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1 1 1 I I i U I , ! /k EXHIBIT No.°7 57*l7 Experimental Lung Cancer Carcinogenesis and Bioassays International Symposium Held at the Battelle Seattle Research Center Seattle, WA, USA, June 23-26, 1974 Edited by Eberhard Karbe and James F. Park With 312 Figures and 144 Tables Springer-Verlao New York Heidelberg Berlin 1974 I 1 i CTR MN 042574

/k 1 i i i 9 The Symposium was sponsored by The Battelle Memorial Institute The Battelle Institute Life Science Program The Division of Biomedical and Environmental Research (AEC, USA) Deutsche Forschu-igsgemeinschaft, W. Germany The program was planned with the cooperation of the Carcinogenesis Program of the National Cancer Institute (NIH. USA) Dr. Eberhard Karbe Chief, Toxicology, Physiology and Experimental Medicine Division, Battelle-Instltut e.V., Frankfurt, W. Germany Privatdozent, Veterinary Pathology, University of Zurich, Zurich, Switzerland Dr. James F. Park Associate Manager. Biology Department, Battelle Pacific Northwest Laboratories, Richland, Wash., USr> tn accord .4n thal Pan of t/y cna/q0 of aa toundr. (•aotdon BanenO. to a0nat ,n tne Iurt/lOr OOucaho- o/ Tan .1 -a tn0 comm'tTant of a.nono to P.ew..qo tne antnlwaon of tn/oaTalan Tn,a s dono n pan ey wopomnq conto..nc.a and T.ol,nqa .nd pq oneo,r.aq.r`q ~O PuDhCaltOn O/ rOprla an0 Pr^qa TOwards tNal OOtlehra. tMa Pupl.eaborl wAdO protecle0 OT cOpqngAl tq- paqVnaT of Ynlan /nlnnOaTOn1. q arblabIa lor IM n.attinq ol rnpto copso, tor acne+anh,P and .oawrcti r to rrIIIo.o IM aoqra." of wonco ano tn. uadut ana. ISBN 0•387•06996•8 Suringer•Verlag New York Heidelberg Berlin ISBN 3•540•05996•t3 Spaoger•Verlag Berlin Heidelberg New York TN,a vOA 41 .u15IF.c/ te ceq+yM ON hqn•t a.e •..r`.e. ..w.el.r tn0 rAolO or pan o/ tn0 n,aur41 a co.c.-.0. a00G1Kyy 1/10ae ol naAaN.04 •OOr-0I•og. •ruM of Inwttapon0. OroadeMenq. rOYoeuetMn Or pnotocpppAq Taeaine 0/ 4nHlr .lorla. aRA Olora•a •- aala sr atl unOr f5• of Uy GO.-aw (NIV'Vm wwNrf OOPwOs re ntM. /N OtAr 1NU1 pn.al0 Ua0. a 10. ,a pa7aaa to the pOel-.hr. In0 anwNnl of nt0 .M t• 0e OO/MrhIn00 Or aqrMTM) .1nl tNe owea,ah0. 0 fay Spneqr•VMaq Sw. rMOrMrq 1974 PnnNd In Gam.nr. TM uu o1 r.q,.,rad nar^... traO.mu11a• alc -n Wa PYEl,pal.On OOea n01 ,.pr 0.00 . h aONmO O/ a apOC,/,C .talOwyrn, tNal aVCA naTOa rtl .t.^'p1 410- tne rOlOranl Prolectlv0 laws anO.OqWalws rV t•rMOrO (ree lr oonorat use OILolPnnnna Jul,ua eont. Mrn.eacnreo•qav BooYbno,n0 awnlocno Wr.rrWOOticarw. G.ob.n. w Grtwanr CTR ~-I~~ 0429 ~ ~

Contents i I Keynote Address: Progress in Respiratory Carc:noqenesls. U. SAFFIJTTI ............................................... 1 SESSION I Methods in Experimental Respiratory Carcinoqenesis and Bioassays. Chairman: S. LASKIN Models in Chemical Respiratory Carcinoqenes:s. S. LASR:y and A. SELLAKUMAR .............................................. 7 The Role of the Host in tie Develo;.ment of :. :iro Models for Carcinogenesis Studies. D.E. WHITMIRE, C.F. DEMOISE, and R.E. KOURI ................................................. 20 The Significance of Aryl Hydrocarbon eyorexylase tnzyme Systems in the Selection of Model Systems f3r Pvsp:ratory Carcino- genesis. R.E. KOURI, C.F. DEMOISE, a-d :.k'. WHITMIRE ....... 4d Pulmonary Carcinogenesis by Two Aryl Ifydrocaroons on Three Mouse Strains. W. H0, K. WILCOX, ana A. F!'RdT .............. 62 Cell-Mediated Immunity after Intratracneal :'xposurc to )-Methyl- cnolanthrene, and its Relationsnip tj T_n.r Transplant Growta in C]H/f Mai Mlce. C.F. DEMOISE, R.i:. I~OL'RI. ino C.E. W1/1T- MIRE ...................................................... 72 The Influence of Carcinogenic Substances Intr N i:eJ Intratra- cheally to Several Generations of l:xscr:no-tal Rats. L.L. GRICIUTE .................. .............................. 81 Large-Volume Intratracheal Instillation of Pirt:cAlate Suspen- sions to Hamsters. D.W. BAXTER and C.D. ............... d6 The Intrapleural Route as a Means for kst:na;:nq Carc:noqeni- city. W.E. SMITH and D.D. HUBERT ........................... 92 Localized Submucosal Bronchial In7ections of Carc:noqens in Dogs. M. OKITA, A.H. COHEN, and J.R. uF.:.FIkL'J .............. 102 Studies of Intrabronchial Particle Deposition Us:nq Hollow Bronchial Casts. R.B. SCHLF:SIVCER, V.R. COHEN, and M. LIPP- MANN ....................................................... 116 i CTR 1-IN 0425>"'S

x i I i I I I The Distribution and Retention of Selected Metals in Rat Tissue after Inhalation of Cadmium Oxide Aerosols. P.D. KAPL:.y, M. BLACKSTONE, and N. RICHDALE ............................... 128 Conception and Mctncds of Experimental Studies in Ger^any tc Estimate the Carcinogenic Burden by Air Pollution in Mar.. 0. SCHM.IHL and K.C. SCHMIDT ............................... 139 Experimental Carcinogenicity and Bioassays of Automobile Ex- haust Condensate and Its Polycycl>,c Aromatic Hydrocarbons. H. BRUNE .................................................. 146 S:'SSION II Multifactcrial Respiratory Carcinogenesis and Related Bioassays. Chairman: P. NETTESHEIM Review and Introductory Remarks: Multifactorial Respiratory Carcinogenesis. P. NETTESHEIM ............................. 157 Morphogenesis of Experimental Lung Tumors in Hamsters: The Effects of Carrier Dust. F. STENBJiCK ...................... 161 Role of Particles in Respiratory Carcinogenesis Bioassay. M.C. HENRY, C.D. PORT, and D.G. KAUFMAN ........................ 173 Effect of Partic::late Benzo(a)pyrene Carrier on Carcinogenesis in the Respiratory Tract of Hamsters. R.L. FARRELL and G.W. DAVIS ..................................................... 186 Chemica: Induction of Lung Carcinomas in Rats. W.H. BLAIR .... 199 Synergistic Effects of Benzo(a)pyrene and N-Methyl-N-vitrc- so;irea on Respiratory Carcinogenesis in Syrian Golden Ham- sters. O.G. KAUFMAN and R.M. MADISON ...................... 207 The Effects of Particulates on Respiratory Carcinogenesis by Diethylnitrosamine. R.L. FARRELL and G.W. DAVIS ........... 219 Respiratory Cocarcinogenesis Studies with Ferric Oxide: A Test Case of Current Experimental Models. D.A. CREASIA and P. NETTESHEIM ................................................ 234 Induction of Pulmonary Tu.Tors in Mice by Oral Administration of a 5-Nitrofuran Derivative. M. KANISAWA ................. 246 Surface Morphology of Tracheal Epithelium in Vitamin A Defi- ct.ency and Reversal. C.D. PORT, D.W. BAXTER, and C.C. HARRIS .................................................... 257 Postinfluenzal Pulmonary Lesions in Vitamin A Deficient Kice. C.G. LOOSLI, J.D. HARDY, and S.F. STINSON ................. 265 Influenza-Virus-Induced Hyperplasla of the Respiratory Tract of the Hamster. C.D. PORT. D.W. BAXTER, D.C. KAUFMAx, and V. GENTA .................................................. 274 Inoculation of Owl Monkeys (ac::a triv:ryz::a) with 7, 12-D,nethyl- benz(a)anthracene and L'crp!ari:aa sa:miri. Induction of Epi- dermoid Carcinoma in the Lung. W.E. GIDDENS, Jr. .......... 280 CTR H~ ~~~~~~

xi SESSION III Bioassays of Respiratory Carcinogens in Tobacco Products. Chairman: W.P. DONTENWILL Review and Introductory Remarks: Bioassays of Respiratory Car- cinogens in Tobacco Products. W.P. DONTENWILL ............. 293 A Modified Method for Locating Labeled Smoke Particles in Or- gans of Syrian Golden and European Hamsters. G. RE2NIK and U. MOHR ................................................... 300 Dotriacontane-16,17-1•C Distribution Pattern in the Respiratory System of Two Hamster Species after Passive Exposure to Ra- dioactive Labeled Smoke. N. KMOCH and 'J. MOHR ............. 309 Cigarette Smoke Inhalation Studies in Inbred Syrian Hamsters. F. HOMBURGER, P. BERNFELD, and A.B. RUSSFIELD ............. 320 Tumorigenic Effect of Chronic Cigarette Smoke Inhalation on Syrian Golden Hamsters. W.P. DON:ENaILL ................... I 331 Effect of Chronic Exposure to Cigarette Smoke on Tumor Incidence in the Syrian Gclden Hamster. A.P. c:EHNER. R.H. BUSCH, and R.J. OLSON ................................................ 360 Carcinoger.icity of Inhaled Cigarette Smoke in the NMU-Pretreat- ed Hamster Larynx. E. KARBE and K. KOSTER ................. 369 Validity of the Sebaceous Gland Tes: and the Hyperplasia Test for the Prediction of the Carc_nogenicity of Cigarette Smoke Condensates and Their Fractions. P. LAZAR and I. CHOUROULINKO'J ............................... ........... 83 Squamous Carcinoma of the Lung from Cigarette Smoke Condensate in Implanted Beeswax Pellets. M.F. STANTCN, M.W. LA1.\RD, and E. MILLER ............................................. 392 SESSION IV Radiation Carcinogenesis and Bioassays. Chairman: J.F. PARK Radiation Carcinogenesis. Introductory Remarks. J.F. PARK .... 401 The Effects of Inhaled Uranium Mine Air Contaminants in Beagle Dogs. R.E. FILIPY, B.O. STUART, R.F. PALMER, H.A. RAGAN, and P.L. HACKETT .......................................... 403 Lesions and Lung Cancers Induced in Rats by Inhaled Radon 222 at Various Equilibriums with Radon Daughters. J. CHAMEAUD, R. PERRAUD, J. LaFUMA, R. MASSE, and J. PRADEL ............ 411 Studies of Pulmonary Carcinogenesis in Rodents Following Inha- lation of Transuranic Compounds. C.L. SANDERS and G.E. GAGLE 422 Lung Irradiation with Static Plutonium Microspheres. E.C. AN- DERSON, L.M. HOLLAND, J.R. PRINE, and C.R. RICHMOND ....... 430 c t° R Nf~ ~c`s~ ~ ~'78

A / 1 I I -xa Respiratory Carcinogenesis in Rats after Inhalation of Radio- active Aerosols of Actinides and Lanthanides in Various Physicochemical Forms. J. LaFUMA, J.C. NENOT, M. MORIN, R. MASSE, H. METIVIER, D. NOBILE, and h. SKUPIySKI ....... 443 PuLmonary Carcinogenesis and Chronic Beta Irradiation of Lung. R.K. JONES, F.F. HAHN, C.H. HOBBS, S.A. BENJAMIN, B.B. BOECKER, R.O. McCLELLAN, and D.O. SLAU:GN ................. 454 Histologic Observations on the Pathogenesis of Lung Cancer in Hamsters Following Administration of Polonium-210. H. LISCO, A.R. KENNEDY, and J.B. LITTLE ............................. 468 Cellular Localization of Intratracheally Administered 212Po in the Hamster Lung Using Autoradiography-of Tli.n Sections from Plastic Embedded Tissue. A.R. KENNEDY and J.B. LITTLE ..... 475 Experimental Respiratory Carcinogenesis: Interaction between Alpha Radiaticn and Benzofalpyrene in the Hamster. R.B. McGANDY, A.R. KENNEDY, M. TERZAGHI, and J.B. LITTLE ....... 485 SESSION V Bioassays of Respiratory Carcinogens. Chairman: M.B. SPORY Review and Introd.:c_o:;• RenarRs: The Role of Organ C•ulture and Cell Cultare Met!%cds in St::dies of Respiratory Carcinoger.e- sis and Ar.ti-Carc:ncqenes:s. M.B. SPO Rv ................... 493 Interactior.s bet•dee^ Radiat:on and Benzo(a)Fyrene in an {•: V::ro Model for Maliqnant Transfo:mation. M. TERZAGHI and J.B. LITTLE .................................................... 497 Dose Dependence of Ca:c:nogen-Induced.Changes in Tracheal Epi- thelium in Organ :i:t-ire. B.P. LANE and S.L. MILLER ....... 507 Topical Application cf Polycyclic Hydrocarbons to Dif.'e:en::- ated Respiratory : t^eliuc in Long-Term Organ Cultures. B.T. ;•SOSSMAN and ...E. CRAIGHEAD ........................... 514 The Bioassay of Carc>.-zqenesis: Effects on the Epithelial Cell Complement of Rat 'racheae Maintained :r v{zr:. D.W. LINDSAY, J.R. JONES, w.J. HIGGI::S, and P.W. BROMrN .................. 521 Effects of Cigarette 5-+oke Condensate (CSC) on Rat Fetal Lang in Organ Culture. I. Ce10C'ROULINKOV and M. MZCHIELS ........ 532 Tracheal Grafts. R.A. GRIESEMER. J. KENDRICK, and P. NETTES- HEIM ...................................................... 539 Studies of Ultras:r•:tt::e, Cytochemistry, and Organ Culture of Human Bronchial Epithelium. B.F. TRUMP, E.M. McDOwELL. L.A. BARRET, A.L. FRANK. and C.C. HARRIS ....................... 54d Studies on Carcinogen Binding in vitro in Isolated Hamster Tracheas. D.C. KAUFMAN, V.M. GENTA, and C.C. HARRIS ....... 564 I c`rR HN 042-579

® The Reversal of Keratinized Squarous Metaplastic Lesions of Vitamin A Deficiency in Tracheobro-r.hial EpitheliLm oy Vitamin A and Vitamin A Analogs in Organ Culture: A Model System for Anti-Carcinogenesis Studies. M.B. SPORN, G.H. CLAMON, J.M. SMITH, N.M. DUNLOP, D.L. NEWTC'., and U. SAFFIOTTI ................................................. 575 Transformation of Nonvirus Producing 8AL9/3T3 Cells by an En- vironmental Extract. M.A. SHERIDAN, D.A. AXLE?, A.J. DENNIS, and R.I. MITCHELL ......................................... 583 Concluding Remarks: Progress in Respiratory Carc:noqenesis 8ioassays. M.B. SPORN ..................................... 590 Subject Index ................................................ 593 List of Contributors ......................................... 605 Ci R t i N 0'-T 2aef 80

/1 Cigarette Smoke inhalation Studies in Inbred Syrian Hamsters* Freddy Homburger', Peter Bernfeld', and A B. Russfield2 'Bio- Research Consultants. Inc. Cambridge. MA 0?td1. USA 2Pathology Department. St. Vincent Hospital. Worcester. MA 0 16 10. USA ABSTRACT Findings were obtained ih 2 lines of tacr••d S;:r:in =•arsters ex-v-s_d to cigarette smcke, and the rvsults will :^_ c:^Fared :i:h _nosa:.- ported by DONTENWILL. Tt:e importance of tne choice of aninal in this type of study will be enphasized. Groups of 102 inbred Syrian hamstcrs of c!- OIC 87.20 and D:0 15.:5 strains (Telaco. Bar Harbor. Maine) •+or.• :•.+icc d~sl;. S da;s a::eek to 8 p.:ffs frcn Fent-icky 1R1 ct::•ir••t:es ef 2 seccn._:a:icn. generated every mir.;,cc in a Malton-~lor::s-aJ Re•:e:se S~cAer T`e sr..:ke exposare to ot:c.,t 1 t: S diluted ercKC laated 15 sec^^ds and was follc•.+ed Gy a il seccnds exposure •: fresh labera::r; air. f0 ani-als of .aci strain were held in cao•'s witmout rar.i;..j :at::- as controls, and 60 a.:Jtticnal control antnals of each strain were treated as tne erreriaencal animals, t•xc.r; that n-3 c =are_:es e:e insert^d into the -,tc`ines. Hr.stopatholoatc stud:c•s •:e:e :e:fer~ed after 45 to 30 «pics :f treatment. In ha-stcrs excosed to s-•cRe. mac:achace clisters czri-aining iron pisnent were seen .n tne psL-o- nar;• paren:t.;ca. espccially fre;•jontly and early :n o'.20 -:-+sters. 2 t::-ors were 'c.ind in the nasopharynges c! s-wke-exposcc ?:0 :S.i6 ar.i-a:s, 1 a`::resarccaa and tne other a cystadencrta. L;•sr:ast:c changes were seen in the larynges aft.r j;.,^roximately :: .ea%s cf str.oke :xpes;tre a nd the :ctal incidence cf such changes in :-; <>-ex- posed ar•.i-ials .as 401 ond 13%, respecti•:elY. Incip:e.^.t:; b.:t still •:ery sz+all. lesi:ns were fo,:nd in tne 1arln_es -. exposed anir.als of each strain after apprcxiaately 80 aeeks c` srck- ing. By 90 wuoks, th~re were mlcrotnvastve carcinomas :n .:? :` .l~e aninals in the more sisceptible BI0 15.16 line and ai in tne less susceptible B:0 87..0 line. This conttr:-s and extends the c_scr•:a- tions of DCNT!!:aILL cn larynceal chanaes. Differences betw^c•+ tne strains used .ill te discussed to emphasize the effect c: ceretic factors on smoke inhalation experiments. * This investi;.+ticn .as supported by a contract from tne `or Tobacco Reseirch - U.S.A., Inc. The vivds expressed in t!-:s ;j=er are those of the au:!%crs and do not necessarily reflect tne =F::+ions of the Ccuncil `or Tcbacco Research. C T R N N 0 42 E:4`31.41

321 Introduction Hamsters are desirable test sub3eccs for tobacco smoke-inhalation studies because they have a much greater' resistance to toxic effects of nicotine than rats or mice (BERNFELJ and UOMBUP.GER, 1972). 2 strains of inbred hamsters (BIO hamsters from Telaco, Bar Harbor, Maine) were used in the present study in order to achieve reproducibility of re- sults and to detect possible strain-related differences in response to tobacco smoke. Materials and Methods 2 strains, the 810 87.20 and BIO 15.16, :+ere selected for chronic toxicity experiments. All animals we:ched 108 : 4 q at the teginning of the chronic studies. Only males were :,sed, r.oused in groups of 6 in 12 x 14 x 6.5-inch polypropylene cages. San-I-Cel, Deodor grade, was used as bedding material. The ani-ai rccm tumperature was kept between 720 and 760F. Fluorescent bulbs were tne only light source and were automatically controlled to operate tetaeen 7:00 a.m. and 7:00 p.m. The animals received ::ayne Mouse Breeder Blox and fresh tap water ad li5it::m. Modified Walton reverse smoking ^ac.`.ines were used, as described elsewhere iHOFFw',:;:: and t:1":CER, 19-01. In :ncse machines, air is pushed through lighted cigarettes by applying positive pressure at the burning end. All animals wore well fitting, perranently-actac.".ed, felt rings around their necks (3/16" thick, 2" outside diamet.r, and 5/8-3/i" inside diameter, depending on the size of the hans:er). The felt rings were reinforced on both sides by thin, ring-snapid aluminum plates of slightly smaller dimensions (1-7/8" outside diameter and 15/16" inside diameter). The heads of the animals were Lnser:,j into the smoking machine by means of these collars, so that only their heads were in contact with the smoke. The bodies remained outside the machine, firr,- ly restrained ty the collars, thus eliminating the need to confine the animals in tubes with attendant excessi:e s.cating and stress. 6 animals could be exposed simultaneously to smcRe inhalation. Since the felt collars prevented the animals :rom groor•.inq their eyes, these were washed with penicillin solution on a co:tcn swat once a day, 5 days/week. Only 1R1 Kentucky Reference cigarettes were used. They are produced by, and were obtained from, the University of Kcntucky, Tobacco and Health Research Institute, Kentucky Researcn Foindation. Compositions of the Reference cigarette (1R1) at 12i moisture were as follows (ATKINSON, 1970): flue-cured lamina, 40.11: fl.+e-cured stem, 14.2t; Burley lamina, 24.9%; Turkish (whole lcaf), 11.61; Maryland lamina, 1.11; glycerine, 2.81; invert sucrose, 5.3%. Smoke was generated from 4 cigarettes burning simultaneously, and was administered to the animals in a 60-second ru.'f cycle. Each cycle con- sisted of a 2-second period of smoKe genuratlon followed by a 15-second period of additional smoke exposure and a41-second period of exposure to fresh air. During the 2-second period of smoke generation, 35 ml of air was push- ed through each of the 4 cigarettes, ;•ivldine) a total volume of 140 ml ~ CTR NN 042=582

smoke which was introduced into the 725 r•1 expes::re cr.arter. A-a3-et- ic stirrer provided instant mixing. The smoke dilu:ian factcr as cal- culated frem the ph;sical characterist:cs :f the maci::ne •.as '25:140 or 5.2 : 1. The smoke concentration inhaled by the hams:ers, in:crrect- eJ for c!:anc^_s due tc exhaled air of the exp-:sed ani-.a:s, •ras :=.ere- f:re approximately 19.:1 of that lea•:ing the r•+outh-end of the cigarette. In corparison, the average concentration cf smoke in the lung cf a human smoker :s a5cut 0.81 (averaae p,.;ff volu•r.e, 35 mI: human f:^c- tional residual capacity, 3.5 liters; human tidal volume, 0.7 liters). It took the smoke less than 1 second to traverse the 6 inches between the mouth-end of the cigarette and the ha-+sters' heads, ass;:rinc that the hamster inhaled smoke no less fresh than that reaching the lungs of the hu:-ar, smoker. Each har.,ster was exposed twice a day, 5 days/week, to 8 consec::ti,!e puff cycles fr---.i the icentucky Reference ciuarettes. T:le first thrc-.jqh eighth puffs from the c:garettes were used, resulting in a butt leneth . slightly longer than 30 mm. The period of exposure ranged :rcn 45 to 100 weeks. Cage-^eld cor•trol animals from each inbred line, of co*parazie ace and :n:tial :eiant. were maintained simultaneo-:sly in the saTe an:-a1 roc-. and were handled in the same way as all other ka^s:ers with re- qard •_- •~P.ekl;• •aely.n.:.^.g, permanently wearing fel:-alurin::m c::lars, e:c.,5u: these control hamsters did not come into contact the smoking ma:::ines. A second ccntrol group consisted of ccr;,araC:e har•sters exnrsed to s~ta:n smoking in tne mac.`.ines, :+hich were caera:ed under standard c:^d:t:cns, except that no cigarettes were :nser:ed into tne-. For each ^f a e 2 lines, BI0 15.16 and 810 B7.:C, 1C2 nam- sters were cxp.csed to smcke, 60 were exposed to sham-smoking cendi- t:o^s, and '.0 •.ere used as cage-held controls. Since it was i-possible to obtain all 222 hamsters required fcr eacn inbred line at :ne sa-e time, animals of the proper in::ial a-e and +eiq^t were ...::oduc^d :nto the experinent in lots of 6, s:acgered over a period of 37 weeks. As far as possible, 1 lot of 6 ant-a:s of eac.`f ef the ccntrols was started at the same time as 2 lots of smoke-exposed hamsters (12 animals). Anir•als •.ere exposed to sr.oke or to control conditions for ~s~-een 75 to 100 •.=e<s. T`ey •aere sacrsf:ced earlier when tncy appeared mcrib,:nd. as :jdzad by consistent weight less or the appearance of ede-a. Some `a^s:ers of all gr=ups were also sacrificed at 45 and 60 weeks. C:-plete autcpsies were performed. The entire resc:ratary tract and any •:tner craans or tissues :;hic.n aF;.eared gress:y a5ncr-rai were s:~d:ed .:s:ol^c::jlll. Lungs were in.':ated and flxc= :n Tel:•:es- nic:ky's :luid in an apparatus especially devised b7 Dr. SACA.MU :ShI- KAiiA /1972). Results 1. Mcr•alit':: As shcwn in Figs I and 2, mortal::f was :•crY :~d in all groups.unti abcct ~e 60th week of the experiwent. a, w-icn ..re :`e animals had a cr,ronol^qical age of approximately 73 :+ee.ks. V~erejftcr, mortality increased rapLdly in all groups. reaching 1C01 ot at:ut 100 weeks. Neither s;rcRe exposure nor sham-smoking had a siq!~i::zan: effect on mortality. There was no difference in survival between the 2 strains. 4J TR / I 1 I 0/' R.v 563

a /1 323 Fig. 1 100 ~ 60 20 l I . ~ i0` 00 Fig. 2 l I 1 I so 60 70 eo NUMBER OF wEEKS L 90 10 2. Changes in Bod wei ht (Figs 3 and 4): In both lines of hamsters, ir3'ividual o y weignts were affected by srr•cke inhalation and, in addition, by mere experimental manipulaticns, s.;ch as sham-smoking conditions and/or stress produced by the latter. Initial veignt in both groups averaged 108 g. The BI0 87.20 ca;e-r.eld controls attained a final weight of 144.3 g, whereas the BIO 15.16 animals attained only 125.0 g. I ~ I L.r f R MN 04,22584

a /It 1 324 150 I u ¢t00 > a 90 0 Fig. 3 Fig. 4 C4GE•NELD 010 15.16 MaYSTERS .0 •5 23 25 30 35 NuM9ER Oir wEEKS ~ _ ... „_ SNaM•SMORING I CIGaRETTE SMOKE - I I 40 45 SO r--~-- . l 90 Exposure to c:;3r--::e s-oRe reduced body weig`: of tc:n :r.ered scrai^s Cy atcjt 10 •; c:r.-i .r.a first 2 weeks of tree:ment. S_csc_uen::;. weights increased a;aln. out se-+e.+hat mcre slo•.ly in :ne 610 15.16 than in the 810 jr:rals. Because of t.•.e low :inal .e:q~: of t-.e cage-held 010 15.:e -aws:e,;s, the weight difference betdeen tne cc,- trols and smo<e--!•:cfed hnmsters was much less in the BIO 15.16 1:^e than :n the B:0 9'.:~ :1,ne. Sham-smok>.ng produced bcdy -e>,qnts :n:er- mediate between :~,zse zf the cage-held controls and t~ie a-+oKe-exFesed animals in line B:0 15.16; it had no effect on body veiq-t in line 810 87.20. ' . CTR . PIN 0,42258=5

a J . . t . • • " . , ~. /1 _.`_ . ~ ' • . .. • . 1 . 323 - i I It thus appears that the long-tera+ response te smoke lnhalation,•, i.e., the failure of the sr..oke-exposed animals ta gair. wei,tit; •.+as' due, at least in large part, to ,3irect e:fects uf smcke :n::alac3qn rather than to nonspecific stress. 3. Larynqeal Lesiois: By far the most important findings in this ex- periment occurred n the larynges (Table 1), ap;:roxicotely two-thir,:s of which were studied histologically (Figs (Tne rvr.,ainder :+~ra transplanted into hamster cheek pouches for ar. experiT.er.t wr.ich wil: be reported separately.) Among the s-o<e-expcse(: hamsters, only 2..,t Cf 48 larynges in the 910 15.16 lir.e +4t) ar.d ? o-t o.` 45 from tne 8I0 d7.20 line (7i) were regarded as h:stol.:,i:ally nor-.a1, as c;.pcsed to 68 to 90% of the larynges from the c4;rresp•:,r.::Lr.q cclcrol a-iTa:s. Tr.e only change seen in the ccntrol _ni7j:s i-.d sr.J:r•-snc..vd anir..als •.as chronic ir.f la-.!~ation. I Table 1. Laryr.geal pathology ~ I --- .'::vrs of animais No of ' 1•r• i. . autopsies i t.p:.r.vlial .lr in:•as:ve .,r..ail Strain Procedure (larlr.x) ~ :crr..sl !•.j n s ca:cer r.illsr.a a - •----~---- - -- -------- , - BI0 15.16 S-iOke 84 (49) 1 2 ja 9 7 S6a--smoke 42 1 36) ~ 2 7 0 0 0 Cace-::eld 40 (25) ~ 17 :. 0 0 Bi0 87.20 Smoke 87 (45) 3 30 2 11 Sham-smoke 44 (30) ~ 27 0 0 0 Cage-held 4d (39) j 31 0 : J () Number of larynges histologically examined. T'.ose not examined •.rere transp:anted. 'Tnese numbers are ir.cludca ...0se animals wi:h epi:he.ial changes. Pathological changes in the smoke-exposed :.jr.st.rs mere classified as follcws: hyperplasia was defined as r pvr:,:ast:c t-.icke:•.tnq cf the squamous epithelium with acanthosis an.: -ilJ -.:cloar dysplasia. Tr.e basal layer remained intact and fairil ;tra:;-.t. :his change was seen in 401 of the larynges of smcke-exwased hl'7 :;.16 nansrers and in 531 of the BI0 87.20 line, but in ncr.o .: : re :_..l rc 1ani-a1 s. Epithelial hyperplasia also occurred with ;,roli!,•raticn of the squarois epithelium in which there was a downgrowtn I I:s i,co tne conr•vc- tive tissue, often in a reticular pattcrn. •..~.~.~r »•;s;,1as,.a and mitotic activity tended to be more intcnse :-.jr. .- :•1yporplasia. This type of change was more frequent in bIJ 1;.10 ir.imals (:Ut) thon in the BIO 87.20 line (13%) and it was atsent in t-e L•cntrols. In many cases of this type of change, the basal layvr .ytJVr"lis was clearly intact. In some, it became very irregular jnJ j;•;.jrvntly isolated cell clumps were seen in the connective tissue, s fr. ;u(qvstinq lymphatic invasion. In advanced stages, this type of r.yFerPl,,sia is excecdinyly difficult to differentiate from early invasive raresncma. This occurrad in 18.81 of the BIO 15.16 animals and in 4% of tnc BIO 87.20 hamsters. It should be emphasized that no distant retastasvs .vre found in any animal. None of these tissue cnanges occurred in :ne :entrols. Small squamous papillomas, similar to those ucc•,.rrinq in sorn of the tracheas and bronchi, were found in the larynqejl vpitnelium in 241 of the 8:0 87.20 and in 151 of the BIO 15.:6 ,ininals erpcseJ to smckc. Occasionally, cnere was pseudoep>.theliomatous ,i...^yrJ•m:h of ce11s .it t.`.e base of pap>.lloma. I I L.r f f"G Hf'i 0422586

/1 Legends see op;.osiee page I i.1 f R HN 0422587

/\ 327 A few larynges of smoke-exposed hamsters also showed chronic inflamma- tion and/or squamous metaplasia of mucus glands. Chronic inflammation also occurred in a few control animals and was associated with sllght epithelial thickening, but never with frank n,vperplasia, pseudoepitne- liomatous change, or papillora formation. There was no significant difference between sham-smoked and cage-he19 controls in this respect. 4. Lun Lesions: 901 of the lungs from BI0 15.16 hamsters and all of t~Tose rom the 8I0 87.20 strain were examined histologically. This revealed both clear-cut strain differences and significant effects of smoke exposure.l Pulmonary macrophages in both strains of hamsters can form small clumps within the alveoli. This tendency is much more pronounced in the BIO 87.20 strain than in the BIO 15.16 strain, as seen from a comparison of the cage-held control groups (44% versus 6i1. It is accentuated by smoke exposure in both strains (929 vers,:s 531, respectively). In neither strain is the incidence of aacrcrr•age cl_rping affected by sham-smoking. There were qualitative as well as quantitative differences in macro- phages clumping among the various groups. Irn nem•atox;lin- and eosin- stained slides of 610 15.16 nax.ster 1.:-g of all crc:ps, and in 810 87.20 hamster lung from t`e ccr.trcl grc,;ps, t`e clumps consisted of comparatively few, s^al;, lc+osely-pacKed macrephayes containing dark brown to black pigment. In lungs of s:^eke-cxpcsej 9I0 67.20 hamsters, the clumps were Iarger, more atundant, and tr.e;. were cc:,posed of very large cells ccntair.ing pale golden picmer•t. :-ese cells were frequent- ly mixed witn polyr••orphonuciear leukoc;tes. Rare smorte-exposed BIO 87.20 hamsters which d:d not have s,~ch cl_:-iped macraphages in their lungs were usually tr.ose found dead of sc:re i::terc:rrant disease. 1The authors are grateful to Drs. STANLEY ROBBINS of Boston University and WALTER BAUER of Washington University. St. Louis..for having re- viewed the histological slides of the most important laryngeal lesions. Fig. 5. Severe epithelial hyperplasia in larynx of male BI0 87.20 hamster exposed to smoke for 78 weeks. Althc::qn some degree of basal cell orientation is retained in the upper pcrcion of this lesion, it is disappearing in the deep portion. ::ote nuclear variation. Hema- toxylin and eosin. X 300 Fig. 6. Severe epithelial hyperplasia in larynx of male 810 15.16 hamster exposed to smoke for 84 weeks. t:ote reticular pattern of growth and complete loss of normal orientation of cells in the deepest portion of the lesion. Hematoxylin and eosin. X 150 Fig. 7. Larynx of male 810 15.16 hamster exposed to smoke for 96 weeks. There is complete loss of normal polarity: nuclei show marked pleomorphisr: 3 mitotic figures are seen. This lesion is difficult to differentiate from early invasive squamous carcinoma. Hematoxylin and eosin. X 190 F~ig 8. Large papilloma nearly occluding the larynx of a male 910 87.20 hamster which had been exposed to smoke for 92 weeks. Hema- toxylin and eosin. X 38 Fig. 9. Cluster of small macrophages in lung of male 810 15.16 hamster exposed to smcice for 45 weeks. Hematoxylin and eosin. X 38 Fig. 10. Multiple large clusters of macrophagos in lung of male 810 87.20 hamster exposed to smoke for 45 weeks. Hematoxylin and eosin. X 38 L..' w/ R  f) i 04f 51...T 8

329 Macrophages of both strains, both the isolated cells ar.d t!:cse occur- ring in clumps, characteristically gave a pos.tive Prussian blue reac- tion for iron. This was intense in the small macropna?es of all 3I0 15.16 animals and of the BIO 87.20 controls. It ~.ras :+eak in the large macrcphages of smoke-exposed 810 87.20 hamsters, which also aipeared to contain yellowish iron-negative pigment. Occasional granules of black, iron-negative pigment consistent with carbon were found in a few..maCro;L~O.Q"•._-....-....~. • ... . Pulmonary parenchyma in both strains contained small foci of ectopic bone not associated with inflammation or other obvious disease pro- cesses. These occurred in 18% of the cage-hald 8i0 15.16 animals, and in 421 of the 810 87.20's. ,Sporadic lungs in both strains showed acute or chronic pneumonitis or contained metastic tumors of some sort, usually adrenal carc:ncma, lymphoma, or leukemic infiltrate. There were no differences in these parameters which could be ascribed to strain or smoke exposure.. Atnormalities of the trachea and bronchi: They were comparatively rare in all groups. A few small patches of squamous metaplasia were seen in 5 to 151 of the BI0 15.16 hamsters, in which the ir.cidence was not significantly affected by smoke exposure. In the cage-held BIO 87.20 animals, squamcus metaplasia was not seen in the trachea and in only 4% of the bronchi. in smoke-exposed BIO 87.20 animals, these figures increased to 261 and 211, respectively. !:o tumors of the air passages were found in control a.^.:r+als of either strain. 3 tenian sqja.-oss papillomas were foun,+j in sroke-exposed BIO 15.16 hamsters, all of which occurred in the trachea. 4 benign squamous papillomas were found in smoke-exposed BI0 87.20 hansters, 2 occurring in the trachea and 2:n the mrin bronchus. 5. Lesions in Xascpharvnx: Sections were taY.en throogh the nasorharynx an3-eclacent struct~:es of the head in all groups of hamsters. Approx- imately half contained only normal tissues. The remainder sh.:+ed a variety of pathological processes, including gingivitis, dental caries, occular inffaaTaticn with p':thisis bulbi, and thromboses cf crt:tal veins. None of these phenomena could be related to smoke exposure. Only 2 tumors were found in the nasophar7.^.x, both occurrir•g :n 3:^- 15.16 anirals •.rrich had been exposed to smoke fcr 60 to 75 weeks. 1 was an adenoid cystic tumor, believed to have originated f:cm -.jc~s glands. The other was a fibrosarcoma that had produced numerous small pulmonary metastases. 6. Heart Lesions: The heart, as such, was not sectioned routinely :n this experiment. However, it was included with the lungs :n 20 to 25% o'f the animals. 251 of the cage-held control eI0 15.16 hearts shc4ed myocardial degeneration, as compared with 171 cf the °IO 8'.20 hearts. This consisted of :^yolysis wlth (in the 8I0 15.16 animals onlyl an abundant infiltration of lynphocytes. Smoke-exposure appeared to in- crease myocardial degeneration in both strains, especially in the 810 15.16 animals. However. the number of animals examined was too small to permit definitive conclusions. Mural thrombi were seen in approximately ).01 of the hearts of both strains. Incidence was not affected by the experimental procedures. CTR NN 042-589

a /1 ® 329 Discussion Chronic exposure to cigarette smoke produces severe hyperplastic changes in the squamous epithelium of the hamster.larynx and is associated with decreased body weight. Smoke-exposure did not in- crease proliferative changes outside of the respiratory tract or the non-neoplastic degenerative c!:ar.ges characteristic of aging hamsters. (Our observations on possible smoke-reiateC changes in the heart are presently equivocal. Further investiga-ion ~iould be highly desirable.) Chronic smoke-exposure significantly reduced survtval :ime in the previously-published DONTENWILL model; no sucn e'!e:t was seen in ours. This difference nay be attributed to t.:e t•:-fold higher carbonmonoxide concentration inhaled by DOt:TENWILL's !%aT.sters than by ours. In fact, previous observations of GG::TE'.W1LL (1970. 1973), showed sustained increase in clood carbex;ne-oq::a::: in his model, while ours produced only transient inc:tases. The point of greatest practical importance to eae:,e from our work is the demonstration of striking stra u: d:f:erca.-ei a-.ong various lines of hamsters with respect to susceptibility to acute toxic effects of smoke, and to hyperplastic resgonse of :ne 1a-ynx to smoke (BERNFELD and HOMBL'RGER, 1972). Animals of tne :r.trec 810 15.16 line have both the highest resistance to smoke or nicot.ne tcxicity and the greatest laryngeal susceptibility - qualities ;reatly increas:ng the sensitivity of the model. Further studies with large n::nbers of animals will be necessary to ascertain the signif:can:e of the laryn- geal strain differences. No tumors of the lung parenchyma following smcke exposure were ob- served either by DONTENWILL or by us. The slightly s:gnificant in- crease in adenomatoid lesions observed by DO'::E':wILL .as not confirmed by us. In both model systems, there was an increase in so-called "smoke cells" in alveoli following smoke exposurt. T`t observation that yellow iron-negative and also black particles ::cur in the macro- phaoes suggests that the particulate pnase of smoke did reach the alveolar wa11 to be taken up by the macrophages. ~:se of the 2 inbred lines in our experiment permitted the conclusion t`at t~ese cells are not directly related to hyperplastic changes in the respiratory tract. They were much more abundant in the BIO 87.20 hansters which had the lower incidence of hyperplastic lesions. References ATKINSON, W.O.: Production of sample cigarettes for tot~acco and health research. Tobacco and Health Conference. 2. 28 (19'Z). BERNFELD, P., HOMBURGER, F.: High nicotine tolerance of Syrian golden ha:nsters. Toxicol Appl. Pharmacol. 22, 324 (1972/. DONTENWILL, W.: Experimental investigations on thL effect of cigarette smoke inhalation on small laboratory animals. In: Inhalat:on Carcino- genesis (M.G. Hanna, Jr., P. Nettesheim, J.R. G:lbert, eds). AEC Symposium Series ho. 18, pp. 389-411. Oak Ridge. Tennessee: U.S. Atomic Energy Commission, Division of Technical Inf. 1970. DDONTENWILL, W., CHEVALIER, H.-J., HARKE, H.-P., LAFRENZ. U., RECKZEH, G.. SCHNEIDER, B.: Investigations on the effects of c`ronic cigarette smoke inhalation in Syrian golden hamsters. J. nat. Ca^cer Inst. 31, 1781-1807 (1973). Ci R. i i N 0 4 ,.!.. v.. .~+~0

/1 330 HERROLD, N.M.: Ind:,ction of olfactcry ncv:oepithelial :-,crs in Syriarr hamsters by diethylnitrosamine. Cancer 17, 11i-121 (195:). HOFFMANN, D., hYNDER, E.L.: Chamber dcvelcrre~: ar.d acresal dispers;or. In: Inhalation Carcinogcnesis (M.G. Hanna, Jr., P.•Ne:tesnei:a, I.R. Gilbert, eds). ACC Symposiam Series no. 18, np. 173-189. oik R:dge, Tennessee: U.S. Atomic Energy Commiss3on, Divlsion of Technical Inf. 1970. ISHIKAWA, S.: Persor.al Cormunlcation 1971. MCCORMICK. A., NICttOLSON, M.J.. 9~%YLIS, M.A., :'tiOEPt:00D, J.G.: Nitro- samines in cigarette smoke condensate. ':ature (New Baol.) 244, 237- 238 (1973). (..r rf f"b iMiN 0'"T' 25..s 1