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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