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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("~