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0 c zx~ ~°~~ A4 (c) i9LI3 Workshops on Stin Paincn8 and in vivo Carcinogenesis Bioassay. Arfington. Va. November 3-6. 1981 Skin Painting Techniques and in vivo Carcinogenesis Bioassays Volume Editor F. Hombur:er. Cambridge, Mass. 53 fifura and 95 ubles. 1983 S. Karger • Buel • Munchen • Paris • London • New York • Sydney / Ih CTR NN 042317

Contents Workshop on Skin Painring Hontbrs>tr.. F. (Cambndje, Maja.): Introduwon . . . . . . . . . . . . . . . . . . . I Bock. f.G. (Buffalo, N.Y.): Comparative Anatomy and Funaion of Skin as Re- lated to ExperimentaI Chemical Cardnopnesis . . . . . . . . . . . . . . . . 5 BrrnJe/d. P. (Cambtidge, Mass.): Lookin8 back on 20 Yeare of, or an Expc:ir.wn• ta1 Protocol on 'Mouse Skin•PsintinS Studio' . . . . . . . . . . . . . . . . . . 16 Hojfmam. D.: K'yn/er, E. L: Ri.r+uom. A.: La Yoie, E.l.: Heehr. S. S. (Valhalla. N. Y.): Skin Bioassays in Tobaeoo Cardnogenesis . . . . . . . . . . . . . . . 43 Ldw.S.C. (Eau :NiJluone. N.J.): Crnde Peooleum nnd Scleaed Fraaions Skin CanarBiounys .................................. 63 Slalo. T. J.: Fuche.. S. M. (Oak Ridse, Tenn.): Scrsia Differencea and Solvent Ef• feaa in Mou.e Skin CarrinoBenaiu Experimentt Using CarenoBctu, Tumor Iniriators and Promoten .............................. !S BernJeid. P.: Howtbwqo. F. (Cambridge, Mass.): Total Exposure of Micx to Porr• dered Tnt Subetaneea (e. j., SLale) . . . . . . . . . . . . . . . . . . . . . . . . 110 Bernjrla. P.: Hombwrja. F. (Cambridge. Mau.): Skin Painting Studies in Syrian Hamaen ...................................... 121 Van Duynn. B. L: Mrlehwnnr. S. (Ner York, N. Y.): Mouse Skin Applieation in Chemical Careinolenesis ............................. ISd BoeE F. G. ( BufTalo, N. Y.): 'Accxlerated' Skin Painting Methods for Cudno{en Evaluation ........... : .......................... 169 Workshop on in vivo Car,aroanesi: Bioas.tay Aorwbrsrsrr. f. (GambridBe, MasL): Introduction: Cananolenesu Bioassay in Hir toncal Pmpecdre ........... . . . . . . . . . . . . . . . . ...... 162 Wrubrrrn, E.1C. (Betbeeda, Md.): History of tbe Bioaauy Prv~ram of the Na- tional Caooer [ntstitfas ............................... Ii7 Adanv. Jl.A.: Hornbwjer. F. (CtmbridBe, Mau.): Design and Lopsties of Life- time Cardnoleaais Bioauly Using Syriaa Hamuen . . . . . . . . . . . . . 202 c"rR H~~ 042318

Contenta VI foa.l. G. (Cambrid8e. Masa.): Intercurrent Diseaae and Eavironmental Variabla in Rodent Toxicolop Studies . . . . . . . . . . . . . . . . . . . . . . . . . . . 208 Ward.l. S(. (Frederick. Md.): Backiround Data and Varfations in Tumor Rua of Conaol Rau and Mice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 241 Konrbrrre..f. (Cambrid8e, Mau.): Backgreuad Data on Tumor Inddence in Coatrol AnimaLs (Syrian Hainsten) . . . . . . . . . . . . . . . . . . . . . . . . 239 Waid.l. M.: Reztiik G. (Frederick. Md.): Refinements of Rodent Pathology and the Patbolo8ist's Contribution to Evaluuion of Carcino8enais Bioassays . . .66 Xith/and.l. (Princeton. N.J.): The Use of in hvo Caralno8enais Bioassay Data in the Development of Policies Aimed at PRotettin8 Public Health ...... :92 Dororr. W. (Arlington. Va.): The AMA's Posttton on Carano8enau Bioasaays .. 301 Subjea Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 311 c-rI"\ N/ ) 042,319

j ' /h NotiCE fllls rtutertal may be protected by copyrlpt IaN (Title 17 U.& Code). Prog. exp. Tumor Res, vol. 26, pp. 125-IS3 (Karger. Basel 1983) SSkin Painting Studies in Syrian Hamsters' Peter Bernjeld. F. Xoniburger Bio-Reaeuc6 lnstitute, Inc-, Cambrid6e, Mast., USA Introduction Skin painting studies in rnice have served as a tool in toxicology for many years. Their purpose is the detection of tumorigenic activity, or the demonstration of the absence thereof, in a wide variety of materials in- cluding, in particular, tobacco smoke condensate and its fractions. For tobacco-smolce-derived test materials acute toxicity is usually the limiting factor in establishing dose levels which can be safely administered to mice. Since Syrian golden hamsters are known to possess a much higher tolerance to nicotine than mice (IJ, it was logical to investigate the useful- ness of the Syrian hamster for skin painting studiei, to determine whether or not this rodent species, in particular its inbred and first-generation hy- brid strains, are suitable for this purpose, and to compare the results ob- tained in harastets with those in mice. The present paper deals with this subjea: some of the results have already been published in a preliminary publication (2j. Methods and Materials CAem,oolr 7,12•DimetLytbestz(alanthraaae (DMBA), benso(alpyreoe (BaP) and ' •mcNyl- eholanthteoe (MC) were purcbased from EEaatman Orpnic Chenucals in iocbester, N. Y. and 12-O-tetradeonoyl-pborbol-l3-acetate (TPA) from Consolidated Midland I Supported by sranu from the National Cancer Institute of the US Public Health Service (CA-2S0E2) and from tbe Fannie E Rippel Foundation. [..r f ~ f`~". 11N ~4...~ "'Y' ~ 3sr.. 04

a Skin Painting Studies in Syrian Hamsters 129 Corp, Brewster, N. Y. Acetone, Reagent, ACS, suitable for use in 'spectrophotometry', Matheson Coleman BeU atalotue No.AX120-0I served for this study. Tobacco smoke condensate (also called 'tar') was kindly supplied by one of the major national tobacco produas manufactvrers as an acetone suspension containin6 300 mg nonaquo- ous ci6arette smoke condensate/ml (W/V). It was derived from a type of cigarette simi- tar to the 2R1 Kentucky reference cigarette. Anuiw(s Syrian 6olden harnsters (Mesoaiarus awanu) were bred at, and obtained from Bio-Rescarth Consultants, Camb.,t.=e, Mass., either as highly inbred or as fint•6enera• tion hybrid animals. All inbred batnster stnins were detived from pedijreed breedin6 stock by brotber•sister matings for from 30 to 75 generations. The fint•6eneruion hybrid hamster stnin most frequently used in this work wu the BIO6 F1 D Alexander line, brsd from B100 87.20 dams and Bt06 15.16 sires. Both parent strains had been maintained as inbred lines for about 45 generations. Skin•tumor•susceptible SENCAR mice were obtained from the Oak Ridge Ani• mal Resources. Oak Ridge, Tenn. through the courtesy of Dr. T.J.Sta=a. The first•{en• eration hydrid CAFI/1 strain (BALB/cJ 9 x A/J d was used as a mouse of average skin tumor susceptibility and was obtained from the Jaekson Laboratory, Bar Harbor. Me. Xosuint and Feeding ojAainwlt All animals were housed in polyeubonate boxes topped with wire serftz lids: 10 mice or S-6 hamsters were kept in one box. San•1-Cel served as bedding matsrial for both species. Boxes were replaced once each week by clean ones containing fresh bed- ding. Mice and hamstcn were always kept in separate rooms. These were continuously ventilated with an air circulation of from 1 I to 2E complete air changes per hour. The animal rooms were lighted by fluorescent lamps, with a arcadian cycle of 14 h light and 10 h daritness. Their temperature ranged between 70 and 76'F. All animals te- csived Purina Lab Chow and fresh tap water from the city of Cambrid6e ad Gbinan. SinruJtaneous and Nontimylcaneour Eqervnerlu The present study consisted of multiple independent experimenu which were started separuely over a time interval of 2 yeats. For this reason each eaperiment in• cluded its own stmulaneous negative and positive control jroups. Neptive control groups were made up of animals receiving either no treatment, or of animals treated topically .nth the vehicle (acetone) only. In the positive oontrol aonpt, hamnm re- ceived a single dose of from 60 to 200 µi DMBA, or mice were administered a single dose of ..S µt DMBA and repetrdve doses of 10 µi T?A/mouse/week. Unless stated otberwise, each table or figure preaented herein persaim to data ob tained simultaneously tn any one di these e+tpenments, while tbe dau of several tables, of several figures or of tables and figures combined may pertain to one and the same esperiment. /1 Lr' "m i f N 4.,,d 4,G. 32,1i.

Bernfeld/Hombur8er 130 Allocation of Anima/s to /ndivtdua/ Groupi In each independent, simultaneous experiment, the animals were assigned to all .a,xrimental and control troups in such a manner that the mean body weights were the same in all groups of one species and sex, and that there was approximately equal body weight distribution in all 8roups of the same species and sex. Except in the exper- iments in which tumori8enic response in ditTerent hamster strains was compared, ani- mab of each independent, simultaneous experiment were of the same a8e, usually be- tween 60 and 70 days. at the time of first treatment. Application oJ ChemicaLr to the Animalr' Skin Haafstera were shaved over the entire area of their backs from the scapular area to the base of the tail and from flank to tlank. A volume of 0.8 ml of a solution of the chemical in acetone, or of acetone alone, was delivered and spread out over an 8 inI area of the shaved back of each hamster, by use of a glass pipette. Frequency of admin- istration ranged from a single application in the beginning of the experiment (in the ase of tumor initiators) to two or thtee applications per animal per week for the dura- tion of the experiment (in the ase of tumor promoter, acetone or tar). When very large doses of BaP or MC (over 1,000 y{/bamster) were used as inieiator; the total dose was split into 1.000 µs portions which were given on S consecutive days of the Ist week (to- tal amouot: 5,000 y{), or on 5 consecvtive days per week during the initial 4 weeks (to- tal amount: 20,000 W. Mice were shaved on an approximately I in= area of the lower part of their backs, adjacent to the tail. Test solutions in acetone, or acetone alone, usually consisting of a volume of from 0.02 to 0.1 ml. were applied on, and were spread over the shaved area of the skin. Test solutions were administered to mice by means of disposable, plastic I-mI tuberculin syringes without needles, in combination with Hamilton repeating dis- pensers which allow to dispense a volume oi 0.02 ml or multiples thereof. Frequency of admitsistruion in mice was the same as in hamsters. Shaving was performed in both species by means of electric animal hair clippers, usually immediately before the first administtadon of test materials and vtSte or twice each week thereafter, as needed. ~ E..hrrw. q[ Skia Trnwa /1l1 aaistvia wers obaerrW for thi presence of the skin tumors at least once each week. Grtus observations of skin tumors were supplemented in many instances by his- topathologic study or the skin after planned sacrifice or spontaneous death of the ani- mals and after their necropsy. The skin response, as judged by 8ross observation, was expressed as the incidence of tumon: i. e., as the numbers of animals with skin tumors in percent of the total num• bera of animals at risk. In addition, the numbers of tumors per animal were counted. Fnally, after some of the initial independent et vriments had been terminated, it was found opportune to determine also the total surface of all melanomas in each hamster. This was aeeompl'uhed by counting the numbers of skin melanomas in each hamster with less than I mm in diameter, then of those with I mm in diameter, with 2 mm, 3 mn. ete, and to calculate the tota) melanoma surface by arbitrarily assuming that (i) each skin lesion had a square shape and (ii) all melanomas of less than I mm in diame- ter had a diameter of 0.05 mtn L.r f f'~. i' i 1f 'i 042322

Skin Painting Studies in Syrian Hamsters 131 Table !. Response to DMBA and/or TPA in mice and hamsters Treatment of animalsI Tumor inndence• after Animal species Animal strain Animal sex initiator (DMBA)=, y.j promoter (TPA)t, µi 12-13 weeks 19-22 weeks 46-47 weeks Mice SENCAR F 25 - 0/S0 0/45 0/45 SENCAR F 25 10 40/49 (81.6) 4s/49 (89.4) 44/47 (93.6) SENCAR F - 10 0/49 1/47 (2,1) 3/43 (7.00) SENCAR F - - 0/50 0/50 0/48 SENCAR M 25 10 28/28 (IC:j 28/28 (100) 28/28 (100) CAFI/1 F 23 10 7/49(143) 24/49(49.0) 39/49s(79.6) Hamsters FI D F 25 - 9/9 (100) 9/9 (100) - FID F 25 10 9/9 (100) 9/9 (100) - FI D F - 10 3/9 (33) 5/9 (55.6) - FID F - - 0/9 0/9 - FID M 23 - 48/49(98.0) 49/49(100) - FID M :S 10 48/48(100) 48/48(100) - FI D M - 10 0/49 20/49 (40.8) - FID M - - 0/49 0/49 I All materials applied on a I in2 area of the back in mice, and on an 8 in2 area of the back in hamsters. In mice, the amounts shown are, therefore, those administered per animal. In hamsten, the amounts given per animal were eight times jrcater than those indicated in this table. 2 Amuunt rer syuare inch uf skin, given once to each animal at the beginning of the e:periment. 3 Amount per square inch of skin. given every week to each animal. 4 Number of animals with skin lesions/number of animals at risk (percent with skin lesions in parcn- theses). s After 4: weeks: animals were sacrificed thereafter. Results Comparison of Tumorigenic Response in Hamsters and Mice As ezpected, mice responded to topical applicaaon of initiator and tumor promoter by developing skin papiQomas and/or epidermoid car- cinomas. In contrast, no such lesions were seen in hamsters which exhibited, however, dermal melanomas as a result of the same treatment. In addition to this fundamental qualitative difference in the response of the two animal species to topically applied carcinogens, there were also marked quantitative differences between hamsters and mice in this regard. From the data in table I it is apparent that: (1) A given dose of carcinogen (25 µg DMBA/in= of skin), even when administered without promoter, caused skin tumors in 98-IOOo/o of the [er i f'ti. . f i >f 'i ti-.+r '""f' 2,r3 2:3

a A 1 Bernfeld/Homburger 132 1o 20 30 40 NuMBERS OF WEEKS 0 / _ _ C eo so Restx;nse of hamsters aod mier to dermal admieistncion of OMBA and/ or TPA. A: male FI D hamsters. after a single dose of :S µi DMBA/in: of skirt. result- in$ in nw8tnanr me/monnar. d: female or male SENCAR miee, after a single dose of :3 ys DMBA/in= of skin and repeotive doses of 10 4 TPA/in=/week. resulting in po- jsJlonsar and/or cassnornas. C: male FID hamsten, after repetitive doses of 10 µs TPA/ie= skin/week, resulting in rrma!" ne+i. D: femak SENCAR mice, after repetitive doses of 10 µi TPA/io-' skin/week, reaultinj in ron pooiUoma.t. E: female or male SENCAR mice, after a single dose of 23 " DMBA/in= of skin. remaining wirhour n- rPotise in tbe skin. hamsters in only 12-13 weeks, while the same dose of DMBA without promoter induced no tumon in any of the SENCAR mice in up to 47 weeks. (2) There was no ditYerence between male and female hamsters in their response to DMBA alone. (3) With a single dose of 25 µg DMBA/in2 of skin, SENCAR mice developed skin tumon only when administration of initiator was fol- lowed by repetitive doses of promoter, i.e.. 10 µg TPA/in2 of shin/weeVc. Such treatment with promoter caused no additional effects in hamsters after topical application of DMBA. (4) Administradon to mice of TPA alone caused papillomas in a small number of >• imals (in 2% after 19-22 weeks). In hamsters, TPA produced a much higher incidence of skin lesions (in 41-56oio of the ani- mals after the same length of time), but in contrast to the malignant mela- nomas obtained with DMBA alone or with a combination of DMBA and L. r TR i-If"'f 0"'! z3 ~""!'

a /h Skin Painting Studies in Syrian Hamstets 133 TPA, the lesions caused by TPA alone were of a benign nature, as will be demonstrated later in this paper. (5) In confirmation of results by DiGiovanni et al. [31 and by Xen- nings et al. [4), SENCAR mice exhibited a considerably higher susceptibil- ity to skin tumor induction than other mouse strains, in the presc.nt case the fust-generition CAFI/J hybrid strain. (6) Although the doses of carcinogen (initiator) and promoter were the same in both species, when expressed in amounts per unit surface area of skin, these same doses were about twice as high in hamsters when referred to the body weights of the animals [2~ This is because hamsters received the same amounts per square inch of skin as -mice but over an eightfold larger surface area. Hence, hamsters were given eight times larger amounts of the chemicals per animal than mice, while their mean body weights were only about four times higher; i, e., hamsters weighed 145 g on the average and SENCAR mice weighed 37 g, 35 weeks after be- ginning of skin treatment. The time course of these developments is illus- trated in fig. 1. Nature of Skin Lesions Skin tumors appearing in both SENCAR and CAFI/J mice as a con- seqrience of initiation with DMP.A and subseqt;er.t promotion with TPA were papillomas and, in many instances, epidermoid carcinomas, as as- certained by histopathological study of selected tissue samples. In con- trast, no papillomas were seen in hamsters where the effects of a single skin application of DMBA alone or of DMBA followed by repetitive TPA treatment manifested themselves by the appearance of black skin le- sions. At first, these latter were small, black or dark brown spots of less than I mm in diameter which were not elevated over the surrounding skin. In DMBA-treated hamsters, these dark spots grew as time pro- gressed, they became more intensely pigmented, then began to protrude and finally reached, in some instances, a size of as much as I inch in di- ameter (fig. '). Histopathological studies showed that this transformation from flat, small, dark spots to elevated black tumors represented a grad- ual transformation from benign melanocyte accumulations (nevi) to ma- lignant melanomas. Lesioas of 2 mm or more in diameter were usually malignant melanomas (rig. 3). Occasionally, a metastitic melanoma was found in the lungs (fig. 4). Skin lesions of less than I mm always con- sisted of benign melanocyte accumulations. These occurred especially in TPA-treated hamsters (curve C of fig. 1). C~"~ ~~ ~~~..~.'~

d 1 2 Betnfeld/Homburter 13s Fij.I. Malignant melanomas and multiple smaller melanocyte accvmulatioro in a male FI D hamster, 35 weeks after having re- ceived a single dose of :00 ug DMBA over an 8 in-' area of the skin. The two black spots symmet- rically loated one on each side of the hamster, are the flank or4ana. i Fij.l. Photomicrograph of a primary skin melanoma in an Fl D hamster, 56 weeks after adminis- tration of a single dose of :S y.g DMBA/in= over an 3 in- area of the back. c"f"R NN 0402-32("~

s Frg.l. Photomierofraph of a metastatie lung melanoma in the ume hamster de• seribed in figure 3, at two diffetsnt maptirications. Fit. S. Subcutaneous melanoma aanaplanted in the second tumor generation. I8 weeks after grafting the tumor from a mak FID into another male FID hamster. C~' ~~ ~~N +~~ ~~~ ;~"

a /h Bernteld/HomburZer 136 30 10 . 10 20 30 40 50 60 ta.'ME?ERS OF WEEKS Fis.6. Average numbers of inelanomas per hamster upon administration of DMBA and/or TPA. F - Male FID hamsten + DMBA + TpA; A - male FID ham- stees + DMBA alone: C - male FID hamsters + TPA alone. The malignant nature of the melanomas was further ascertained by transplanting large tumors into recipient hamsters of the same Fl hybrid strain and of either sex, where these gnfts grew to large subcutaneous tu- mors of more than I inch in diameter (fig. 5). Such transplantable mela- nomas are now being carried in the fourth tumor generation. Mulriplieity and Size of Melanontat and Nevi in Xanurert The total number of darkly pigmented skin lesions in the S in= treated area of the hamsters' skin increased rapidly as time progressed. At 30 weeks after DMBA administration it reached an average number of about 30 lesions per hamster (fig. 6). At that time, most of these lesions wers 2 mm in diameter and, hence, were malignant melanomas. There were no significant differences in the numbers of melanomas per hamster when DMBA was administered alone or in conjunction with TPA as a potential promoter. Melanoma counts in individual hamsters exhibited a near normal distribution, with maximurn values of 43 and 50 melanomas per hamster (fig. 7). Crl ) 1 )W 7 1 1 0423.24.d'

Skin Painting Studies in Syrian Hamsten 137 a I t I L A 0 10 20 30 40 50 00 0 10 20 30 40 50 60 NUMBERS OF MELANOMAS PER HAMSTER Fej.7. Numbers of inelanomas per male Fl D hamster. 30 weeks after dermal ad- ministration of (a) a single dose of 200 y.B DMBA, and /D/ a single dose of 200 µ8 DMBA and repetitive doses of 80 y.i TpA/week. Amounts of DMBA and TPA pven are per hamster, i, e., per 8 in- uea of skin. Hamsters treated with TPA only had much fewer lesions (curve C in fig. 6), but their incidence reached 100oio in another experiment (curve C in fig. 1). Some of these latter black spots were very small, and their count was, therefore, quite inaccurate. ConsequentJy, their average num- ben per hamster differed somewhat among nonsimultaneous experi- ments and reached the value of ten spots per hamster in one experiment. Such small pigmented spots would even be detected in FID hamsters treated with acetone only, but never in hamsters which were shaved and received no skin treatment. For the most representative evaluation of the skin response in ham- sters, it was deemed necessary therefore, to take into consideration not only the incidence of pigmented skin lesions (percent animals with skin lesions) and the multiplicity of these lesions (numbers of lesions per ham- ster) but also their size, resulting in the measurement of the total mela- noma surface for each hamster (see Methods and Materials). The exam- ple given in table II demonstrates the usefulness of calculating group means of the total melanoma surface, where neither the melanoma inci- dence alone, nor the mean values for melanoma multiplicity alone, were apt to distinguish between the response of three groups of hamsters. To- tal melanoma surface was not determined in our earlier experiments. dLJ t~ TR MN 042- '~21= ~

Bernfeld/Homburser 138 Table 11. Detetmination of total melanoma surface in male FI D hamsters 31 weeks after be8innin8 of skin treatrnent Skin treaunent, Melanoma incidenceT Melanoma multiplicityJ Melanoma size (ran8e)4 mm Total melanoma surfaceJ. mm= 100 µ8 DMBA6 6/6 (100) S.S (2-10) < 1-4 14.1 (9.2) 20 µ8 TPA7/week 13/ 13 (100) 8.3 (2-20) < 1-2 2.4 (1.9) 032 ml acetone3/week 15/19 (78.9) 2.9 (0-I I) < I-I 0.30 (0.93) I Amounts per hamster, i.e., per 8 inl area of skin. I Number of animals with melanomas (or nevi)/number of animals studied: percent in parentheses. 3 Mean numbers of inelanomas per hamster, ranges in parentheses. 4 Diameter. s Meana, standard deviations in parentheses. 6 One sin8)e dnse. I In 0.32 ml acetone, every week. s Every week. a 20 30 40 50 a NUMBERS OF WEEKS ftt.d. Dose-response relationship for melanoma formation in male FID hamsters after dermal administration of DMBA. Amounts of DMBA shown are per hamster. i.e- per 8 in= area of skin. [...x TR f f f''{ 0""f' s:3:.910

. Skin Painting Studies in Syrian Hamsters 139 I r ~ 5 [ 33.3'q DMBA ~ F ~f ~10 DMBA R M /100,q DMBA ~ 0 [ 333.q DMBA I a 0 10 20 30 40 50 NUMBERS OF WEEKS f71.9. AveraBe numbers of melanomas per male FID hamster after administra- tion of increasing doses of DMBA. Amounts of DMBA shown are per hamster, i.e., per 8 in: area of skin. 00 O V > 42 ~ Q i20 J10 `, `~ 2 123 +c 33 3 10C 333 Xooo :O,DOSEOFCMpA .G :Iv'5:. PEp kAM$TER ) Fit.l0. Dose•response relationship for melanoma formation in male FID ham- sters after appiiauon of DMBA to their skin. Curves A and B were obtained in non- simultaneous experiments. The data of curve A and those of figures 8 and 9 oripnated from ooe and be se.e e.periwent; th~o.a of wrve B aad ol table 11 us derived from another experiment. Ncvertheless, the data for tbe tya values from the expenment of curve B agree well with those of curve A(data not shown). Scale on ordinate to the left peruins to curve A; scale on ordinate to the right to curve B. ~ ~ r 1 * Z n 8~ '9 S 2 C~~R H~~ ~~~~~~ 1

Bernfeld/Homburger 140 ToDk 111. Melanoma formation in Ft D male and female hamsters upon skin administration of DMBA' i Sex of Skin treatment3 Number of Number of hamsters with rumonJ after hamsten DMBA3, µs Aoetooe'iweeh. ml hamsters ! weeks 10 weeks 12 weeks 14 weeks Males 200 - 13 2 7 12 13 200 032 13 0 1 7 12 0.32 13 0 0 0 0 Females 200 - 13 0 2 2 8 200 0.32 13 0 3 6 l2 - 0.32 13 0 0 0 0 I Experiment not simultaneous with any other experiment reported herein. 3 Amounts per hamster. 3 Ooe single dose. 4 Every week. s Melanomau or nevi. DosrRuponse Relarionship There was an excellent dose•response relationship of melanoma for- mation in hamster sftin, when the response was evaluated (i) by the tumor incidence (percent of animals with melanomas or nevi; Gg.8), or (ii) by the tumor multiplicity (average numbers of melanomas or nevi ,per ham- ster; fig. 9), (iii) by the total melanoma surface per hamster (curve B of rig. 10). In the latter case, a near-stnight line, relationship existed be• tween the logarithm of the dose of DMBA and the logarithm of the re- sponse (melanoma surface per hamster). A straight-line relationship was also apparent between the logarithm of the dose of DMBA and the recip- rocal value of t. the time when S0oio of the hamsters had tumors, an ob- servation directly derived from the tumor incidence (curve A in fig. 10). Melanoma Formation in Hamsters of Both Sexes Male FI D hamsters were slightly more susceptible than females to melanoma induction after a single topical dose of DMBA (lines 1 and 4 of table III). When DMBA administration was supplemented, however, y by repetitive applications of acetone to the shin, both sexes appeared to be equally susceptible to tumor formation (lines 2 and 4 of table 1I1). This phenomenon may simply be due to an increased penetration of the carcinogen in the female hamster, as the consequence of acetone treatments. C.r f R f f N 0"'f' 2..:3 31.=

Skin Painting Studies in Syrian Hamsters 141 Table IV. Absence of promoting effects of TPA on DMBA•induccd skin melanomas in FI D hamsters' Sea Number of hamsters Skin trutment= h a~1e~ udied Skin response melanomaa total incidence after melanoma6 DMBAs. TPA/weelc4, acetone/week. 6-9 10-12 22-21 surface', mm, Vi µi ml weeks weeks weeks Males 200 80 48 - 48 48 - 200 - 0.32 49 - 48 49 - - . 80 - 49 - 0 20 - - - 032 49 - 0 0 - Females 200 80 - 9 3 9 9 30.6 200 - 0.32 9 1 9 9 47.1 - 80 - 9 0 4 S 0.25 - - 032 9 0 0 1 0.08 Males 33 20 - 6 3 6 6 1.75 33 - 032 6 4 4 5 0.75 10 20 - 6 2 6 6 1.13 10 - 032 6 . 0 0 3 0.17 - 20 - 18 6 16 17 1.64 - - 0.32 24 1 13 14 0.70 Males 5 80 - 6 1 4 6 - 5 20 - 6 0 3 3 - 5 4 - 6 0 2 4 - 5 - 032 6 0 0 3 - - 80 - 6 3 5 6 - - :0 - 6 0 I 3 - - 4 - 6 0 0 0 - - - 032 6 0 0 0 - Males 1 40 - 9 0 6 9 1.0 1 - 032 9 2 2 04 0 - 40 - 9 2 6 6 0.47 - - 032 9 2 . 4 2 0.19 I The raults sbown in this table were obtained in five nonsimultaneous experiments. Each simultaneous expenment is separated from the nonsimultaneous experiments by a new entry io the first oolutm. =.trnounts indinted are per hamster; i.e, per 8 int area of skin. 3 One single dose at beginning of experiment. ' In 032 ml of acetone. amount administered every week. s After deductin8 the oumber of animals that had died premuurely. ' Nelanomas or nevi. After weeks; not available for two of the earlier experiments. s Two nevi had reQesaed t.d' ~' }~e . i i i 7 042333

Bernfeld/Homburger 142 Table V. Failure of tumor-promoting et7ccts by dermal administration of TPA in male FID hamsters after tumor initiation by gastric intubation of DMBA Tre•ement of hamstersI Skin response after 23 weeks DMBA by gastric = TPA by dermal J melanoma' incidence3 total melanoma' Melanoma' diameter, mm intubauon , mg application . µs surface, mm= mean maximum 10 40 9/9(100) 23.9 1.1 3 10 - 9/9 (100) 31.3 1.1 4 - 40 6/9 (67) 0.5 - - 0/9 (0) 0 - - ~ Amounts per hamster. ~ One single dose at beginning of expenment. ~ Administered every week, on a surface area of 8 in-' of skin. ' Mclanomas or nevi. s Numbers of animals with lesions per numbers of animals studied: percent in paren- theses. Absence of PPOmoring Effects by TPA Because of the effecu of acetone administered repetitively, as de- scribed in the preceding paragraphs, and since promoter (TPA) is usually administered in acetone solution, it is imperative that promoting expen- ments in the hamster include appropriate negative control groups in which the administration of initiator is followed by repetitive acetone treatments. In a series of five consecutive experiments, in which the dose of in- itiator (DMBA) was raised over a 1-200 range and that of promoter (TPA) over a 1-20 range, it was demonstrated that TPA did in no in• stance cause promotion of DMBA-initiated tumor formation in male or female Fl D hamsters (table IV). These findings are in contradiction with data published by Goerrr/er et al. (SJ who observed tumor promotion in hamsters by topically admin- istered TPA (37 µg/hamster/week, given in three portions of 20 nmol each) after having given these animals a single dose of 10 mg DMBA as initiator by gastric intubation. A similar experiment performed by us with male FID hamsters (in lieu of Goerrde.i female hamsters from th• Ger- man Cancer Research Center, Heidelberg, FRG) yielded dermal melano- rnas in the control group without TPA treatment, but did not show any additional tumorigenic effects upon skin administration of 40 µg TPA/ CTR _ e e N 042~`~ 34

Skin Paintin; Studies in Syrian Hamsters 143 hamster/week (table V). The results in table V reflect the skin response after 23 weeks. At no time before or after that duration was there any evi- dence for TPA acting as a promoter. Consequently, in our hands, TPA has never acted as a tumor promoter in the hamster. Influence of the Generic Background of Hamsters on DMB.t-lnduced Melanoma Formation Susceptibility to DMBA-induced melanoma formation was studied, in addition to the FID hybrid line, in 14 13106 inbred ctrains and in one other Fl hybrid strain (table VI). In contrast to all other results presented herein, the data in table VI were not obtained in a simultaneous experi- ment and the animals were of a rather wide age spread (from 3 to 6 months of age) at the time of treatment with DMBA. This lack of simul- taneiry of experimentation and the large age spread applied to a compar- ison of conditions both among the various strains and within some of the strains. Hamsters were used for this series of experiments as they became available from our breeding colony. There was no indication that these factors (age of the hamsters and simultaneity of experimentation) influ- enced in any way the results obtained. The results in table VI clearly show that the genetic background of the hatr,steiz greatly modified their susceptibility to tumor formation. Three inbred and one F1 hybrid strain were more susceptible than the FID strain, while at least 12 strains were less susceptible, including two strains which appeared to be resistant to the effects of topically applied DMBA. In the more susceptible strains, there was mainly an increased melanoma surface, especially in the PD4 strain, while the rate of tumor formation was about equal to that in the FI D hybrids. In hamster strains with lower tumor susceptibility there was, however, not only a markedly smaller tumor surface, but also a considerable lengthening in the time needed for tumor formation. There was no difference between the tumor susceptibility of male and female 87.20 hamsters and only a small but not significant difference between male and female FID hybrids, according to the data of table VI; both sexes of F1 D hybrids also exhibited approximately the same re- sponse in another experiment (table III). In contrut, female 1.5 hamsters were markedly more susceptible to melanoma inducement by DMBA cttan maks of that inbred strain (table VI). The coat color of the hamsters did not appear to be related to the susceptibiliry of the strains to skin melanomas. A white coat color ap- CTR MN 04'2335

Bernfeld/Homburter 144 Table V1. Response of diverse inbred and FI •hybrid strains of Syrian hamsters to skin administration of a sin8le dose of 60 µs DMBA Strain desig• nationt Coat color Sex Numbc, of hamsters Melanoma= incidence (o.b) after(weeks) Total melanoma= surface(mm=) after (weeks) studied 6-10 I1-IS 20-30 39-45 11-IS 20-30 39--t5 PD4 acromelanic white M I I 36 100 :.3 162 FI BJ a8outi F 6 83 100 32.5 72.=9T anophtAalmic white M 6 100 _8.5 1.5 acromelanic white F I S 17 60 93 25 FIBJ asouti M 6 67 100 24 FID• aeouti M 6 83 100 4.5 IS 82.73 lilac M 3 33 100 12 87..0 rust F II 27 100 II 87.20 rust M 8 25 100 9.3 tPI rust M 5 40 100 3.5 •.:2 a8outi M 4 S 1.5 acromelanac white M 8 S0 63 4 121 t cteam M 12 0 17 _. 1037 piebald M 8 88 1.5 15.16 tawny M 6 33 100 0.:5 4.U a8outi M 8 25 7.88 atun M 4 25 41.56 cream banded M 4 0 0 86.93 white ~1 6 0 0 1 8100 inbred strains unless stated other.nse. listed approximately in order of decreasing rumor suscep- tibiliry by, primanly, total melanoma surface. 1 %/elanomas or nevi. s Fnt•8encraaon hybrid strain (87.20 dams x 1.5 sires). ' First•8encntion hybrid strain (87.»0 dams x 15.16 sires). pears both among the most and lhr k= sticc.eptih.e sctains, as well as amons a strain of intermediate susceptibility. Nonpigmented skin tumors were observed in two of the inbred ham- ster strains; i.e., in PD4 and 1.5 hamsters. Some of these tumors were rather large (3-4 mm in diameter) and were i'entiC~ed as amelanotic ma- lignant melanomas by histopathologic study. AlI of these amelanotic mel- anomas eventually became darhly pigmented, a transition which hap- pened within a few days of their detection in some instances, but which lasted up to several weeks in other cases. Pigmented and nonpigmented CTR IIN 0 42-~~3 6

Skin Painting Studies in Syrian Hamsters 145 Ta61e Vll. PiBmentation of inelanomu in two hamster strains' Hamsters Pigmentation of Total number of inelanomas Total melanoma surface;. mm= strain sex total number involved melanomas (all hamsters) all hamsters mean per lesion PD4 males 6 dark 14 243 17.4 6 colorless 4 38 9.5 ts darlc and/or la 281 15.6 1.S females 15 colorleut dark 18 127 7.0 IS colorless 17 . 253 14.9 13 dark and/or 3$ 380 10.9 colorless3 I Nonsimultaneous experiments. • t 28 weeks after administration of 60 µi DMBA to PD4 hamsters, and 20 weeks after administruion of 60 y.8 DMBA to 1.5 harnsten. 3 AII melanomas combined, regardless of pi8mentatioa tumors usually occurred simultaneously in the same animals. In PD4 male hamsters, only L' o/b of the melanomas were not pigmented (4 out of 18, table VII); the total surface of amelanotic lesions in all hamsters un- der investigation was only 13.5 0ro of that of all skin tumors (38 out of 281 mm=), and their mean surface area per lesion was barely more than one half of the surface of the pigmented melanomas (9.5 vs. 17.4 mm2). In contrast, as much as one half of all skin lesions were amelanotic melano- mas in female 1.5 hamsters (17 out of 35), their combined surface in all hamsters involved was two thirds of the surface of all skin tumors, and they were twice as large on the average as the pigmented melanomas in that strain. Effects of Carcinogens Other than DMBA on Hamster Sadn Topical administration of BaP to the skin of male Fl D hamsten was studied over a l:6,000 range of the dose of carcinogen, that of MC over a 1:1,500 range. Promoter (TPA) was administered weekly to additional groups of hamsters at seleaed dose levels of initiator. For the purpose of comparison, the effect of DMBA was studied simultaneously at two dose levels. The results of this experiment are shown in table VIII in which each line represents one group of 6 hamsters, except for the negative con- Cf R MN 04'~..~3'"?

Bernfeld/Hombur8er 146 Table Nlll. Effects of BaP and MC. oo the skin of male FlD haauters Skin treatmend Total melanoma surface per DMBA=, µ8 BaPs, µ6 MC2, µi TPA/weeks, µi hamster after 31 weeks, mm= - 0.a0• 10 2.4 333 - 1.2 100 - 14.1 3-33 - - 0.80 10 - - 0.65 10 - 10 2.45 33.3 - - 0.50 100 - - 2.0 100 - 10 2.3 333 - - 2.4 1.000 - - 0.88 1,000 - 10 2.1 s,00o+ - - 0.45 20,0000 - - 4.8 - 10 - 0 - 10 10 2.1 - - I00 - 0.67 - - 100 10 1.7 - - 1.000 - 0.54 - - 1,000 10 1.15 - - 5,000s - 0.50 I Amounts per hamster; i. e., per 8 in= of skin. I Amounts given at bepnning of experiment only. 3 Amounts pveo each week. 4 Assimals of this aoup were trested witb 032 ml acetone/hamster/week. $APDlied in rive portions of 1.000 µi on 5 consecvtive days. • Applied in 20 portions of 1.000 µi durin8 4 consecutive weeks (on 5 days/week). trol groups, i.e., 24 hamsters receiving no skin treatment (line l) and 18 hamsters receiving promoter (TPA) only (line 2). Treatment with carcin- ogen in all groups of this experiment was performed or started simultane- ously. Melanoma or nevr. incidence was monitored at weekly intervals and total melanoma or nevus surface was determined every 6-9 weeks. Only the final data on melanoma surface are shown in table VIII. At that time CTR 1`°'~~ 0~#`~~'~..~8

Skin Paintin` Studies in Syrian Hamsters 147 (after 31 weeks) melanoma or nevus incidence had reached 100% in vir- tually all groups including the control group, contributing nothing to the tumorigenic effects of the chemicals studied. The data in table VIII show that, while 100 µg of DMBA caused a significant response after 31 weeks (14.1 mm2 of melanoma per hamster), up to 50 times higher doses of either BaP or MC induced no melanomas or other skin tumors. A weak and somewhat ambiguous response was ob- served after administration of 20,000 µg BaP where, in addition to a total of 63 black spots of less than I mm in diameter, one darkly pigmented tu- mor of 2 mm and one nonpigmented tumor of 3 mm diameter were seen in 6 hamsters. Consequently, the potency of BaP to cause melanomas in hamsters is more than 200 times smaller than that of DMBA. According to the data in table VII1, the potency of MC is not much different from that of BaP. In the average mouse strain, DMBA is only about three times more tumorigenic in the skin than BaP (6J. Administration of TPA did not cause any promotion in hamsters primed with either BaP or MC. Effects of Tobacco Tar on Hamster Skin - An experiment on the effects of cigarette smoke condensate, also termo-d tobacco tar, on hamster and mouse skin, with and without prim- ing the animals with DMBA, was conducted simultaneously with the first experiment on the effects of DMBA in these two species. The results are shown in table IX, and they are comparable to those in table I; some of the results of table I were repeated in table IX for easier com- parison. The most striking result, apparent from the data in table IX, is the relatively high tolerance of hamsters to tobacco tar. Between 86 and 90o/b of the female SENCAR mice and 75oio of the male SENCAR mice had died within the initial 12-13 weeks of treatment with tobacco tar, while only from 8 to 200/0 of the female hamsters had died in almost twice that length of time (19-2.' weeks). This is in spite of the fact that the amount of tar applied per animal was eight times greater in hamsters than in mice, or about twice as high per unit of body weight (see above for mean body weights of the animals). The observation of skin melanomas in hamsters whose skin has been treated with large amounts of tobacco tar was rendered impossible, be- cause of a heavy buildup of residual, nonabsorbed tobacco tar on their skin. Consequently, tobacco tar treatment had to be suspended for I C~°R MN ~4~~~~9-

Tabk IX. S:'- response in FIl) hamsters and SEN('AR mice to lopical administration of tobacco tar with and without priming by nMllAr Animal Animal Animal Number of Treatment of animalsl Tumor incidcnces afler species Strsin tea animals f)MBAs,pt tobacco 12-1) 19-22 46-47 atudied tar'i, m8 weeks weeks weeks Mice SENCAR F 10 - ISO 0/5 0/5 1/3()3ah) SENCAR F 50 25 ISO 0/7 0/7 2/6()3aib) SENCAR F So 25 - 0/50 0/I5 0/I5 SENCAR F 50 - ! o/so 0/50 0/4e SENCAR M 28 25 ISO 0/7 0/7 1/7(14%) Hamsters flf) M S0 - ISO N()' 0/46 0/16 FIt) IL1 SO 25 ISO NOr 10/I5 (88.9aib) 0/3)0 F11) M SO 25 - 18/19(98a.e) 49/49(100%) - F11) M S0 - ! 0/49 0/49 - t For comparison, lines 7, 4, 1 and 9 of this table were Iaken from table I, lines 1, 4, 11 and 14, respectively. These caperiments were con- ducted simultaneously. s Materials applicd on a I int area of the back in mice, and on an I in area of the back in hamsters. In mice, the amounts shown are, therefore, those administcred per animal. In hamstcrs, the amounts given per animal were eight times 8reater than those indicated in this table. s Amount per square inch of skin, given once to each animal at the beginning of the eaperiment. 4 Amount per s4uare inch of skin, 8ivat evcry week to each animal. s Number of animals with skin lesions /nuotbcr of animals at risk (percent with skin lesiuns in parentheses); papillomas (or carcinomas) in mice, me(anomas or ncvi in hamstcrs. • No acetone treatment in the cunuol 8roups. r No observations madc, residual tar ptcvcntcd detection of inelanumas. s After 37 wccks; animals sacnficcd thcreaftcr. ~ OIL W

a /\ Skin Painting Studies in Syrian Ham-ters 149 week prior to the intended skin observation. In spite of the larger amounts of tobacco tar administered to hamsters than to mice (twice as much per unit body weight, eight times as much per animal, but equal per square inch area of skin), no melanotic lesions or nevi could be de- tected in hamsters after 37 weeks. In comparison, from 14 to 33% of the few surviving SENCAR mic%. treated with tobacco tar had papillomas af- ter 46-47 weeks. Priming of tobacco-tar-treated mice with DMBA revealed no pro- moting activity of the tar, but the numbers of applicable surviving mice were much too small to conclude that no promotion actually did occur. In hamsters, the effectu of DMBA overshadowed the detection of any possible promoting effects of tobacco tar. Thus, the hamster is not an appropriate species to study the tumori- genic effects of tobacco tar by skin tests, in spite of the high tolerance of the hamster to the acute toxic effects of nicotine (1), confirmed herewith. Diuursion Our findings on the formation of malignant skin melanomas in Syr- ian hamsters upon topical administration of DMBA confirm various p;:bli;ttcd reports (7-11J and further demonstrate the differences in re- sponse to DMBA application between hamsters and mice. Urethan is an- other carcinogen found apt to cause skin melanomas in hamsters (12-14), and skin melanoma causation by DMBA in other species was reported in the guinea pig (1S) and the gerbil (16). Despite our awareness of the differences in pathological response to DMBA and to other carcinogens between mice and hamsters, it has been pointed out only recently that the hamster is exceedingly more suscepti- ble to skin tumor induction than the mouse (2, 17J. The latter authors at- tributed the observed phenomenon mainly to promotion by croton oil, (i, e., by TPA), an effect which we cannot confit4t, however. This high susceptibility of the hamster manifests itself by (a) an extremely rapid re- sponse which is clearly demonstrable within l I weeks of a single skin ap- plication of 333 µg DMBA (4.17 µg DMBA/in= of skin) and (b) by a re- sponse to extremely small doses of DMBA; i.e., to a single application of 3.33 µg DMBA/hamster (0.417 µg DMBA/in2 of skin). As a comparison, an amoumt of 25 pg 0443lVmovee (25 yq DMBA/in' of skin), applied to SENCAR mice, the most skin-tumor-susceptible mouse strain known (3), CTR t~~~ +c~~ ~..~41

Bernfeld/Homburger ISO did not cause any tumon in as much as 47 weeks, unless promoter (TPA) was given in addition to the initiator. Much of the previous work on skin melanoma induction in hamsters had been done with Syrian white hamsters (11, 14J, and it has been sug- gested that nonpigrnented dermal melanocytes with nascent pigment, as occur in the white hamster, are moro susceptible to the carcinogenic ef- fects of DMBA than melanocytes in which the gtanules are saturated with melanin, as in the golden hamster (181. Our results do not appear to support that view, since they place the white hamster strains both on the top and on the bottom of the scale of susceptibility of hamster strains, and since they show only one out of the four white hamster strains tested to possess a markedly higher melanoma susceptibility than many of the golden hamster strains. In spite of reports in the literature on an alleged promotion of the DMBA-induced melanoma formation in hamsters by croton oil (171 or by !PA (SZ, our data clearly demonstrate the absence of such promoting ef- fects. The first of these reports (17) does apparently not include control groups without promoter and, hence, there is no evidence that promotion actually took place. In the work claiming the existence of tumor promo- tion in the hamster by TPA (31, the initiator (DMBA) was given in mas- sive doses (10 mg) by gastric intubation, as opposed to dermal applica- tion of about 100 times smaller amounts. This manner of administering DMBA did not, in our own hands, cause any tumor promotion, however. We assume that the marked difference in promoting effects between the work of Goerrtler et al. (SJ and our own is due to the use by the German workers of a hamster strain, the genetic background of.which is not de- scribed and is unknown to us. It is conceivable that in some of our own inbred hamster strains exhibiting very low susceptibility to dermal mela- noma formation (i.e., in those in which DMBA alone did not induce any melanomas), TPA might well cause tumor formation. This hypothesis was not tested, however. While it has been established that skin application of DMBA, a: wet! as of urethan, causes detinal tumon in the hamster, other carcinogens, such as BaP and MC, proved to be completely refractory up to doses as high as 5 mg/hamster, and an amount of 20 mg BaP/hamster caused only a very weak and somewhat equivocal response. Although being much more prone to dermal tumor induction than the mouse, when DMBA is the carcinogen, the hamster is, on the other hand, considerably more resistant to the tumorigenic effects of BaP and MC than the mouse. C `t°I ! I 1N 04234.n..e

Skin Patntin; Studies in Syrian Hamsters ISI The reasons for these species differences are not known to us and may be manifold. Among the more plausible explanations may be the absence in hamster skin of certain of the specific enzyme systems (arylhydrocarbon hydroxylase. AHH) which convert the polycyclic hydrocarbon adminis- tered into the ultimate carcinogen. The same reasoning may apply to the failure of tobacco tar to cause skin tumors in the hamster, even though the amounts of tar tolerated by the hamster by far exceed those that can be administered to mice. Summary 14 Inbred strains and two first-=eneration hybrid strains of Syrian hamstert, rang- ing in coat color from white to $olden, were given single applications on the skin of their backs of DMBA. or of BaP. or of MC. without or with weekly treatment with tu- mor promoter (TPA). Cigarette smoke condensate (tar) was also applied to the skin of some hamsters. without or with initiator. The tumoriSenic effects of these treatments were observed and were compared with those obtained in SENCAR or CAFI/1 mice. The hamster responded to topical administration of DMBA with the formation of dermal melanoma,s, as opposed to the appearance of papillomas in mouse skin. The susceptibility of the hamster to DMBA-induced skin tumors was considerably Breuer than that of the moct susceptiblc'mouse str.ir,, the SENCAR mouse. A single dose of only 3.33 µs DMBA/animal, or 0.417 µi/in2 area of skin, caused melanomas in ham- sters, and 12.3 ys DMBA/in- area of skin produced melanomas in 504e of the ham- sten after only 9 weeks, while 25 µs DMBA caused no skin tumots in SENCAR mice in 47 weeks, unless promoter was also administered. Melanoma formation was preceded by the appearance of small, pigmeated, be- nign nevi which Frew rapidly to tumors of up to I inch in diameter, became malignant when : mm in stu, as judged by histopathologic studies, and Qew to highly pig- mented. subcutaneous melanomas when transplanted into other hamsters. Occasional pigmented lunj metutatic melaeomu were seen. There was an excellent dose-re- sponse relauonship with re1ard to the average latency period in hamsters. to the num- ben of melanomas per hamster (up to 50 tumors per animal) and to the total surface area of all melanomas per hamster. The response of male and female harnsten to DMBA was about the same. There were marked difTercrteea in susceptibility to DMBA-induced skin tumors among various inbred hamster straitu, two of the strains remained refracsor,r to the efTeas of DMBA and some produced only a weak response. The coat color of the hamsters was unrelated to the DMBA-induced skin tumor su:cep tibtlity. Unlike in mice. TPA caused no skin tumor promotion in hamsters at any of a wide range of DMBA iniuating doses and at various ptomotet doses "se.6 ;fael+ef two polycyclic hydrocarbons, other than DMBA, proved to be more than 1,500 times less tumorigenic in hamster skia than DMBA- Doses of 5.000 ya BaP or MC per ani- mal caused no skin tumors in hatttsten in 31 weeks, and the response to a dose of I CT R M N 0 4 2- 3 4 3

Bernfeld/Hombur8er 152 20,000 µi BaP was weak and doubtful. Attempts at promoting the tumor response to BaP or MC in hamsters with TPA remained unsuccessful. Hamsters tolerated skin appliwtions of tobacco tar much better than mice, and a dose of 150 mg tar/in2 area of skin killed from 75 to 904 of SENCAR mice in 12 weeks, while 90% of the hamsters survived 22 weeks and 32-664 survived 47 weeb. In spite of this low acute toxicity of tobacco tar in hamsters, there was no tumori8enic raponx of tobacco tar in hamsten after 47 weeks. . Rejerenett I Berefeld,P.: Homburter,F.: High nicotine tolerance of Syrian golden hamsters. Toxicol. Appl. Pharmscol. 22: 324 (1972). 2 Bernfeld. P.: Homburger, F.: Rapid skin earcino8enesis in the Syrian hamster. Proc. 72nd Annu. Meet. Am. Au. for Cancer Research. 1981, p. 81. 3 DiGiovanni,J.; Slata,T.J.; Boutwell,R.K.: Comparison of the tumor-initiating aaiviry of 7,12-dimethylbensia)attthracene and benzo(a/pyrene in female SEN- CAR and CD• l mice. Caraino:enesis 1: 381-389 (1980). 4 Henninp, H.; Devor. D.; Wenk, M. L; Slaµ T.J.; Former. B.: Colburn, N. H.; Bowde0.G.T.; EISjo,K.; Yuspa.S.H.: Comparison of rwo-stage epiderrnal ar- clno8enesis inidated by 7,12-diamethylbengs*rtthtaane or N•metltyl-N'-oitro-N- nirroaoguanidine in newborn and adult SENCAR and BALB/c mice. Cancer Res. 41: 733-779 (1981). 3 Goetttler, K.; Loehrte, H.: Schweizer.J.: Hesse. B.: Two-stage tumori8enesis of detatal melanocytes in the back skin of the Syrian golden hamster using systemic initiation with 7,12-dimetdylbenx(a)anthraeene and topical promotion with l:-O- tetradeonoylphorbol-13-acetate. Cancer Res. 40: 1 SS-161 (1980). 6 SIaBa.T.J.; Fscher.S.M.; Triplett,LL: Nesnow,S.: Comparison of complete unrino8enesis in mouse skin: the induction of papillomas by tumor initiation- promotion a reliable short term asuy. J. Am. Co1L Toxicol. 1: 83-99 (1982). 7 Della Portt.G.; Rappapoti H.: Saffiotti, U.: Shubik P.: Induaion of melanotic lesions durin8 skin earsanoBenesis in haauters. Archs. Path. 61: 305-313 (1956). 8 HorninB,E.S.: lndumon of pituitary tumors and melanomas in the golden ham- ster. Ciba Fdn. Coll. Endoa.12: 22-29 (1958). 9 Ghadially, F. N.; Barter,J. F.: Histogenesis of experimentally induced melanouc tumorn in the Syrian hamster (Cncenu ourvnn). J. Path. Baet. 19: 263-27I (1960). 10 illman,O.: Ghadially, F. N.: Coat colour and experimental melanotic tumour pro- duetion in the haauter. Br.1. Cancer 14: a83-488 (1960). I l Rap9aport,H.: Pietra,G.: Shubik,P.: The induction of melanotic tumors resem- bling cellular blue nevi in the Syrian white hamster by cutsneous applicauon of 7,12-di,metbylbeaija{aatbncene. Caneer Res. 21: 661-666 (1%1). 12 PSetra,G.: Shubik. P.: Induction of inelanotic tumors in the Syrian golden hamster after adminisrration of etbyl arbamate. J. aatts. Cancer Inst. 2J: 627-630 (1960). 13 Toth. B.: Tomads, L: Shubik P.: Multipotential carclno8enesis with urethan in the Syrian golden hamster. Cancer Res. 21: 1336-1 Ss 1(1961). 14 Vesaelinovitclt,S. D.: Mibailovicls. N.: Richter, W. R.: The induction of malignant C °~R PIN 0~' ~".,~44

Skin Painting Studies in Syrian Hamsten 153 melanomas in Syrian white hamsters by neonatal exposure to urethan. Cancer Res. 30: 2513-2547 (1970). I S Pawlowski. A.: Haberman. H. F.: Aravindakshan Menon, l.: Skin melanomas in• duced by 7.12-dimethylbenuethrscene in albino guinea pip and its similarities to skin melanomas of humans. Caacer Res. I0: 3632-3660 (1980). 16 Quevedo. W. C.: Fleischmann, ft D.: Dyckman,l.; Jimbow. K.: Bienieki.T. C.: iJl- trastruetunl observations on DMBA-induced dermal hyperpi8rnentation and blue nevus•like tumors in the Mongolian gerbil. Proc. Soa e:p. Biol. Med. 163: 461-A68 (1980). 17 Berhelhammer,J.; Hook. R R.; Smith. G. D.: lnduaion of inelanomas in hamsters with 7,12-dimethylbenr(.)anthracene and croton oil. Fed. Proc. 40: 745 (1981). 18 Nakai. T.: Rappapon, H. A.: A study of the histogenesis of experimental mela- notic rumors resembling cellular blue nevi: the evidence in support of this neuro- genic origin. Am.J.. Path. 43: 175-199 (1%3). P. Bernfeld, Ph.D., Bio-Research Iostitute. lnc., 9 Commercial Avenue. Cambrid8e. MA 02141 (USA) cri'! Ii N 042ti3"7` 5