TOTAL RESPIRATORY TRACT 1.4 0.7 BLOOD 2.9 2.0 LIVER 1.3 0.5 KIDNEY 0.4 0.2 STOMACH 0.7 0.1 REMAINING IYicRRAL TISSUES 1.2 3.8 URINE 92.2 91.2 FECES 0.2 1.5 a 3H-Catec^ol was given as an aerosol as described in text. The H-Catecnol given in cigarette smoke was as described •in Hwang et al., (24). Cata are given for 120 min after exDosure. 287 CTR CONTRACTS 029485 11249235 CT R H N 0 44: ' 7EE5

Final Repur•t Contrdct: C11t-U03U TAULL 149 Uli'U;1IlUl1 At(U UlS11tlUU(1UN (lF ALRUSOLIZLD 3N-12-0-1ETRAULCUNYLPUURUOL-13-ACETATE (TPA) IN UC3F1/CU11 M1CEa NANOGRAMS OF TPA TISSUES AEROSOL CONCENTRATION (ug/L) LUNG LARYNX NEAD STOMACN LIVER SKIN OTHER INTERNAL IISSUES TOTAL 800Y 54 195 (0.23)b 25 (0.80) 29 (0.3 1) 26 (0.46) 4 (0.25) 68 (0.65) 12 (0.31) 360 76 226 (0.20) 40 (0.65) 43 (0.2 8) 13 (0.31) 8 (1.00) 125 (0.38) 16 (0.25) 471 255 (0.29) 4% (0.09) 26 (0.3 1) 7(0.29) 3 (0.33) 42 (0.45) 11 (0.45) 391 0 z 7U a ~ ~ Mice were "nose-only" exposed to aerosolized TPA at the concentrations Indicated for 6 minutes. After exposure, ...{ mice were sacrificed by C02 asphyxiation, and tissues removed, and radioactive content determined. Radioactivity was converted to nanograms TPA using the specific activity of the aerosolized TPA collected on•a Cambridge filter ` ~ Ul sampled during exposure. Data are the means from 4 mice per condition. K ~• b Coefficient of variation. ~.~.. N in A ~ OD . ;0

fln.l kcyort COntra[t: C1N-0030 1A01.E 151 D[PUiI(lull ANU OISPUS11I0N Of RADIOAC11YIlr (D11/) IN INtERNAE IISSUES Of SClfl/C1N NICE AT YARIOUS IINES AfftR fIfOSURE 10 10 u9 AEROSOlIlEO AN-IEIRAOECAN01lN10R101 /}CETAIE (iM)/E . GENERAlEO fRUM A 0.21 IIA SUIUiION (SP.ACT.• 1)  IU~ Ifr/.9) / tINE Af1ER EIIIOSURf (NOURS) IISSUES 0 0.5 1.0 4.0 8.0 24.0 19.0 tuNc 21.109 (^.1Q) 6.9i0 (0.2l) 4.111 (0.31) 916 (0.50) 309 (O.4S) e& (0.45) L11 (0.11) IUNIIINAIL 5,001 ('1-16) 1,691 (0.16) 912 (0.16) 440 (0.56) 169 (0.5:) •1 S1 (1.05) IkAC1tEA 1.766 (0.15) iRb (0.19) 42 (0.62) •1 q •1 •1 IARTNl 1.262 (o.)s) UO (0./0) 104 (0.11) 46 (0.50) 31 (0.U) 9 (0.50) 21 (0.1s) tolAl RISPIRAIORT ERAtI 29.110 9,71] S.]0S 1.443 510 91 166 ~ ~ buao 4.410 (0.31) R.102 (0.10) •.9s2 (0.61) 62s (0.I1) 242 (0.12) 191 (0.L7) z92 (0.60) N ESOMNWS 021 (0 f!) 1 60/ (0 06) 22R (0 26) 20) ISI (1 29 (1 61) <1- •1 A . . . . . . ~ SEONACN 6" (0 8 81) 064 (0.S]) 1 7 926 (0.34) ]21 (0.71) 6 070 (0.24) 7 50) 902 (1 1 se) 242 (0 N ta V ~ tl/IDOE/t . . 1 . 6 (1.13) , 7 (11.61) . 1 (1.121 . <1 . . <4 . 2 (0.91) S1iEEN 110 (0.S1) 100 (0.41) m (0.41) .) (0.56) 35 (0.50) 10 (1.21) 11 (0.19) N n R10Nii 306 (0.31) 950 (0.31) 1.010 (0.11) 186 (0.46) s1 (0.31) 0(1.1s) 30 (0.61) tD a Q IANCR[AS 32 (0.N) 114 (0.31) 1" (0.60) 7s (0.42) 141 (1.61) 0(1.60) 1? (0.)0) IEARt 109 (O.SO) 199 (0.14) 144 (0.21) 13 (0.30) 46 (0.22) 12 (0.50) 14 (0.00) ONA1N 122 (0.0)) 3 (0.14) 41 <1 •1 •1 1 (4.SI) ADRENMS 1 (21.6i) fo (0.66) q (0.OS) •1 •1 41 •I M OYAAIES 9 (0.66) •1 Is (0.66) <1 z6 (0.Se) I N (0.l0) •1 •°st C) DIERui ]2 10.6/) 61 (0.50) ls (0.]0) 41 (0.62) 7 1 (1.)2) 3 (r.15) u (0.12) ~ l1YER 1.271 (0.40) 621 (2.1S) 5.969 (0.20) 2.133 (0.33) 1.099 (0.11) 1)l (O.SIU 141 (O.SS) ..3. ~ Smu INIaSIlU4 lJ1kLE IOTESf1/tE 303 (1.02) 22l (0.14) 1.504 1.019 (0.92) (0.11) ).21s (0.60) 1,610 (0.96) 6.059 6.226 (0.09) (1.00) 9.291 (0.OS) s.s0o (0.69) •04 (0.11) 5.556 (0.6s) 1.019 (0.91) 1.698 (1.06) IolAl. INIEaIAI •0,142 29,091 31.615 22.378 24.1181 0,6z6 3.112 N uR UIE _ 912, 1,269 ],945 • 4,065 e,S4s 27.069 6 1 b21 6S fECES 101 2 ,696 , nu1E 16_O1S (1.1)) 1.911 (0.45) J./6S (1.00) ],-024 (0.66) 1_49e (0_2]) 141 (0.60) 560 (0.17) ' S.rylc tocluJts ur/ue .u1leCtcJ 1.elrec• 0•oJ )0 .Itc.tes. u S..plc Imalw4ts ItLtf tullecltd 6cl.w.• 0 cud 0 Iwrrs.

-% 1 < . CTR CCNTRACTS 0294ee 11249238 CTR MN 044•278

VII. RE?EREaCES 291 CTR CaNTRACTS 029489 11249239 CTR HN 044279.,

rinai Report Contract: CTR-0030 REFEREHCES REFERENCZS 1. Pike, a.C. and Henderson, B.E.. Epidemiology of Polycyclic Hydrocarbons: Quantifying t::e Cancer Risk from Cigarette Smoking and Air Pollution Ef:ects. In: H. Celboin (ed.), Polycyclic Hydrocarbons and Cancer, Academic Press, N.w York, pp. 3:7-334, 1981. 2. Doll, R. and ?eto, R.. The Causes of Cancer: Quantitative Estimates o: Avoidable Risks of Cancer in the United States Today. J. :7at1.-Cancer Inst. 66:1193-1308, 1981. 3. Kouri, R.E., 3illups, L.H., Rude, T.H., idhitmire, C.E., Sass B., and *4enry, C.J. Correlation of Inducibility of Aryl Hydrocarbon Hydroxylase with Susceptibility of 3-Hethylclolanthrene-Induced Lung Cancers. Cancer Lett. 9:277-284, 1980. 4. Henry, C.J., Hillups, L.H., Avery, M.D., Rude. T.H.,.Dansie, D.R. Lopei, A., Sass, 8., iv'hitmire, C.E., and Kouri, R.E. Lung Cancer model System Using 3-Methylcholanthrene in Inbred Strains of Mice; Cancer Res. 41:5027-5032, 1981. 5. Hall, W.C., Lubet, R.A., Henry, C.J., and Collins, M.J. Sendai Virus - Disease Processes and Research Complications; In: :'. Haeaa (ed.), Complications of Viral and nycoplasmal Infections in Rodent Toxicology Research and Testing, Hemisphere Publications, Waahington, D.C. (In press), 1983. 6. Parker, J.C.. The Possibilities and Limitations of Virus Control in Laboratory Animals. . Speigel, S. Erichsen, and H.A. Solleveld (eds.), In: Animal Quality and Models in Biomedical Research, 7th ICLAS Symp., Utrich 1979, Gustav Fischer Verlag, Stuttgart, Nev York, pp. 161-172, 1980. 7. Henry, C.J., Caton, J.E., Stokely, J.R., Guerin, M.R., Lopez, A., Avery, M.D., Dansie, D.R., Henderson, G.M., Cayle, T., Whitaire, C.E., and Kouri, R.E. Deposition and Distribut:on of the Total Particulate Matter of Cigarette Smoke in Mice Using a Large-Capacity Smoke Exposure System. Toxicol. appl. Pharmacol. 58:399-409, 1981. 8. :loneyhun, J.H., Stokely J.H., and Plorant, L. Process and Instruraents Corporation Automatic Smoke Exposure Machine - SEtS II. M.R. Guerin, J.R. Stokely, and C.E. Higgins (eds.), Ini Tobacco Smoke Inhalation Bioassay Chemistry, DOE Report ORetL-5424, Cak Ridge National Laboratory, NTIS, Oak Ridge, Tenn. pp. 19-33, 1979. 9. Higgins, C.E., Gayle. T.M., and Stokely, J.R. Sensor fos Detection of Tobacco Smoke Particulates in Inhalation Exposure System; Beitr. Tabakforsch. 9:185-261, 1978. 292 CTR CQNTRRCTS 429494 11249240 C T F Z P-1 N 04 1, 2 '0

FittaL Report Contract: CTR-0030 RF.pERENCZS 10. Holmberg, R.W. Determination of Particle Size in Tobacco Smoke in Inhalation Exposure Devices Using Methylcyanacrylate Fixation and Electron Microscopy. In: M.R. Cuerin, J.R. Stokely and C.E. Higgins (eds.), Tobacco Smoke Inhalation Bioassay Chemistry, DOE Report ORNL-5424. Oak Ridge National Laboratory, NTIS. pp. 103-118, 1979. 11. Henry, C.J., Billups, L.H., Avery, M.D., Rude, T.H., Dansie, D.R., Lopes, A., Sass. 8., ahitmire, C.E., and Kouri, R.E. A Lunq Cancer Model System Using 3-tiethylcholanthrsne in Inbred Strains of Mice. Cancer Res. 41:S027-5032, 1981. 12. Kouri, R.E., 8illups, L.H., Hall, W.C. and Henry, C.J. A Mouse Lunq Carcinoma Model System. Proc. of the 12th Conference on Environmental Toxicoloqy. AFAMRL-TR-81-149, pp. 35-49, 1982. 13. Mitruka, B.M. and Rawnsley, H.M. Clincial Biochemical and Hematological Reference Values in Normal Experimental Animals. Masson Publishinq U.S.A., Inc., New York, 1981. 14. Rasmussen, R.E., Boyd, C.H.. Dansie, D.R., Kouri, R.E., and Henry, C.J. DNA Replication and Unscheduled DNA Synthesis in Lungs of Mice Exposed to Cigarette Smoke. Cancer Res. 41:2583-2588, 1981. 15. Dinowitz, M., Nims, R., 8hooshan, 8., Kouri, R.E., and Henry, C.J. Induction of Ornithine Decarboxylase (ODC) by 12-0-Tetradecanoylphorbol-l3-acetate (TPA) in Pulmonary Tissue: A Model System for Tumor Promotion in Mouse Lungs. Proc. Am. Assoc. Cancer Res. 21:31, 1980. 16. Benedict, W.F., Banerjee, A., Kanaqalingam, K.K., Dansie, D.R., and Henry, C.J. Increased Sister Chromatid Exchange in Mice Exposed to Whole Cigarette Smoke. Mutation Res. (In Press) 1983. 17. +toessner, J.l., Jr. The Determination of Hydorxyproline in Tissue and Protein Samples Containing Small Proportions of this Imino Acid. Arch. Biochem. Biophys. 93:440-447, 1961. 18. Jick, H., Porter, J., and Morrison, A.S. The Effect of Smoking on the Ag• of Menopause. Lancet 1:1354-1355, 1977. 19. Mattison, 0. and 'Thorqeisson, S. S.. Ovarian Aryl Hydrocarbon Hydroxylase Activity and Primordial Oocyte Toxicity of Polycyclic Aromatic Hydrocarbons in Mice. Cancer Res. 39:347L-3475, 1979. 293 CTR COh4TRRCTS 029491 11249241 CTR I-IN 0"442-81

Final Report Contract: CTR-0030 RETERENCES 20. 21. Ingram, D.L. In: S. Zuckerman (ed.) , Press, New York, pp. 247-273, 1962. The Ovary, Academic Kuroda, 0., Henry, C., Felton, J. Nightingale, M., and Mattison, D. Effect of Cigarette Smoke Inhalation, Intratracheal, or Intraperitoneal Benzo(a)pyrene Treatment on Oocyte Number in C5781, C3H, and (CS7BLxC3H) F Mice Proc. of the Third Annual Meetinq of the Am. Colleqe o1 Toxicology, December 8 - 10, 1982. 22. Henry, C.J., Whitmir.e, C.E., Lopez, A., Dansie, D.R., Avery, M.D., Caton, J.E., Stokely, J.R., Holmberg, R.W., Guerin, H.R. and Kouri, R.E. The Dosimetry and Distribution of Whole Cigarette Smoke Particulates in Inbred Strains of Mice: Comparison of a Larqe Smoke-Exposure Machine (SEM) with a Small-Capacity Smoke Exposure Machine (Walton). In: C.L. Sanders, F.T. Cross, G.E. Daqle, and J.A. Mahaffey (eds.), Pulmonary Toxicology of Respirable Particles. Technical Information Center, U.S. Department of Energy, NTIS. pp. 177-192, 1980. 23. Henry, C.J., Lopez, A., Dansie, D.R., Avery, M.D., Wt:itmire, C.E., Caton, J.E., Stokely, J.R., Guerin, M.R.., Curren, R.D., and Kouri, R.E. Distribution and Clearance of Three Cigarette Smoke Constituents, Dotriacontane (DTC),' Nicotine (NIC) and Benzo(a) pyrene (BP), after Exposure• of Mice to Whole Cigarette Smoke. The Toxicologist 1:139, 1981. 24. Hwang, K.-K., Sonko, 0, Dansie, D.R., Kouri, R.E., and Henry, C.J. Studies on the Deposition and Distribution of Catechol from Whole Cigarette Smoke in BC3F1/Cum Female Mice. Toxicol. Appl. Pharmacol. 64:405-414, 1982. 25. Matsushima, S., Ishiguro, S., and Sugawara, S. Composition Studies on some Varieties of Tobacco and Their Smoke. I. Major Components in Smoke Condensate. Beitr. Tabakforsch. Int. 10:32-38, 1979. 26. Mold, J.D., Peyton, M.P., Means, R.E., and Walker. T.B. Determination of Catechol in Cigarette Smoke. Analyst 91:1189-1193. 1966. 27. Van Duuren, B.L., Katz, C., and Goldschmidt, B.M. Cocarcinogenic Agents in Tobacco Carcinoqenesis. J. Natl. Cancer Inst. 51:703-705, 1973. 28. Hecht, S.S., Carmella, S., Mori, H., and Hoffman, D. A Study of Tobacco Carcinogenesis. XX. Role of Catechol as a Major Cocarcinogen in the weakly Acidic Fraction of Smoke Condensate. J. Natl. Cancer Inst. 66s163-169, 1981. 29. Morimoto, K. and Wolff, S. Increase of Sister Chromatid Exchanges and Perturbations of CsIL Division Kinetics in 294 ' CTR CC3NTRRCTS 423492 11249242 CTR NN 04-4~af-':k

Final Report Contract: CTR-0030 REPERENCES Human Ly:ophocytes by Benzene Metabolites. Cancer Res. 40:1189-1193, 1980. 30. Hwang, K.-K., 3hooshan, B., Kouri, R.E., and Henry, C.J. Synthesia of Triti:Ln Labelled Catechol. J. of Labelled Compounds and Radiopaarmaceuticals. 19:35-38, 1982. 31. Hay, K.R. The Collison Nebulizer - Description, Performance and Application, J. Aerosol. Sci. 4:235-243, 1973. 32. Hwang, R:1C., Sonko, 0., Dansie, D.R., :SuLlinax, D., Kouri, I.E., and Henry, C.J. Chemical Deposition of Aerosolized H-Catechol in BC3F1/Cum Female Hice. The Toxicologist 3:108, 1983. 295 C.TR COHTRACTS 029493 11249243 CTR !!N V• 4, 1/ E+u.8rrt..F

CTR CQNTRRCTS 029494 11249244 CTR HN 044284

r Final Report Contract: CTR-0030 pQ8LIG1TI0K,S VIII. PQBLICATIONS 296 .CTR CONTRRCTS 029495 11249245 CTR VIf 'I 04''4265

Pinal Report Contract: CTR-0030 ppBLICATIONS .. POBLICaTIONS PROM MICROBI'OGOGICAL ASSOCIATES SDPpORTED BY CTR January 31, 1984 CHAPTER, BOOKS Demoise, C.F., Kouri, R.E. and Whitmire, C.E. Cell-Mediated Immunity After Intratracheal Exposure to 3-Methylcholanthrene and its Relationship to Tumor Transplant Growth in C3Fi/f Mai Mice. In: E. Karbe and J.F. Park (eds.), Experimental Lung Cancer, Springer-Verlag, Yev York, pp. 72-80, 1974. Kouri, R.E., Demoise, C.r. and Whitmire, C.E. The Significance of the Aryl Hydrocarbon Hydroxylase Enzyme Systems in the Selection of Model Systems for Respiratory Carcinogenesis. In: E. Rarbe and J.P. Park (eds.), Experimental Lunq Cancer, Sprinqer-Verlaq, New York, pp. 48-61, 1974. Whitmire, C.E., Demoise, C.1P. and Kouri, R.E. The Role of the Host in the Oevelopment of In Vivo Models for Carcinoqenesis ' Studies. In: E.Karbe and J.F. P-asTTc (eds.), Experimental Lung • Cancer, Springer-Verlag, New York, pp. 20-47, 1974. Curren, R.D., Kouri, R.L. and Schechtman, L.lt. Studies on Metabolic Activation of Chemicals for Mammalian Cell Transformation and Mutagenesis. In: N. Mishra, V. Dunkel and M. Mehlman (ads.), Advances in Modern Environmental Toxicology Vol. 1, Mammalian Cell Transformation by Chemical Carcinogen, Senate Press Inc., Princeton Junction, WJ, pp. 319-353, 1980. Henry, C.J., Whitmire, C.E., Lopez, A., Dansie. D.R., Avery, M.D., Caton, J.E., Stokely, J.R., Holmberq, R.W., Guerin, M.R. and Kouri, R.E. The Dosimetry and Distribution of Whole Cigarette Smoke Particulates in Inbred Strains of Mice: Comparison of a Larqe Smoke-Exposure Machine (SSM) with a Small-Capacity Smoke Exposure Machine (Walton). In: C.L. Sanders, P.T. Cross, C.L. Daqle and J.A. Mahaffey (eds.), Pulmonary Toxicology of Respirable Particles. Technical Information Center, Q.S. Oepartment of Energy, aTIS. pp. 177-192, 1980. zouri, R.E.. Schechtman, L.M., and Nebert, D.w. Genetic Control of Carcinogen Metabolism. Int R.C. Kouri (ed.), Genetic Differences in Chemical Carcinoqenesis. CRC Press, Boca Raton, Florida, pp. 21-26, 1980. 297 C.TR CaHTRACTS 423436 11249246 CTR HN 044,2286

Final Report Contzact: CTR-0030 PUBLICATIONS Nayat, 1C.T., 0'Nsi11, B. and Kouri, R.E. Inheritance of Endoqenous RyA Viruses. In: R.E. Kouri (ed.), Genetic Differences in Chemical Carcinogenesis. CRC Press, Boca Raton, Florida, pp. 93-128. 1980. Schechtman, L.H., Henry, C.J. and Kouri, R.E. Exposure, Up-take and Distribution of Chemical Carcinogens. In: R.E. Kouri (ed.), Genetic Dittsrsnces in Chemical Carcinoqenesis, CRC Press, Boca Raton, Florida, pp. 1-20, 1980. Kouri, R.E., McRinney, C.E. and Henry, T.J. Genetic Control of Breast Cancer Susceptibility in Animals. In: Henry T. Lynch (ed.), Genetics in Breast Cancer, Van Nostrand Reinhold, New York, NY, pp. 14-48, 1981. Schechtman, L.M. and Kouri, R.E. Correlations Between Mutaqenesis and Neoplastic Transformation in In Vitro Systems. In: T. Suqimura, S. lCOndo, and H. Takebe (e sa :), Environmental Hutaqens and Carcinogens. Univ. Tokyo Press, Tokyo, pp. 209-216, 1982. Henry, C.J., Hwanq, K.R., Kanaqalinqam, K.K. and Kouri, R.E.' Recent Developments in Inhalation Toxicology: Evaluation o!• Selected Short-Term Endpoints Follovinq "Nose-Only" Exposure of Rodents. Int F. Homberqe= (ed.), Safety Evaluation and Regulation of Chaaicals. S. Karqer, New York, pp. 233-242, 1982. Rouri. R.E., Billups. L.H.. Hall, W.C. and Henry. C.J. A Mouse Lunq Carcinoma Model System. Proc. of the 12th Conference on Environmental Toxicology, AFAKRL-TR-81-149, pp.35-49, 1982. Henry, C.J. and Kouri, R.L. Specialized Test Article Administration etose-Only Exposure and Intratracheal Inoculation. In: H. Salem (ed.), Inhalation Toxicology Marcel Dekker, Nev York, (Submitted) 1983. 298 CTR cohlTRACTS 029497 11249247 C.r f R HN 044 2 8 r21

linal Repott Contract: CTR-0030 pOBLICATIONS ABSTRACTS !Couri, R.E., Ratrie, H. and Whitmire, C.E. Genetic Control of Susceptibility to Cancers Induced by 3-Kethylcholanthrene. •Proc. XI Int. Cancer Conqress, p. 77, 1974. Billups, L.H., Henry, C.J., Whitmire, C.L., and Kouri, R.E. Pathogenesis of Chemically Induced Lung Lesions in Mice. Amer. Assoc. Cancer Research No. 565, 1978. Nayar, K.T., Rouri, R.E. and Levy, J.A. Xenotropic Virus Expressioa and Susceptibility to Chemically Induced Cancer. Proc. Am. Soc. Hicrobiol. p. 272, 1978. Henry, C.J., Billups, L.a., Avery, H.D., Dansie, D.R., Lopes, A., Breth, L.A., Rude, T.H., and Kouri, R.E. - Lung Cancer Model Systems in Inbred Strains of Mice. Proc. Am. Assoc. Cancer Rea. 20:242, 1979. Rasmussen, R.E., Boyd, C.H., Henry, C.J., and Kouri, R.E. DNA Repair is Reduced in Lungs of Mice Exposed to Ciqarette Smoke. Proc. Am. Assoc. Cancer Res. _ t242, 1979. Henry, C.J., Lopez, A., Whitmire, C.E., Caton, J., Henderson, C.M., and Kouri, R.E. - Deposition and Distribution of Total Particulate Matter (TPDt) in BC3F1 Mice After Exposure to Cigarette Smoke. Toxicol. Appl. pharmacol. 48:A5, 1979. Dinovitz, ti., Nims,'R., Bhooshan, B., Kouri, R.E., and Henry, C.J. Induction of Ornithine Decarboxylasa (ODC) by 12-0-Tatradecanoylphorbol-13-acetate (TPA) in Pulmonary Tissue: A Model System for Tumor Promotion in ltouse Lungs. Proc. Am. Assoc. Cancer Res. 21:31, 1980. Henry, C.J., Avery, M.D., Dansie, D.R., Lopez, A., Breth, L.A., Billups, L.H., and Kouri, R.E. The Effect of Exposure to Whole Cigarette Smoke on 3-Methylcholanthrene (MCA) Induced Lunq Tumors in BC3l1/Cum Mice. Proc. Am. Assoc. Cancer Res. 21:126 1980. Henry, C.J., Billups, L.H., Dinovitz, N., Rasmussen, R.B. Avery, M.D., Dansie, D.R., Lopez, A., Breth, L.A., ltulliIIax, H.D., and'Kouri, R.E. Th•:Cttect of Exposure to Whole Cigarette Smoke oa Pulmonary-Mixed Function Oxidase, Ornithine Oecarboxylase, DNA Repair Capacity and• on 3-ttethylcholanthrene (MCA) Induced Lung Tumors in BC3F1/Cum Mice. Symposium oa CoCarcinoqenesis and Biological Effects of Tumor Promoters, Castle of Elmau, D-8101 Klais/Bavaria, Federal Republic of Germany, p. 56, 1980. 299 CTR CL]NTRRCTS 029498 11249248 CTR NN '..,f446*2>88

Yinal Report Contract: CTR-0030 PQBLICATI0H5 Henryr C.J., Lopez, A., Dansie, D.R., Avery, M.D., Whitmire, C.Z., Caton, J.E.. Stokely, J.R., Cuerin, M.R., Curren. R.D., and Kouri, R.E. Distribution and Clearance of Three Cigarette Smoke Constituents. Dotriacontane (DTC), Nicotine (NIC) and Benzo(a)pyrene (BP). after Exposure of Mice to Whole Cigarette Smoke. The Toxicologist 1:139, 1981. Henry, C.J., Collins, M.R., Hall, W.C., Putman, b., Lubet, R.A., Dansie, D.R., Avery, M.D.. McKinney, C., and Kouri, R.E. Effects of Sendai Virus and Vaccine on Short-Term Toxicological and Immunoloqical Markers in Strain A/J Mice. 32nd Annual Meeting, Am. College V.t. Path, p. 127, 1981. Henry, C.J.,* Billups, L.H., Hall, W.C.. Avery, M.D., Dansie, D.R., Mullinax, H.D., and Kouri, R.E. The Effect of Lifetime Exposure to Whole Cigarette Smoke in BC3l1/Cum Mice. Proc. 13th international Cancer Congress, 52, 1982. Henry, C.J., Kanaqalinqam, K.K., Hwanq, K.R.. Dinowitz, M., Lubet, R.A., Hall, W.C., Rasmussen, R., Benedict, W., and Kouri, R.t. The Effect of Exposure to Whole Cigarette Smokee on Short-Term Endpoints ia BC3l1/Cua Mice. Proc. 13th, International Cancer Congress, 51, 1982. Hwanq, K.K., Sonko, 0., Dansie, D.A., Rouri, R.E* and Henry, C.J. Studies on the Deposition and Distribution of Catechol from Whole Cigarette Smoke in BC3l1/Cum Mice. The Toxicoloqis t, 2:46. 1982. Hwanq, K.K., Sonko, 0.. Dansi*, D.R., Huang, S.S., Kouri, R.E., and Henry, C.J. Identification of Urinary Catechol and Its Structurally Related Chemicals from Whole Cigarette Smoke in lSice. Proc. Am. Chem. Society, 74, 1982. Kanaqalinqam, K.K., Reed, S.M., Dansie. D.R., Hall. W.C., Kouri, R.E., and Henry, C.J. Autoradiographic Analysis of DNA Synthesis in Pulmonary Tissues of Mice Exposed to Whole Cigarette Smoke. The Toxicologist. 2:46, 1982. Kanaqalinqam, R.K., Banerj.e, A., Reed. S.K.. Dansie. D.R., Benedict, W.l., Kouri, R.E., and Henry, C.J. Cigarette Smoke Increases DNA Replication in Lunq and Sister Chromatid Exchange (SCE) in Bone Marrow of Mice. Proc. Am. Assoc. Cancer Res. 23:56, 1982. Schechtman, G.H., Gallagher, M.A., Henry, C.J., Kouri, R.E., Gubet, R.A. induction of Neoplastic Transformation and Single Strand DNA Breaks in C3H 10T 1/2 C.lls by Polycyclic Hydrocarbons and Short Chain Alkylatinq Agents. Proc. Ted. Am. Soc. for Experimental Biologists, 641, 1982. 300 CTR CaNTRACTS 029499 11249249 CTR I~IN 0,44:~813-

rinaL Repott Contract: CTR-0030 POHLICATIONS Hwang, 1I.K., Sonko, 0., Dansie, D.R., Mullinax, D.. Kouri, R.E., ind Henry, C.J. Chemical Deposition of Aerosolized H-Catechol in 9C3F1/Cum Female Mice. The Toxicologist, 3:108, 1983. Kuroda, D., Henry, C., Fleton, J. Nightingale, M., and Mattison, 0. e.ttect of Cigarette Smoke Inhalation, Intratracheal, or Intrap.ritoneal Benzo(a)pyrene Treatment on.0ocyee Number in CS7BL, C3H, and (CS7BLxC3H) F Mice. Proc. of the Third Annual Meeting of the Am. CL. of Toxicol., December 8-12, 1983. 301 .CTR C0NTRACTS 029500 11249250 CTR HN 04-4'` S-410

Final Report Contract: CTR-0030 paDLICATIONS MANUSCRIPTS Whitmire, C.E., Salerno, R.A., Rabs-tdt.n, L.S., Huebner, R.J. and Turner, H.C. RNA Tumor-Virus Antigen Expression in Chemically Induced Tumors. Virus-Genome-Specified Common Antigens Detected by Complement Fixation in Mouse Tumors Induced by 3-Methylcholanthrene. J. Natl. Cancer Inst. 47:1255, 1971. Whitmire, C.E. and Huebner, R.J. Inhibition of Chemical Carcinogenesis by Viral Vaccines. Science 177:60, 1972. Whitmire, C.E. -and Salerno, R.A. RNA Tumor Virus Antigen and Tumor Induction by Various Doses of 3-Methylcholanthrene in Various Strains of Mice Treated as Weanlings. Cancer Res. 32:1129, 1972. Kouri, R.E., Ratrie, H. and Whitmire, C.E. Evidence for Genetic Relationship Between Susceptibility to 3-Methylcholanthrene Induced Subcutaneous Tumors and Inducibility of Aryl Hydrocarbon Hydroxylase. J. Natl'. Cancer Inst. 51:197, 1973. '- Kouri, R.E., Salerno, R.A. and Whitmire, C.E. Relationships. Between Aryl Hydrocarbon Hydroxylase Inducibility' and. Sensitivity to Chemically-Induced Subcutaneous Sarcomas in Various Strains of Mice. J. Natl. Cancer Inst., 50:363-369, 1973. Rhim, J.S. and Huebner, R.J. In Vitro Transformation Assay of Major Fractions of Cigarette Smoke Condensate (CSC) in Mammalian Cell Lines. Proc. Soc. Exptl. Biol. & Med. 142: 1003, 1973. Salerno, R.A., Ramm, G.M. and Whitmire, C.E. Chemical Induction of Subcutaneous Tumors in BALB/c and Swiss Mice Infected with Wild Type C RNA Viruses Derived from BAGB/c Tissues. Cancer Res. 33:69, 1973. Whitmire, C.E. Virus-Chemical Carcinogenesis: A Possible Viral Immunologic Influence on 3-Methylcholanthrene Sarcoma Induction. J. Natl. Cancer Inst. 51:473, 1973. Whitmire, C.Eo and Salerno, R.A. Influence of Preinfection of C578L/6 Mice with Graffi Leukemia Virus on 3-Methylcholanthrene Induced Subcutaneous Sarcoma. Proc. Am. Sec. Exptl. Biol. & Med. 144:674-679, 1973. Rouri, R.E., Ratrie, H. and Whitmire, C.E. Genetic Control of Susceptibility. to 3-Hethylcholanthrene-Induced Subcutaneous Sarcomas. int. J. Cancer, 13:714-720, 1974. 302 CTR CONTRACTS 029501 11249251 CTR VIN 044.'12291

Pinal Report Contract: CTR-0030 POBLIC31T20NS Rouri, R.B., Whitmire, C.E. and Benedict, W.F. In Vivo and I Vitro Effects of Cigarette Smoke Condensate Fractions. Proc. m. ssoc. Cancer Res., 16:173, 1975. Kouri, R.E., Rude, T.H., Thomas, P.E. and Whitmire, C.E. Studies on Pulmonary Aryl Hydrocarbon Hydroxylase Activity in Inbred Strains of Mice. Chem. Biol. Interactions, 13:317, 1976. Levy, R.L., Barrington, M.H., Lerner, R.71., Griffin, G.F. and Whitmire, C.E. Immunosuppressive Effects of 3-Methylcholanthrene Given Intratracheally in Various Strains of Mice. Cancer Res. 37:3892, 1977. W'hitmire, C.E. and Lopez, A. Comparison of the Effects of Beeswax:Trioctaaoin and Trioctanion Vehicles on 3-Methyl- cholanthrene, Benzo(a)pyrene, and 7,12-Oimethylbenz(a)- anthacene Subcutaneous Carcinogenesis in Three Strains of yice and One Hybrid. J. Natl. Cancer Inst. 61:1107, 1978. Bremner, T., Reddy, P., Nayar, R.T* and Xouri, R.E. Nucleoside Phosphorylase (NP-2) of Mice. Biochem. Genet. 16:1143, 1979. Kouri, R.E., Rude, T.H., Curren, R.O.. Brandt, R.R., Sosnowski, R.G., Schechtman, L.M., Benedict, W.r., and Henry, C.J. Biological Activity of Tobacco Smoke and Tobacco Smoke-Related Chemicals. Environ. Health Perspectives, 29:63-69, 1979. Levy, J.A., Joyner, J., Nayar, 1C.T* and Kouri, R.E. Genetics of Xenotropic Virus Expression in Mice: I. Evidence for a Single Locus Regulating Spontaneous Production of Infectious Virus in Crosses Involving NZB/BINJ and 129/J Strains. J. Virology 30:754-758, 1979. Kouri, R.E., Billups, L.H., Rude. T.H., Whitmire, C.E., Sass, B. and Henry, C.J. Correlation of Inducibility of Aryl Hydrocarbon Carbons Hydroxylase with Susceptibility to 3-Methylcholanthrene-Induced Lung Cancer. Cancer Letters 9:277-284, 1980. Nayar, 1C.T., Levy, J.A., 0'Neill, B. and Kouri, R.E. Xenotropic Virus Expression and Susceptibility to Chemically-Induced Cancer. Cancer Res. 40:4364-4367, 1980. 303 CTR CQNTRACTS 029502 11249252 CTR VIN 044229-11"221

Final Report Contract: CTR-0030 POBLICATIONS Henry, C.J., Caton, J.E., Stokely, J.R., Guerin, M.R., Lopez, A. Avery, M.D., Dansie, D.R., Henderson, G.M., Gayle, T., Whitmire, C.E., and Kouri, R.E. Deposition and Distribution of the Total Particulate Matter of Cigarette Smoke in Mice clsing a Large Capacity Smoke Exposure System. Toxicol. Appl. Pharm. 58:399-409, 1981. Henry, C.J., Billups, L.H., Avery, M.D., Rude T.H., Dansie, D.R., Lopez, A., Sass, B., Whitmire, C.Z., and Kouri, R.E. Lung Cancer Model System Osinq 3-Kethylcholanthrene in Inbred Strains o! Mice. Cancer Res., 41:5027-5032, 1981. Henry, C.J., Billups, L.H., Avery, M.D., Rude. T.H., Dansie, D.R., Lopez, A., Sass, B., Whitmire, C.E., and Kouri, R.E. A Lung Cancer Model System Using 3-Methylcholanthrene in Inbred Strains of Mice. Cancer Res. 41:5027-5032, 1981. Hwang, K.-K., Bhooshan, 8.. Kouri, R.E., and Henry, C.J. Synthesis of Tritium Labelled Catechol. J. of Labelled Compounds and Radiopharmacuticals. 19:35-38, 1982. Rasmussen, R.L., Boyd, C.H., Dansie, D.R., Kouri, R.E., and Henry, C.J. DNA Replication and Onscheduled DNA Synthesis in• Lunqs of Mice Ezposed to Cigarette Smoke. Cancer Res. 41:2543-2588, 1981. Hwang, K.-K., Sonko, 0., Dansie, D.R., 1Couri, R.E., and Henry, C.J. Studies on the Deposition and Distribution of Catechol from Whole Cigarette Smoke in BC3F1/Cum Female Mice. Toxicol. Appl. Pharmacol. 64:405-414, 1982.. Kouri, R.E., MclCinney, C.L., Slomiany, D.J., Snodgrass, D.R., Wray, N.p., and McLemore, T.L. Positive Correlation Between High Aryl Hydrocarbon Hydroxylase Activity and Primary Lung Cancer as Analyzed in Cryopreserved Lymphocytes. Cancer Res. 42:5030-5037, 1982. Benedict, W.r., Banerjee, A., Ranaqalinqam, K.K., Dansie, D.R., and Henry, C.J. Increased Sister Chromatid Exchange in Mice Exposed to Whole Cigarette Smoke. Mutation Res. (In Press) 1983. Henry, C.J., C.lhard, J:G`., Gosnell, S., Knipscher, R.C., Lopez, R.S. A., Dansie, O.R., Billups, L.H., Hall, W.C., and Kouri. Pulmonary Localization of Particulate Constituent of Cigarette Smoke in Mice. (In preparation) 1984. Henry, C.J., Breth, L.A., Gerhart, J.N., Dansie, D.R., Mullinax, H.D., Whitmire, C.E., and Kouri, R.E. Carboxyhemoqlobin LAVels as a Measure of Cigarette Smoke Exposure in Mice (In preparation) 1984. 304 CTR CaHTRRCTS 029503 11249253 C T R I - I N 0 4 4 ,229.. - 3

?inal Report Contract: CTR-0030 ?pBLIC.ITIONS Henry, C.J., Gayle, T., ?lorant. G.. Dansie, D.R., Greenspan, J., Guerin, a., Holmberq, R., K.K. Xanaqalinqam, and <ouri, R.E. Chronic Iahalation Studies in Mice: I. Facilities and :quipment =or "Vose-0nly" Exposures to Cigarette Smoke. (Submitted) 1984. Hwanq, Sonko, 0., Dansie, O.R., Jaqusiak, M., Kouri, R.E., and Henry, C.J. tdentitication of Urinary Metabolism in aice of Catechol in Whole Cigarette Smoke. (In preparation), 1984. Lubet, R.A., Herscowitz, H.B., Kanaqalingam, K.K., Dansie, D.R., Kouri, R.E., and Henry, C.J. Lack of Significant Isimunosuppression Following Exposure of BC3F1/Cua Mice to Whole Cigarette Smoke. (In preparation) 1984. 305 CTR CChlTRRCTS 029504 11249254 CTR NN 0`-~A2~`~ .