epidermalcarcinogenicityofbis(2,3 …cancerres.aacrjournals.org/content/39/5/1718.full.pdf ·...

[CANCER RESEARCH 39, 171 8-i 725, May i 979]0008-5472/79/0039'0000$02.00

Epidermal Carcinogenicity of Bis(2,3-.epoxycyclopentyl)ether,2,2-Bis(p-glycidyloxyphenyl)propane,and m-Phenylenediaminein Male and Female C3H and C57BL/6 Mic&

J. M. Holland,2 D. G. Gosslee, and N. J. Williams

Biology Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37830 (J. M. H.], and Computer Sciences Division (D. G. G.j and Y-12 Plant Laboratory(N.J. WI, UnionCarbideCorporation,NuclearDivision,OakRidge,Tennessee37830

combination with either the resin monomers or the mixture.To calculate relative carcinogenic potency, we compared the

dose-response relationship obtained for the unknowns withthat obtained for a reference skin carcinogen, benzo(a)pynene.Our index of comparison was the concentration applied perweek of unknown, relative to benzo(a)pyrene, that would benecessary to elicit a common risk or probability of tumoroccurrence within a 24-month exposure period.

Potential strains response variability was assessed by conducting the experiments in 2 inbred mouse strains that wereobserved to differ considerably in their susceptibility to topicallyapplied epidermal carcinogens. The index of relative potencywas estimated with confidence limits by fitting weighted leastsquares regression lines to the dose-response relation for bothsexes for both strains.

MATERIALS AND METHODS

Animals. Experimental animals used in these studies wereobtained by chance mating inbred stocks no more than 5generations removed from pedigree inbred lines maintained bythe central animal facilities of the Biology Division, Oak RidgeNational Laboratory. Equal numbers of male and female miceof 2 inbred strains [C57BL/6Bd (hereafter called B6) andC3Hf/Bd (hereafter called C3)] were used. Fecal pellets obtamed from production cages were routinely cultured for Salmone!!a sp. and Pseudomonas sp. Retired breeders weretested for serological evidence of Sendai and pneumonia virusof mice as well as ectromelia, minute virus of mice, mousehepatitis virus, Theiler's GD VIII, reovirus type 3, and lymphocytic choniomeningitis. All samples obtained from breeders thatproduced the mice used in these experiments were negativefor these agents as well as for endo and ecto parasites.

Experimental animals were housed 5/cage and randomlyassigned to treated or control groups at 10 to 12 weeks of age.The experiments were carried out in closed-colony, limitedaccess facilities. Mice were fed pasteurized Purina 5010-Claboratory chow and given hyperchloninated (15 ppm) andacidified (HCI, pH 2.5) water ad !ibitum. Bedding consisted ofsterilized ground corncobs (SanoCel).

Test Materials. Materials assessed for epidermal carcinogenicity and percutaneous toxicity were commercial grade andwere obtained from the sources indicated: I and II, UnionCarbide Corp., Chemicals and Plastics Division, Hackensack,N. J.; Ill, Aldrich Chemical Co., Milwaukee, Wis. The referenceskin carcinogen, benzo(a)pyrene, was practical-grade materialobtained from Sigma Chemical Co., St. Louis, Mo. Chart 1 liststhe chemical structures and Chemical Abstracts Registry numbers for each of the materials tested for epidermal carcino

1718 CANCER RESEARCH VOL. 39

ABSTRACT

The systemic toxicity and skin carcinogenicity of bis(2,3-epoxycyclopentyl)ether, 2,2-bis(p-glycidyloxyphenyl)propane,and m-phenylenediamine were assessed relative tobenzo(a)pyrene by application to the skin of male and femaleC3H and C57BL/6 mice three times/week for 24 months. Thebis(2,3-epoxycyclopentyl)ether and the 2,2-bis(p-glycidyloxyphenyl)propane were also applied as an equal parts mixture todetermine whether the materials would interact as skin carcinogens. A comparison of dose responses showed thatbenzo(a)pyrene was 107 x 1O@,161 x 1O@,and 51 x 1O@more potent as a skin carcinogen than was bis(2,3-epoxycyclopentyl)ether, 2,2-bis(p-glycidyloxyphenyl)propane, andtheir mixture, respectively. The m-phenylenediamine was notcarcinogenic in skin of either strain at the maximum exposurerate allowed by systemic toxicity.

The two inbred strains used in these studies exhibited aconstant ratio of sensitivity to induction of epidermal cancer bydiverse chemical agents. Statistical analysis of this differencefor various compounds at different dose rates revealed thatC57BL/6 mice were 2.4 times more sensitive to epidermalcarcinogenesis than were C3H mice under identical circumstances of exposure. It is suggested that when carcinogenicityis assessed on a relative basis, the observed constancy in theratio of response would enable these two inbred strains to beused interchangeably. The choice of strain would affect significantly the sensitivity of the assay, but this would becomeimportant only in the assay of extremely weak carcinogens.

INTRODUCTION

Epoxy resins are versatile compounds for which new applications continue to be found. Despite the technical and economic importance of these materials, we are aware of only afew reports relating to their potential carcinogenicity (1, 5, 16,17). The purpose of this study was to quantitate the epidermalcarcinogenicity of 2 different epoxy resin monomers, bis(2,3-epoxycyclopentyl)ether (I) and 2,2-bis(p-glycidyloxyphenyl)propane (II), by use of mouse skin as an assay system. Anequal-parts mixture of I and IIwas applied to determine whetherthey would interact as skin carcinogens. m-Phenylenediamine(Ill) occasionally used as an amine cross-linker or polymerizingagent was also evaluated, but no attempt was made to test it in

1Research sponsored by the Division of Biomedical and Environmental Research, United States Department of Energy, under Contract W-7405-eng-26with the Union Carbide Corporation.

2 To whom requests for reprints should be addressed.

Received November 16, 1978; accepted February 8, 1979.

on June 13, 2018. © 1979 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

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Epiderma! Carcinogenicity of Epoxy Resins

daily, 5 days/week, for 2 weeks. Mice were weighed at thebeginning and at the end of this period and were monitoredthroughout for signs of acute local or systemic toxicity (chemical dermatitis, depression, and dehydration).

Benzo(a)pyrene dose levels were arbitrarily set at 0. 1 and0.01 % (w/v) in both strains. For the 24-month study, I and IIand the mixture were applied to C3 mice at 50 and 10% (v/v).Due to its toxicity, I was applied at 25 and 5%, whereas II andthe mixture were applied at 50 and 10% in B6 mice. MaterialIll proved extremely toxic in both strains and for this reasonwas applied at the maximally tolerated 2 and 0.4%. Vehiclecontrols were shaved and handled the same as was any othergroup.

All mice dying during or killed at the end of either the acuteor chronic study, with or without skin tumor, were necropsied.The viscera were examined, and the calvania was opened.Gross lesions, including all skin tumors, were subjected tohistological examination. To detect metastasis, we histologically examined the regional lymph nodes of animals bearingskin tumors.

Statistical Methods. Comparisonsof average body weightbetween treated and vehicle control were made by use ofDunnett's method for multiple comparisons with a commoncontrol. Distributions of deaths were analyzed by computationof the odds ratio of mortality observed throughout the study forsuccessive 50-day intervals, from which a maximum likelihoodsummary odds ratio was calculated. This statistic furnished anexact one-sided test of the hypothesis that the mortality distnibutions for a given treated group and the vehicle control wereidentical. Details of the approach used and other examples ofthis method can be found elsewhere (6).

Quantitation of the carcinogenicity of each material was donein 2 stages. (a) Histologically confirmed skin tumor latencieswere analyzed by the procedure of Kaplan and Meier (8) andThomas et a!. (15). This method estimates proportions of micesurviving without tumors and it adjusts for animals that die orare killed without skin tumor. (b) These latencies were fitted toa 3-parameter Weibull distribution in which the rate of appearance of new skin tumor-bearing animals is proportional tobk(t â€”w)k - I In this equation, t is the number of days frominitiation of treatment until a skin tumor is detected, w is thelength of the minimum latent period (in days) and may be acharacteristic of the animal species used for the test, k determines the shape of the distribution and may be related to thenumber of discrete steps or stages necessary for expressionof a skin tumor (10), and b is a scale factor directly related tothe chemical dose. Maximum likelihood procedures were usedto estimate a common k and w for all groups and a b for eachgroup. We are grateful to Dr. D. G. Thomas of the NationalCancer Institute for providing computer programs for the calculations in both of these stages (15).

Relative carcinogenic potency between those materials capable of inducing skin tumors and benzo(a)pyrene was estimated by considering log b to be a response which is linearlydependent on log dose. Weighted least-squares regressionlines were fitted to the data with the restrictions that the linesbe parallel and that the strain effect remain constant for allcompounds. The approximate variance of log b Is inverselyproportional to the number of tumor-bearing animals in eachgroup. Therefore, each weight is proportional to the number oftumor-positive animals observed. Full elaboration of the meth

I Bs(2 ,3 -epoxycyclopentyl)ether 2386-90-5

0@@o@o

H 2,2 -Bs(p - @yc)dytyoxypbenyI)propone 1675-54-3

H@@CHCH20*C*OCH2C@@CH2

CH3

@ m-Phenylenediamne 08-45-2

NH2

@NH2

@ Benzo(ojpyrene 50-32-8

JI@I@

Chart 1. Structural formulas and Chemical Abstracts Registry numbers forthe materials tested for epidermal carcinogenicity.

genicity. Resins I and II were miscible with acetone, while Illshowed a limiting solubility of 0.674 g/mI at 20Â°.A 2% (w/v)acetone solution of III contained 0.6% (w/w) of an insolubleresidue judged by IA spectroscopy to be a polymeric form ofthe compound. The purity and identity of I and II were determined by IR and nuclear magnetic resonance analysis of fnactions isolated by gel permeation chromatography and by vacuum distillation. By these criteria, II contained 10% (w/w),additional material Identified as an epoxidized polyglycol (M.W.>500), and a small amount of phenyl glycidyl ether. No impurities were detected in I by nuclear magnetic resonance. Acetone solution of analytically pure III was colorless, while thecommercial grade III used in the present study produced darkbrown solutions, suggesting either decomposition or impurity.

Sufticient amounts of each material were obtained to permitanimal exposure for the duration of the study from the samebatch or lot. Stock materials were kept in well-stoppered amberglass containers at room temperature. Undiluted I had a tendency to separate Into solid and liquid isomenic forms at roomtemperature and therefore had to be warmed in a water bathand mixed before aliquots were removed for preparation of testsolutions. Fresh dilutions of each unknown and of referencecarcinogen were made biweekly. The solutions were kept inglass-stoppered Erlenmeyer flasks within a fume hood.

Animal Exposure. Groupsof 80 (40 femaleand 40 male)C3and 40 (20 female and 20 male) B6 mice were given 50 @tIofeach material Monday, Wednesday, and Friday for 24 monthsor until the groups's skin tumor response exceeded 90%.Application was to the shaved dorsal skin by use of a micropipet. For each of the unknowns, 2 dose levels were used: thehigher dose was that which could be tolerated on the basis ofeither local or systemic toxicity, or physical characteristics ofthe material (solubility and viscosity); the lower dose wasarbitrarily set at one-fifth of the upper dose. In this experimentâ€â€˜dose' â€˜refers to the dose rate (in mg/week).

To assess potential cumulative toxicity, we exposed groupsof five 10- to 12-week-old male and female C3 and B6 mice

MAY 1979 1719



Averageterminalbodyweightand cumulative percentain B6 micege

ofmortalityCompoundDose/wk

(mg)Body

wt (g)Cumulative%

mortality at 24mos.

FemaleMaleFemaleMaleI37.5

7.523Â±o.6@ 25 Â±1.2

26Â±0.8 31 Â±0.62135

1520II75

1524Â±0.825Â±1.3

24Â±1.1 28Â±1.03525

2515Mixture75

1518Â±1.0 18 Â±1.2

24 Â±0.7 26 Â± 1.18080

3025Ill3

0.624Â±0.4 28 Â±0.6

23 Â±0.9 28 Â±0.610 35

3510Benzo(a)pyrene0.1

50.01523

Â±0.3 30 Â±0.524Â±0.4 30Â±0.4

Acetone 0.15 25 Â±0.6 29 Â±0.5 250aMean Â± S.E.

CompoundDose/wk (mg)Bodywt

(g)Cumulative%

mortality at 24mos.

FemaleMaleFemaleMaleI75

1521Â±o.7@

25Â±0.824Â±0.8

27Â±0.76245

5538II75

1524Â±0.8

26 Â±0.625Â±0.7

27 Â±0.84855

4842Mixture75

1525Â±0.6

26 Â±0.927Â±0.8

28 Â±0.75242

4250Ill3

0.626Â±0.7

26 Â±0.627Â±0.7

28 Â±0.74542

5048Benzo(a)pyrene0.1

50.01526

Â±0.327 Â±0.530

Â±0.332 Â±0.3Acetone0.1526

Â±0.827 Â±0.74245a

MeanÂ±S.E.

J. M. Ho!!and et a!.

odology and approach used in these calculations is beyond thescope of this paper, and additional details are given in Petoand Lee (10), Peto et a!. (11), and Pike (12).

RESULTS

In the short-term toxicity tests, daily applications of 50 @tlof50% acetone solutions of I and II were tolerated by C3 micewithout mortality or signs of toxicity. Under the same conditions, 5 of 9 B6 mice died after 4 doses of I. Daily applicationsof Ill at 10% concentrations or greater, resulted in death ormorbidity in both strains. Gross necropsy of mice followingcutaneous application of either I or Ill revealed a pale swollenliver and kidney which was indicative of hepatorenal toxicity.

Focal dermatitis, presumed to be of allergic etiology, wasobserved sporadically in mice exposed topically to I, II, or themixture in the first 6 months of the 24-month study. Seldomwere more than a few animals in any group affected, and thelesions were self-limiting. Local reactions were characterizedby a thickening of the surface epidermis, which was indicativeof hyperplasia and hyperkeratosis. The lesions occasionallybecame exudative but gradually healed without scar formation.To determine whether cumulative systemic toxicity developedover the 24-month exposure, we weighed the surviving miceand compared the average weight of treated animals with thatof the acetone control. In C3 mice (Table 1), weight losssignificantly greater than in the acetone control was observedin both sexes exposed to I (p < 0.05). In B6 mice (Table 2),significant weight loss was observed in males exposed to I, II,and the mixture (p < 0.05). Whether weight loss was indicativeof systemic toxicity or was a consequence of the debilitatingeffects of epidermal neoplasia was not established. A reflectionof overall differences in mortality rate is provided by cumulativepercentage of mortality at 24 months. Comparison of treatedand acetone control groups revealed that significantly greatermortality occurred in C3 female mice at the highest dose of I(Table 1) and in B6 mice (both sexes) at the highest doses ofthe mixture (Table 2) (p < 0.05). No mortality comparison was

Table1Averageterminalbodyweightandcumulativepercentageof mortality

in C3 mice

Table2

made for benzo(a)pyrene-treated mice because they werekilled on the basis of their skin tumor frequency, which, at thedose rates used, reached nearly 100% in the first 12 monthsof exposure.

Although skin carcinogenesis was the primary focus of thisstudy, we were also interested in whether the unknown matenals penetrated the skin or were inhaled or ingested in amountssufficient to alter nonepidermal tumor risk relative to the acetone control. The reason we considered this a secondary endpoint was that the systemic dose was unknown, and the expenimental protocol was sufficient to detect only macroscopictumors. The incidence of histologically confirmed tumors observed at death is given in Table 3 for C3 mice and in Table 4for B6 mice. Tumor incidence fluctuated greatly and either wasuncorrelated with dose or was comparable to that in the acetone control with one possible exception, lung tumors in C3mice. According to Fisher's exact test (4), the frequency oflung tumors in C3 mice (sexes pooled) was significantly increased following treatment with I at 75 mg/week (p = 0.004).All other comparisons were not significant (p > 0.05). Lungtumor risk was not significantly increased in any of the B6groups(p> 0.05).

The incidence of histologically benign, malignant, and metastatic epidermal tumors is given in Table 5 for C3 mice and inTable 6 for B6 mice. For the C3 strain, it was apparent that I atthe highest dose was weakly carcinogenic. In this strain neitherIll nor II at the doses tested elicited skin tumors. An unusualaspect of this experiment was the striking potentiation of carcinogenesis when the 2 resins were combined. Based on crudefrequencies, the effect resulted in approximately a 10-folddifference in tumor yield.

The same general pattern was observed in B6 mice (Table6). In addition, the greater genetic sensitivity of this inbredstrain to skin carcinogens was demonstrated. This sensitivityprobably accounts for the detections of weak carcinogenicitydue to II. As was the observation in C3 mice, material Ill alsofailed to induce skin tumors in B6 mice. Synergistic carcinogenesis was noted for the mixture, with tumor frequenciesapproaching 100% in males.

1720 CANCERRESEARCHVOL. 39



Incidence of B6 micea with neop!astic disease in tissues other thanskinCompoundDose/wk

(mg)Incidence

of tumor-bearing animals by organ or histologicaltypeSexReticulum

cell sarcoma and Other en

Lung Uver Kidney Bladder lymphoma Ovary docrine BreastConnectivetissueOtherapi

thelialOthermeson

chymalI37.5

37.57.57.5F

MFM1

0 0 0 7 0 0 01 1 0 0 3 00 0 0 0 5 0 0 01 3 0 0 4 00

4011

0001

00

1II75

751515F

MFM0

2 0 0 10 1 0 11 0 0 0 8 00 1 0 0 7 0 1 01 5 0 0 7 00

0000

0100

01

0Mixture75

751515F

MFM1

1 0 0 2 0 0 00 1 0 0 1 00 1 0 0 12 1 1 04 0 0 0 5 00

0001

0010

00

0Ill3

30.60.6F

MFM0

0 0 0 3 0 0 00 0 0 0 5 0I 1 0 0 7 0 1 00 2 0 0 8 00

0020

0100

22

1Benzo(a)pyrene0.1

50.150.0150.015F

MFM0

0 0 0 0 0 0 00 0 0 0 0 00 0 0 0 0 0 0 00 0 0 0 0 01

0000

0001

00

0Acetone0.15

0.15F M03 0 0 6 2 0 0

1 1 0 0 3 00 02 010a

TwentyanimaIsof eachsex were usedfor eachmaterialanddose.


Table3Incidence of C3 miceawith neoplastic disease in tissues other than skin

Incidence of tumor-bearing animals by organ or histological type

Reticulumcell sar

Dose/wk coma and Other en- Connective Other epi- Other mesonCompound (mg) Sex Lung Liver Kidney Bladder lymphoma Ovary docrine Breast tissue theilal chymal

75 F 9 14 0 0 6 13 0 5 5 0 075 M 5 16 1 0 3 1 0 1 015 F 3 3 0 0 3 14 1 6 8 2 015 M 4 28 1 0 2 1 0 0 0

75 F 6 3 0 0 3 15 2 4 9 0 175 M 3 22 0 0 3 1 1 0 015 F 1 5 0 0 5 18 0 10 9 0 515 M 1 21 2 0 11 0 0 1 2

Mixture 75 F 1 8 0 0 5 15 2 8 10 0 275 M 3 14 1 0 5 0 1 2 015 F 2 5 0 0 6 14 0 6 6 3 315 M 6 19 0 0 6 0 4 0 0

3 F 5 3 0 0 7 19 0 3 11 0 43 M 4 19 0 0 4 0 3 0 10.6 F 5 4 1 0 11 13 1 6 6 2 30.6 M 3 21 0 0 13 0 0 0 2

Benzo(a)pyrene 0.15 F 0 0 0 0 0 0 0 0 3 0 00.15 M 0 0 0 0 0 0 0 0 1 0 00.015 F 0 0 0 0 0 0 0 0 0 0 00.015 M 0 5 0 0 0 0 0 0 0 0 0

Acetone 0.15 F 1 6 0 0 6 17 0 11 8 1 00.15 N 2 20 0 0 4 0 1 0 1

a Forty animals of each sex were used for each material and dose.

Table4

MAY 1979 1721



Fortyanimalspersex,dose,andcompound.CompoundDose/wk

(issg)SexPapillomaCarcinomaLocalizedMetastaticTotalI75

751515F

MFM1

1002

1000

0003

20

0II75

751515F

MFM0

0000

0000

0000

00

0Mixture75

751515F

MFM12

13346

19181

01219

325

14Ill3

30.60.6F

MFM0

0000

0000

0000

00

0Benzo(a)pyrene10.1

50.150.0150.015F

MFM1

26

1139

3825280

01140

4032

40Acetone0.15

0.15F M1 00 00 01 0

CompoundDose/wk(mg)SexPapillomaCarcinomaLocalizedMetastatlcTotalI37.5

37,57.57,5F

MFM0

1001

3000

0001

40

0II75

751515F

MFM1

0011

6000

0002

60

1Mixture75

751515F

MFM2

01013

11000

61115

172

1Ill3

30.60.6F

MFM0

0000

0000

0000

00

0Benzo(a)pyrenea0.1

50.150.0150.015F

MFM2

1651

71911141

01120

2018

20Acetone0.15

0.15F M0 00 00 00 0

J. M. Ho!!and et a!.

Table5Skin tumorincidenceby histologicaltypein C3mice

a Benzo(a)pyrene was applied for 220 days at the 0.1 5 level and for 375 days at the 0.015

Table6

level.

Skin tumorincidenceby histologicaltypein B6 miceTwenty animals per sex, dose, and compound.

a @nz@a)pyrenewasappliedfor220 daysat the0.15 levelandfor310 daysatthe0.015level.

1722 CANCERRESEARCHVOL. 39



GraphStrain

MaterialLogdose/ wkLog bpoint (Chart3)C3I1.875â€”18.502

Â±0.194aaMixture1.875â€” 1 7.445 Â±0.065bMixture1.176â€”17.839

Â±0.097CBenzo(a)pyreneâ€”0.824â€”1 1 .736 Â±0.049dBenzo(a)pyreneâ€”

1 .824â€” 1 4.628 Â±0.052eAcetoneâ€”0.824â€”19.257

Â±0.434B6I1.574â€”18.244

Â±0.194fIII.875â€” I 8.020 Â± 0.154gII1

.1 76â€” 19.000 Â±0.434hMixture1

.875â€” 1 6.596 Â±0.077iMixture1.176â€”18.480

Â±0.251jBenzo(a)pyreneâ€”0.824â€”1 0.51 8 Â±0.069kBenzo(a)pyreneâ€”

1 .824â€” 1 3.503 Â±0.0701aMean

Â±S.E.


Gross, microscopic, and biological features of these tumorswere varied, but a few patternsdid emerge. The tumorsthemselves differed in size and gross appearance from 1- to 2-mmfocal lesions to complex growths often involving the entireback. Since, with the exception of the benzo(a)pyrene controlgroup, tumor-bearing mice were not killed until the end of theexperiment, many of the more malignant lesions were metastatic to both regional lymph nodes and lung. Metastasis appeared to be positively correlated with tumor residence time,thus accounting for more frequent metastasis in mice treatedwith test materials as well as the lower doses of benzo(a)pyrene.

Tumor malignancy was determined histologically on the basisof cellular characteristics as well as infiltrative local extension.In instances in which tumors were multiple, diagnosis of theanimal's tumor reflected the most advanced tumor stage. Forexample, if a mouse had several papillomas, a few locallyinfiltrative carcinomas, and a metastatic solitary carcinoma, forpurposes of the data In Tables 5 and 6 this animal would havebeen diagnosed as having a metastatic carcinoma and wouldnot be counted in either of the other 2 categories. In the caseof the test materials, tumor multiplicity was not considered aninterpretable parameter because long residence times as wellas malignancy of many tumors contributed to confluency.

We have calculated an index of relative potency for thosematerials inducing more than one skin tumor relative to the skintumor response obtained with benzo(a)pyrene by fitting a function to the data that interrelates the susceptibility of the animaland the dependent(time to tumor) and independent (dose-rate)experimental variables. The Weibull distribution has been usedto make comparisons between compounds when animals areexposed to constant, repetitive doses, and the measured response variable is time to tumor (10).

For proper interpretation of the parameter b of the Weibullmodel, a good fit to the data for each group and constancy ofk and w among groups is necessary. A comparison of the fitted(dashed) lines and actual data (solid!ines) for C3 and B6 (Chart2) are given for high and low doses of both benzo(a)pyreneand the mixture of I and II. Common values (Â±S.E.)of k and w

0 ,- - , I I C I C C0 100 200 300 400 500 600 700 800

TIME (days)Chart 2. Kaplan-Meier fits to the data relating probability of survival without

tumor to duration of exposure. The corresponding Weibull fits, with k and wassumed constant, are superimposed.

estimated from these 8 groups were 6.2 Â±0.4 and 48.5 Â±6.0, respectively.

Tests of the assumption of constant k and w appear valid atthe 5% level of significance for all except the B6 group at thehighest dose of the mixture. Including the data for this groupforced statistical rejection of the hypothesis of constant k andw (p < 0.001 ). Since this group exhibited the poorest fit to theWeibull model, we elected to assume k and w constant on thestrength of the other 7 groups. Table 7 lists the logarithm ofthe parameter b for these groups and, in addition, all othergroups in which more than one tumor occurred. The assumption of linearity could not be tested, since only 2 nonzero doselevels were used for each compound.

Eight lines were fitted to the data in Table 7 with restrictionsthat the lines be parallel and that the strain effect be constant.The 12 points deviated significantly from the weighted, leastsquares parallel lines, but when the response of C3 mice at thelowest dose of the mixture, which deviated extremely from itsfitted value, was deleted, the remaining 11 points did notdeviate significantly from the newly fitted lines. The weightedsum of squares of the deviations is approximately distributedas x2 since the weights were the reciprocals of the variances.On this basis, the tests of goodness of fit yield a x2 value of43.8 for 1 2 points (6 degrees of freedom) and a value of 2.56for 11 points (5 degrees of freedom). The lines obtained afterPoint c was excluded are given in Chart 3. Even though theextreme deviation of Point c could be explained by the restriction of parallel lines, we suggest thatthe biological mechanismsunderlying the response of mouse skin to topically appliedcarcinogens is sufficiently similar with different materials thatthe assumption of parallelism is warranted unless substantialevidence is offered to the contrary.

We calculated an estimateof relative carcinogenicpotencybased upon the ratio of doses of a test material, relative to thatof benzo(a)pyrene, necessary to elicit a common instantaneoustumor risk. The corresponding graphical procedure would beto draw a line parallel to the abscissa of Chart 3 that intersectsthe dose-response lines for benzo(a)pyrene and each of theunknowns. At the points of intersection, lines could be drawnperpendicular to the abscissa. The corresponding doses atwhich they intersect the log dose axis provide the numerator

Table7Therelationshipbetweenskin carcinogenicityandcarcinogendosein

C3 and B6 mice whenbothsexes werepooled

KAPLAN MOSER(â€”). WEIBULL --- -), LOW OPEN@ HIGH (CLOSED);

I â€˜II,C3H(.,O)ondC37BL,6)@,O); &o3P, C3H(a,A)o.dC57BL6)s,o

0

Iâ€”

F-

0IF-

z0F-ci:0ci0ci

10

0.8

06

04

02

1723MAY 1979

A@..\a@oR

t@ @.â€¢1@

ii@ I%@ s@a''



J, M. Holland et a!.

response to the resins separately. The possibility that a reactionproduct or impurity was present in the mixture that was notalso present in the separate resins could be excluded on thebasis that gel permeation chromatography, fractional distillation, and IA and nuclear magnetic resonance spectroscopyfailed to demonstrate impurities or constituents in the mixturethat were not also found in the materials as supplied. Otherpurely speculative possibilities include interaction at the mechanistic level or mutually enhanced penetration. Resin II is aviscous, essentially nonvolatile material that persists for severalhr after acetone has evaporated from the mouse's skin. ResinI, on the other hand, is considerably less viscous and more

volatile and does not persist on mouse skin. It is possible thatan equal-parts mixture of the 2 may enhance skin penetrationof either or both.

At the mechanistic level, if we postulate that I is a weaktumor initiator and that II is a tumor promoter, then the combination would be expected to show synergistic interaction, andit is possible that one or both of the resins must be metabolically

activated after gaining entry to the target cell. If one resinenhances the metabolism of the other or if metabolism ismutually stimulated, this also could be a factor contributing tothe greater effect obtained with the mixture.

The increased sensitivity of B6 mice to both toxic and carcinogenic effects of the chemicals was striking and was a cleardemonstration of the need to exercise caution in drawing toogeneral conclusions from test results based upon a singlestrain or species. This is especially true when effects areminimal, as was the case with II. If the experiment had beendone exclusively on C3 mice, then we would have concludedthat II, at the doses tested, was noncarcinogenic; but this wasnot the case, as was clearly demonstrated in B6 mice.

A finding of this study of possible general significance wasthe constant ratio of skin tumor susceptibility observed in C3and B6 strains exposed to distinctly different chemical carcinogens. This suggests that comparison of different carcinogenson the basis of relative potency may be independent of thestrain of mouse used. The choice of strain would, in thiscontext, more critically influence the scale or range of differences that could be measured. In other words, both C3 and B6mice are satisfactory for assessing the carcinogenicity of I, themixture, and benzo(a)pyrene, but B6 mice (or any other strainof equal or greater sensitivity) are necessary for precise measurement of relative risk for II.

Previous data concerning the toxicity and carcinogenicity ofIll in animals reveal that this compound is toxic as well asallergenic (7, 9, 13). Long-term animal tests to determine thecarcinogenicity of II have been limited, and the data are conflicting. Saruta et a!. (14) described an injection site sarcomainduced in one of 5 Wistar-King rats after 11 months of injectionat 9 mg/kg every other day. In the same experiment, III givenby the same method failed to induce any tumors at 18 mg/kg.Burnett et a!. (2) applied hair dyes containing an unspecifiedamount of Ill once weekly to groups of 100 mice for 18 months.Under these conditions, none of the materials produced evidence of systemic toxicity or carcinogenicity.

Our data indicate that at a dose approaching the maximumtolerated level, Ill neither induces skin tumors nor substantiallyincreases the incidence of tumors in other tissues, in spite ofevidence that skin penetration occurred readily with this material, thus supporting the observations of Burnett et a!.

(30-j

-20@- 2 0 - I 5 -tO 0 5 0 0.5 1 0 1.5 2.0 2.5

LOGWEEKLYDOSE(mg)

Chart 3. The relationship between log b and log weekly doee(in mg) for eachpositive compound In each strain. Letters, points for which coordinates are givenin Table 7. 0, B6 strain; â€¢C3 strain. Point c was excluded from the analysis.

and denominator of the potency ratio or index of relativecarcinogenic risk. These comparisons reveal that benzo(a)pyrene was 107 x 1O@,161 x 10@,and 51 x 10@timesmore potent a skin carcinogen than I, II, and the mixture,respectively. The lower 95% confidence limits (3) for thesepotency ratios were 68 x 1O@,99 x 1O@,and 43 x 1O@for I,II, and the mixture.A similarcalculationfor strainsrevealedtheB6 mouse to be 2.4 times more sensitive than the C3 mouse(95% confidence limits, 2.9 to 2.0).

DISCUSSION

Weil et a!. (17) conducted lifetime skin carcinogenesis testsin male C3H mice with I and II tested separately. In 2 expenments with II, they observed a single skin tumor after theanimals had been exposed for 16 months to the undilutedmaterial. Skin tumors were not observed in mice exposed to Iat a 30% concentration in acetone. Both of these experimentswere complicated by poor survival after 16 months of exposurethat significantly reduced the number of animals at risk in thelatter third of the experiment. Hine et a!. (5) also conducted lifespan carcinogenicity tests on an unspecified Shell bisphenol Aresin similar to II. Groups of 30 male C3 mice were painted 3times weekly with 0.2 ml of a 5% acetone solution of thismaterial. After 24 months, 4 of 13 surviving animals haddeveloped malignant skin tumors (3 carcinomas and one fibrosarcoma).

On the basis of these published observations, we concludethat there is consistent evidence that II is a weak carcinogen,a finding confirmed in the current study. The only publishedaccount of tests with I failed to demonstrate activity; however,for the reasons mentioned, the negative results of this test areunconvincing. Our data show clearly that not only is I an activeskin carcinogen but may be more potent than II.

An important observation was the demonstrated increase inskin tumorigenicity of the resins when applied as a mixture.Skin tumors occurred in greater numbers of mice and withshorter latencies than would be expected on the basis of the

1724 CANCER RESEARCH VOL. 39




In conclusion, we have demonstrated that 2 different epoxyresin monomers are weakly carcinogenic in mouse skin. Themagnitude of their carcinogenicity was assessed by comparison with a reference carcinogen benzo(a)pyrene. For an equalprobability of tumor occurrence, benzo(a)pyrene was 107 x103, 161 )( 1O@,and 51 x 1O@times more potent a carcinogenthan were I, II, and a mixture of them, respectively. This impliesthat for a unit dose of benzo(a)pyrene sufficient to increase therisk of tumor by a given degree, 107 x 1O@times that unit dosewould be necessary to achieve an equivalent risk for I, 161 x103 for II, and 51 x 1O@for a mixture of them. In addition toskin carcinogenicity, the materials also elicited systemic toxicity, as evidenced by weight loss and mortality. It is possiblethat a portion of this systemic effect was due to the presenceof debilitating epidermal tumors. However, the observation ofweight loss in groups exhibiting a low incidence of skin neoplasmsis consistentwith systemictoxicity.

ACKNOWLEDGMENTS

The authors are grateful to many people who contributed to this project,especially L. C. Gipson and M. J. Whitaker for treatment and observation ofanimals, E. Leach for computer programming, J. G. Dorsey for the nuclearmagnetic resonance analysis, and C. Rains for secretarial assistance.

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1. Bourne, L. G., Mimer, F. J. M., and Alberman, K. B. Health problems ofepoxy resins and amine-curing agents. Br. J. md. Med., 16: 81-97, 1959.

2. Burnett,C., Lanman,B., Giovacchinl,R., Wolcott,G., Scala, R., and Keplinger, M. Long-term toxicity studies on oxidation hair dyes. Food Cosmet.Toxicol., 13:353-357, 1975.

3. FIeIIer,E. C. A fundamentalformulaIn the statisticsof biologicalassay,andsome applications. 0. J. Pharm. Pharmacol., 17: 117-1 23, 1944.

4. FIsher, R. A. The logic of inductive Inference. J. R Stat. Soc. Set. A, 98: 39-54, 1935.

5. Hine, C. H., Guzman, R. J., Coursey, M. M., Wellington, J. S., and Anderson,H. H. An investigationof the oncogenicactivityof two representativeepoxyresins. Cancer Roe.. 18: 20-26, 1958.

6. Holland, J. M., and Mitchell, T. J. The relationship of strain, sex, and bodyweight to survival following sublethal whole-body X irradiation. Radlat. Roe.,66: 363â€”372,1976.

7. Kachalay, D. P., Orlov, N. S., Myannik, L. E., Ivanchenko, A. M., Venetskaya,F. V., and Grushevekaya,N. Vu. Effectsof m-phenyienedlamineon internalorgans and the nervous system. Farmakol. Toksikol. (Kiev), 8: 180-183,1973.

8. Kaplan. E. L, and Meter, P. Nonparametricestimationfrom Incompleteobservation. J. Am. Stat. Macc., 53: 457-481 , 1958.

9. Oriov, N. 5. AllergIccystitisof chemicaletiology(Rus.). Urol. Nefrol., 39:33-36, 1974.

10. Peto, R., and Lee, P. N. Weibull distributions for continuous-carcinogenesisexperiments. Biometrics, 29: 457â€”470,1973.

11. Peto, R., Lee, P. N., and Palge, W. S. Statistical analysis of the bioassay ofcontinuous carcinogens. Br. J. Cancer, 26: 258â€”261, 1972.

12. Pike, N. A suggested method of analysis of a certain class of experimentsin carclnogenesis. Biometrics, 22: 142-161 , 1966.

13. Rusakov,N. V., Varshavskaya,S. P., Sardarova,G. L., and RakhamtUlaev,N. N. Experimentaluses of proceduresof studyingallergic propertiesofchemicals during their enteral administration into the organism. In: S. N.Cherklnskl, S. N Pervgl (ode.), Organlzm Sreda, Mater. Nauch. Konf. Gig.Kafedr, Sixth, Vol. 1, pp. 79-81 , (Russ.) Moscow, U.S.S.R.: Mosk. Med.Inst.1970.

14. Saruta, N., Vamaguchi, S., and Matsuoka, T. Sarcoma produced by subdermal administration of metaphenyienedlamlne and metaphenylenediamlnehydrochloride. Kyushu J. Med. Sd., 13: 175â€”180,1972.

15. Thomas, D. G., Breslow, N., and Gart, J. J. Trend and homogeneity analysesof proportions and life table data. Comput. Blomed. Roe., 10: 373-381,1977.

16. Van Duuren, B. L Carcinogenicepoxides, lactones, and halO-Othersandtheir mode of action. Ann. N. V. Aced. Sd., 163: 633-651 , 1969.

17. Well, C. S., Condra, N., Haun, C., and Striegel, J. A. Experimental carcinogenicity and acute toxicity of representative epoxides. Am. md. Hyg. Assoc.J., 24: 305â€”325,1963.

MAY 1979 1725



1979;39:1718-1725. Cancer Res J. M. Holland, D. G. Gosslee and N. J. Williams in Male and Female C3H and C57BL/6 Mice

-Phenylenediaminem-glycidyloxyphenyl)propane, and p2,2-Bis(Epidermal Carcinogenicity of Bis(2,3-epoxycyclopentyl)ether,

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