nitrosamines, aromatic amines, dyes, coumarins, and...

[CANCER RESEARCH 26 Part 1, 619-625, April 19661

Summary

The carcinogenic activity of 50 compounds of various chemicalclassifications was determined 6 months following the p.o. administration of a single experimentally determined maximum tolerated dose of each compound in sesame oil to young femaleSprague-Dawley rats.

Histologically confirmed cancer of the breast, absent in controls, was observed in rats following the feeding of: 7 , 12-dimethylbenz[a]anthracene; 4H-cyclopenta[defjphenanthrene; 2 ,3-dihydro-3-ethyl-6-methylcyclopenta[ajanthracene; 2-anthramine;dimethyl-p-styrylaniline; 4'-fluoro-4-biphenylamine; N-(7-chloro-2-fluorenyl)acetamide; 2 ,7-fluorenediamine; 3-methyl-2-naphthylamine hydrochloride; N-hydroxy-N-2-fluorenylacetamide; N-6-(3 ,4-benzocoumarinyl)acetamide; 5 ,5-diphenylhydantoin ; 1-chloro-2 ,4-dinitronaphthalene; and p-ureidobenzenearsonic acid. These results confirm previous observations thatmammary tissue in rats is remarkably sensitive to exogenouschemical influences, and further suggest that certain coumarins,as well as hydrocarbons and aromatic amines, are capable of in

ducing cancer of the breast of rats under the experimental conditions used.

Carcinoma of the kidney was seen in 1 rat each, of 20, withcyclopentaphenanthrene, 2-anthramine, 2' ,4' ,6'-trimethylacetanilide (of 40 rats), 4 ,4'-oxydianiline, 3-methyl-2-naphthylaminehydrochloride, N-hydroxy-N-2-fluorenylacetamide, and ColourIndex Acid Blue 9 and was present in 1 control rat, of 89. Carcinoma of the lung was noted in 1 rat fed dinitrochloronaphtha

lene and in 1 control animal. Precancerous change in the ovaryoccurred in a rat fed 4 ,4'-sulfonyldianiline and in none of thecontrol animals.

Introduction

The determination of the carcinogenicity of chemicals cuStomarily involves chronic administration of the material at suitabledosages and observation of the animals for 2 years, or sometimeseven for their entire life-span. This approach is not only tediousbut also quite expensive. Thus, many attempts have been madeto shorten the time required to detect carcinogenic properties

inherent in a chemical structure. Some approaches hinged on relating some chemical or physical property to the carcinogenicitybut little general success can be claimed. Other modes included

1 Research performed under Contract No. P1143-64-66 from the

NIH.Received for publication August 9, 1965.

in vitro systems which again applied to only restricted series orclasses of chemicals. The search for new, different species to supplement the customary mice, rats, hamsters, and dogs likewisehas not had unqualified success.

Genetically mediated differences in responsiveness to a givencarcinogenic treatment are well known. It would appear that 1

system already existing and promising, because of exquisite sen

sitivity of the mammary gland in female Sprague-Dawley rats,has been discovered as a result of the work of Huggins (andothers) who, extending the findings of Shay (31), has observed arapid development of mammary cancer under certain specificexperimental conditions (2, 16, 33). Even a single dose of a com

pound has given rise to mammary carcinoma in less than 6months. The initial reports with this system, referred to herein asthe Huggins system, have involved mostly carcinogenic aromatichydrocarbons and a few of the aromatic amines (14â€”16).In orderto assess the value of such a rapid system with other types of

chemical agents, the following study reports the results of administering a single maximum tolerated dose (MTD2) of 50 compounds of a variety of structures to female Sprague-Dawley ratsunder the precise experimental technics developed by Huggins.

Materials and Methods

Animals

On the basis of our previous experience and the report of Huggins and collaborators (2, 15, 16, 33), female Sprague-Dawley rats

obtained from Sprague-Dawley, Inc., Madison, Wisconsin, wereused in all evaluations. The animals were approximately 45 daysof age upon receipt and were fed compounds for toxicity determinations and carcinogenicity studies at 50â€”55days of age. Ratswere housed in stainless steel cages, 16 x 16 x 5 inches, no morethan 5 rats/cage, and were fed Purinalaboratory chow ad libitum.

Chemicals

SOURCES. Many of the compounds were commerical products.

Samples of the following materials were supplied as follows:p-ureidobenzenearsonic acid by Whitmoyer Laboratories, Myers

town, Pennsylvania; griseofulvin by Schering Corporation,Bloomfield, New Jersey; chlorpromazine by Smith, Kline and

2 The following abbreviations are used: MTD, maximum tol

erated dose ; CCNSC, Cancer Chemotherapy National ServiceCenter; DMBA, 7,12-dimethylbenz[a]anthracene; CRD, chronicrespiratory disease.

619APRIL 1966

On the Carcinogenicity of a Single Intragastric Dose of Hydrocarbons,Nitrosamines, Aromatic Amines, Dyes, Coumarins, and Miscellaneous

Chemicals in Female Sprague-Dawley Ratsl

D. P. GRISWOLD,JR.,A. E. CASEY,E. K. WEISBURGER,J. H. WEISBURGER,AND F. M. SCHABEL,JR.Southern Research Institute (D. P. G., F. M. S.) and Birmingham Baptist Hospital (A . E. C.), Birmingham, Alabama, and National Cancer Institute (E. K. W., J. H. W.), Bethesda, Maryland

Research. on February 5, 2020. © 1966 American Association for Cancercancerres.aacrjournals.org Downloaded from

http://cancerres.aacrjournals.org/

D. P. Griswold, Jr. , A . E. Casey, E. K. JVeisburger, J. H. Weisburger, and F. M. Schabel, Jr.

French Laboratories, Philadelphia, Pennsylvania; 7-dimethylamino-4-rnethylcoumaiin, 6-acetamidocoumarin, and N , N-di

methyl-p-styrylaniline b@v Cancer Chemotherapy National

Service Center (CCNSC), National Cancer Institute, Bethesda,Maryland ; 1-chloro-2 ,4-dinitronaphthalene, 1 , 2 ,3 ,4 , 5 ,6-hexa

chloro-7-nitronaphthalene, and 3-methyl-2-naphthylamine hydrochloride by Fundamental Research Company, Berkeley,California; N-6- and 7-(3 ,4-benzocoumarinyl)acetamide, N-(7-

chloro-2-fluorenyl)acetamide, and N ,N'-(l ,3 ,6 ,8-tetrachloro

fluorenylene)bisacetamide by T. L. Fletcher, University ofWashington, Seattle; yellow No. 3 and acid blue 9 through A. A.Nelson of the Food and Drug Administration, Washington, D.C.;4 ,4'-oxydianiline by Dow Chemical Company, Midland, Michigan; 3 ,3' ,4 ,4'-biphenvltetramine by NARMCO Research andDevelopment Division, Whitaker Corporation, San Diego,California ; the cvclopenta[alanthracenes by Seymour Greenberg,

Whittier, California; and the nitrosourea.s were synthesized byT. P. Johnston, Southern Research Institute. We are indebted tothese individuals, whose cooperation made this project possible.

The compounds were checked for purity and, where necessary,purified by appropriate means.

RATIONALE FOR SELECTION. Hydrocarbons: 7 , 12-Dimethyl

benzla]anthracene (I)MIIA), the reference carcinogen for thisseries of experiments, has been employed widely to produce breast

tumors by the Huggins method. Therefore, a good deal of back

ground information on the characterstics and structure of thelesions is available.

41/-Cyclopenta[defjphenanthrene or 4 ,5-methylenephenanthrene is found in coal tar, is the basic skeleton of various naturalproducts, and combines the structures of phenanthrene and fluo

rene.Acenaphthene, although reported to be noncarcinogenic in

various tests, produces mitotic abnormalities in plant cells (4).

All the remaining hydrocarbons are analogs of 7-methylhenz[a]anthracene, itself positive in the Huggins test, in which thearomatic ring in the a [)osition has been replaced by a saturated5-membered ring. These compounds were originally made asanalogs of certain steroids (S. Greenberg, personal communication).

Nitrosoamines: 1 ,4-Dinitrosopiperazine, which is quite potentin causing esophageal and liver tumors (9), is an intermediate inorganic synthetic processes. A closely related compound, 3,7-dinitroso-1 ,3 ,5 ,7-tetraazabicyclo-[3 .3 . 1]nonane (dinitrosopen

tamethylenetetramine), has a similar structure, except that thenitroso groups are on opposite sides of 2 fused saturated rings,instead of on 1 . It is a foaming agent in plastics and rubber man

ufacture (37). N-Nitrosomethylurethan and N-nitroso-N-methylaniline, both known carcinogens for the stomach and liver,respectively, are intermediates in synthetic processes (30). 1 ,3-

Bis(2-chloroethyl)-1-nitrosourea and 1-(2-chloroethyl)-3-cyclo

hexyl-1-nitrosourea combine the features of the nitrogen mustards and nitrosoureas, both of which have been found

carcinogenic. Originally, these compounds were prepared bySouthern Research Institute for a CCNSC program and success

fully applied in intracerebral leukemia (Ref. 29 and unpublishedobservations of the authors).

Aromatic amines and derivatives: The largest group of substances come under this classification. Dimethyl-p-styrylaniline

(4-dimethylaminostilbene), a known carcinogen, has previouslybeen found positive in the Huggins test and serves as an additional reference.

Octafluorobenzidine is one of the benzidine derivatives withincreasing use as a starting material in the polymer industry.

Furthermore, with all the ring positions blocked by the fluorosubstituent, detoxification would be hampered. 3 ,3' ,4 ,4-Tetraaminobiphenyl, also a benzidine derivative, and 4 ,4'-oxydianilineare experimental intermediates in the manufacture of plastics.4 ,4'-Sulfonyldianiline is of value as a hardener in curing resinsand for its medical and veterinary uses in treatment of leprosy,tuberculosis, mastitis, and malaria.

Diphenylamine has been reported to cause bladder papillomasor renal cysts in rats (34) . This substance is used as a poultryfeed additive and in preventing scab on fruit trees.

The following amines or derivatives are known experimentalcarcinogens: 2-anthramine for skin and liver; 4'-fluoro-4-biphenylamine for kidney ; 2 ,7-fluorenediamine for liver andstomach ; 3-methyl-2-naphthylamine3 for mammary gland (32)and intestinal tract. N-Hydroxy-N-2-fluorenylacetamide, anactive metabolite of the well-known carcinogen N-2-fluorenylacetamide, attacks many organs in long-term feeding trials buthas not yet been studied after a single dose.

2' ,4' ,6'-Trimethylacetanilide and 3' ,4'-dimethylacetanilide arederivatives of useful dyestuff intermediates ; there are preliminary

reports that they are weakly carcinogenic (22). N-(7-Chloro-2-fluorenyl)acetamide and 1 ,3 ,6 ,8-tetrachloro-2 ,7-diacetamidofluorene, although derivatives of the active fluorenamine, havenot yet been tested adequately. 2-Chrysenamine can he considered as a derivative of 2-naphthylamine, a known carcinogen.

Dyestuffs: Auramine 0, (4 ,4'-imidocarbonyl-bis(N ,N-di

methylaniline)), a dye of varied uses, has given hepatomas inmice and rats on feeding (36). It is a structural analog of certainof the amines examined in this experiment. o-Aminoazotoluenehas caused hepatomas in mice, while 2 ,2'-azonaphthalene is morelikely to yield cholangiomas. 1-Phenylazo-2-naphthylamine hasnot been found carcinogenic thus far (13). The remaining dye,Colour Index Acid Blue 9 (FD & C Blue No. 1), is a representative of the triphenylmethane dyes.

Coumarins: In view of the carcinogenicity of various lactones,including the aflatoxins and f3-propiolaetone, a small series of substances of this class were included. N-6- and N-7-(3 ,4-Benzocoumarinyl)acetamide, synthesized for a cancer chemotherapy

program, are structural analogs of the weak carcinogen, N-3-

fluorenylacetamide, and of the potent carcinogen, N-2-fluorenyl

acetamide, respectively. In these cases, the central 5-memberedring of fluorene has been replaced by a 6-membered lactone ring.The other 2 similar coumarins were chosen to determine whichstructural elements affected the activity.

Miscellaneous: Diphenylhydantoin is a cyclic amidine or imid

azole derivative. Although a widely used medicinal, there are reports of liver damage following prolonged use (26) . The samereason, namely reports of liver dysfunction in l)atients after clinical use, holds for chlorpromazine, a well-known tranquilizer.

Aminotriazole, safrole, and phenazine have diverse heterocyclicstructures and have caused thyroid, liver, and bladder tumors,

3 Weisburger, E., Weisburger, J., Hadidian, Z. , and Frederick

son, T. Unpublished observations.

620 CANCER RESEARCH VOL. 26



Tests for (â€˜hcmicalCarcinogens

respectively (19, 21 , 28). Thioacetamide and ethionine are hepatocarcinogens (10, 11). Since the N-oxide group often enhances@)hysiologicaction, phenazine and phenazine-N-oxide, which are

basic skeletons for drugs and have been proposed as cloud-nucleating agents, were tested.

The well-known insecticide, dieldrin, reportedly has a mildtumorigenic effect in mice (7). Dinitrochlornaphthalene, whichhas a nitro group in the important 2-position of naphthalene, isof some value as an insecticide, fungicide, and intermediate insynthetic organic processes. A related compound, hexachloronitronaphthalene, an industrial intermediate, has most of thepositions blocked where detoxification would normally occur.

Carharsone (p-ureidobenzenearsonic acid) , a remedy for amel)iasis, appeared to give a low incidence of hepatoma in trout(20).@Likewise, a valuable antibiotic for dermatologic disorders;griseofulvin, Ul)011oral administration caused mouse hepatoma(18). Additionally, dietary griseofulvin aLo had a cccarcinogeniceffect on methylcholanthrene-induced cutaneous tumers in mice

(1).MODE OF AI)MINISTRATION. Each compound, prior to P.O. ad

ministration, was dissolved or suspended in sesame oil with theaid of (a) a motor-driven Thomas tissue homogenizer (ThomasTissue Grinder, Arthur H. Thomas Company, Philadelphia,Pennsylvania) or (b) in most instances a sonic homogenizer(Sonifer, high intensity, model L575, Branson Instruments, Inc.,Stamford, Connecticut) in a volume of 1 ml/dose. Occasionally,volumes were increased to as much as 3 ml/dose in order to provide a good suspension. Compounds were administered by gastricintul)ation using a No. 8 French Davol rubber catheter tip andreloading syringes for each dose.

TOXICITY DETERMINATIONS. The MTI) for each ccrnpound was

assessed in accordance with a predetermined plan. Initially, 2 ratswere fed each of the following doses of a compound : 100, 50, 10,5.0, 1.0, 0.5, and 0.1 mg/rat.. If mortality was observed within30 days at a (lose, then closer titrations in this range were carriedout. For examl)le, if 1 or both animals given 50 mg died within30 days postfeeding and both of those on 10 mg survived, then 2rats were treated with each of the fcllowing doses: 50, 40, 30,and 20 mg/rat, and observed for 20 days. In instances where nomortality occurred within 30 days of the initial feeding, doses of500 and 300 mg/rat, when availability of drug permitted, werefed to each of 2 rats per dose. The dose just I)elow that causingmortality (1 or 2 rats) in 30 days was accepted as the carcinogenicity test dose. When no mortality was observed, a maximumdose of 500 mg/rat was used.

CARCINOGENICITY STUI)IES. Following determination of an

MTD, 20 rats were fed this level of each compound. Compoundswere examined for carcinogenicity in 9 groups of 4â€”7compounds,classified by chemical type, e.g., hydrocarbons, aromatic amines,etc. Included in each of 4 of the groups was a DMBA-fed positivecontrol, and in each of the other 5 groups was a sesame oil-negative control. In certain instances where excessive mortality occurred following administration of the carcinogenic test dose(dinitrosopiperazine, 4'-fluoro-4-biphenylamine, trimethylacetanilide, 2 ,7-fluorenediamine, and ethionine), additional groups

4 Carbarsoiie at several dietary dose levels for a 2-year period

failed to induce cancer in rats (Eoff, H. J., personal communicatioli).

of rats were given the same dose or a lower dose of compound inorder to make available a comparable number of animals forautopsy at the end of the 6-month observation period. The ratswere weighed and thoroughly inspected weekly for appearanceof mammary tumors or other grossly apparent tumors. Recordsof animal body weights provided a useful indication of possibleapproaching death. If animals appeared ill for any reason whichallowed the prediction of approaching death, they were examinedtwice daily and sacrificed just prior to likely death.

At the time of death or sacrifice, all animals were carefullyautopsied. The l)ituitary, adrenals, kidneys, spleen, and liver wereweighed. Any grossly tumorous or otherwise diseased tissue, aswell as mammary tissue, intestinal tract (duodenum, jejunum,ileum, cecum), pituitary,5 liver, both ovaries, and both adrenals,were fixed in 10% formalin. Paraffin-embedded and H & Estained sections were studied microsco@)ically without knowledgeof the observer (A. E. C.) of the experimental history of the donorrat. In addition, representative pieces of thyroid, lungs, thymus,sl)leen, kidneys, urinary bladder, stomach, and heart were @@avedfor later examination if indicated.

Results

The results of toxicity determinations as well as mortalityfigures resulting from the administration of the carcinogenic testdose may be seen in Table 1. Three compounds, 6-methylcyclopenta[a]anthracene, 3-ethyl-6-methylcyclopenta[a]anthracene,and 3 ,6-dimethylcyclopenta[a}anthracene, were administered forcarcinogenicity studies at doses of 100 mg/rat. These levels werenontoxic in preliminary trials, and their selection was based onthe limited supply of each chemical. Fluorescence of the feces, 24hr postfeeding, of rats which had been fed acenaphthene andcvclopentaphenanthrene (300 mg /rat) suggested maximum absorption had occurred at this dose and obviated the need of higherdoses. Similar considerations held for acid blue 9 and 2 ,2'-azonaphthalene, where dye appeared in the feces following feedingof300 mg/rat.

A histologic description of tissues which were considered to beneol)lastic or suggestive of preneoplasia is listed in Table 2. Hyperplasia or metaplasia of the mammary glands, tubo-ovariandisease, a high incidence of lung lesions, as well as miscellaneousdiseased tissues, were found in the course of these studies ; however, such tissues in which disease entities not directly suggestiveof neoplasia were found were not included in this tal)le.

Chronic respiratory disease (CRD) of laboratory rats was recognized as a serious potential problem in the use of these animalsfor long-term studies, but this disease problem does not necessarily preclude the use of rats when care is taken to differentiatethe CRD lesions from any chemically induced lesions. Descriptions of this disease entity, including histopathologic details, areavailable in the literature (12, 23, 24).

The organ weights (pituitary, adrenals, kidneys, spleen, andliver) were similar in the experimental and control groups.

5 The pituitary was removed and preserved when possible. In

the few instances where it was not possible to conduct an autopsyimmediately following death of the animal (not so in cases of s:tc@rificed animals) the pituitary was often too decomposed for sat isfactory identification and removal.

APRIL 1966 621



Classification and agent7@3O-Dmortali3y6-MonthCoumarinsâ€”ContinuedN-7-(3

,4-Benzocoumarinyl)acet5001/201/20amide6-Acetamidocoumarin2003/204/207-Dimethylamino-4-methylcou

1503/203/20mariaMiscellaneous5,5-Diphenylhydantoin1500/200/20Phenazine3000/202/20Phenazine-N-oxide3000/204/20Safrole2000/201/203-Amino-1H-1,2,4-triazole5001/201/20Thioacetamide302/207/20Ethionine5008/378/371,2,3,4,5,6-Hexachloro-7-nitro

5001/201/20naphthaleneDieldrin4.05/407/401-Chloro-2,4-dinitronaphthalene5000/201/20Chlorpromazine

hydrochloride200/203/20p-Ureidobenzenearsonicacid5001/201/20(Carbarsone)Griseofulvin5001/201/20ControlSesame

oil3/895/89

D. P. Gi@iswold,Jr., A . E. Casey, E. K. Weisburger, J. H. Weisburger, and F. M. Schabel, Jr.

TABLE 1â€”ContinuedTABLE 1

MORTALITY IN 50- TO 55-DAY-0u FEMALE SPRAGUE-DAWLEY

RATS FOLLOWING A SINGLE ORAL DOSE OF COMPOUND

30-Daymortality

(dead/total)

0/200/200/201/13

0/20

2/20

14/204/203/20

0/20

12/20

1/20

0/20

1/201/20

0/200/203/20

16/402/20

18/402/202/200/200/20

8/400/201/20

0/20

2/20

1/20

1/200/20

0/200/202/20

0/20

6-Monthmortality

(dead!total)

4/201/200/203/13

0/20

3/20

14/207/204/20

0/20

20/20

2/20

2/20

2/201/20

0/202/205/2017/40

2/2019/402/202/200/200/20

Dosage(mg/rat)

18300@

300a

100k

bOb

bob

40

30

90

40

4.0

2.05.0

2.530

500

bO5.030

500

350

300150500

500

100150

150

100

300100

150

300

30@

30@

150

500

Classification and agent

aFluorescenceof feces24hr postfeedingsuggestedno needtouse larger doses.

b Doses were based on the limited supply of each compound.

C Dyes in feces 24 hr postfeeding suggested no need to use larger

doses.

The DMBA-fed animals were included as a control measure.Huggins has reported the incidence of breast cancer to be as highas 100% in rats following DMBA feeding, and 89% of these rats

were reported to have fibroadenoma (17). Daniel and Prichard

have reported an incidence of 80% mammary tumors, consisting

of 3 tumor types (5). Previous experience in our laboratories at

8/40 Southern Research Institute has shown an equally high incidence0/20 of mammary tumors following a single feeding of DMBA. Thus,2/20 in the present study, tissues from only 1 of the 4 groups on

DMBA were subjected to complete histopathology which con5/20 firmed our prior results and expectations (Table 2). Most of the3 20 large, palpable tumors were carcinomas. With DMBA, many1/20 rats bore an average of 2â€”3tumors. In total, in the 4 groups of 20

rats each, 178 palpable lesions were counted during the 6 months;1/20 25 had regressed at the time of autopsy.1/20 Various incidences of spontaneously occurring mammary tu

mors have been reported in older female Sprague-Dawley rats1/20 (8, 17, 35). Huggins, however, has reported that only 2 spontane0/20 ous mammary cancers were observed in about 20,000 untreated2/20 female rats less than 8 months of age (15).

An experimental period of 6 months was selected because0/20 Huggins (personal communication) indicated that the majority

of the lesions induced by chemicals would be mammary adeno

_____carcinomas,aswasindeedthecase.Later,thisstrainofratsalso

Hydrocarbons7, 12-Dimethylbenz[alanthracene4H-CyclopentaldefjphenanthreneAcenaphthene

2,3 - Dihydro - 3,6 - dimethyl - 1H-cyclopenta[alanthracene

2,3 - Dihydro - 6 - methyl - 1H-cyclopenta[alanthracene

2,3-Dihydro-3-ethyl-6-methyl-IHcyclopenta[ajanthracene

Nitrosamines1,4-Dinitrosopiperazine

3,7-Dinitroso-1,3,5,7-tetraazabicyclo-[3 .3 . llnonane

N - Methyl - N - nitroso ethyl carbamate

1,3 - Bis(2 - chloroethyl) - 1 - nitrosourea

1-(2-Chloroethyl)-3-cyclohexyl-1-nitrosourea

N-Nitroso-N-methylanilineAromatic amines

Octafluorobenzidine2-Ant hramineN ,N-1)imethyl-p-styrylaniline4'-Fluoro-4-biphenylamineN-(7-Chloro-2-fluorenyl)acetamide2',4',6'-TrimethylacetanilideDiphenylamine4 ,4-Oxydianiline2-Chrysenamine1,3,6,8-Tetrachloro-2,7-diacet

amidofluorene2 ,7-Fluorenediami ne3' ,4'-J)imethylacetanilide3-Methyl-2-naphthylamine hydro

chlorideN-Hydroxy-N-2-fluorenylacet

amide3 ,3' ,4 ,4'-Tetraaminobiphenyl4 â€˜4'-Sulfonyldianiline

DyesAuraniine 0Yellow 5 (1-phenylazo-2-naphthyl

amine)Acid Blue 92 ,2'-Azonaphthalene4-o-Tolylazo-o-toluidine (o-amino

azotoluene)Coumarins

N-6- (3 ,4-Benzocoumarinyl)acetamide

CANCER RESEARCH VOL. 26622



Agent6- MonthsurvivorsDiseased

tiSSUCaDescription@@

TABLE 2

HIsToPA@risoLoGIc DESCRIPTION OF DISEASED TISSUES SUGGESTIVE OF FRANK NEOPLASIA

OR PRENEOPLASIA

7 , 12-Dimethylbenzanthracene@'

Cyclopentaphenanthrene

Lobular carcinoma, Grade 0â€”Il(13) ; fibroadenoma (1) ; fibroadenomatous hyper

plasia (1) ; moderate hyperplasia (1);suppurative and plasma cell mastitis (1);

undetermined (1)Lobular carcinoma, Grade IIColumnar cell carcinoma, Grade II mixed

typeDiffuse fibroadenomatous hyperplasiaLobular carcinoma, Grade I (1) ; epidermal

cyst, large; squamous metaplasia, adenomatous hyperplasia, acidophilecells (1)

FibroadenomaDiffuse fibrous hyperplasia

Epidermal cyst; diffuse hyperplasiaLobular carcinoma, Grade 0â€”I(6) ; diffuse

lobular hyperplasia, marked inspissatedmaterial, ducts (1) ; 5-gm massâ€”unde

termined (1)Columnar cell carcinoma, Grade 0â€”I,

acinar typeEpidermal polyp, epidermal cyst (squa

mous epithelium) (1) ; hyperplasia,mucosa (1)

Lobular carcinoma, Grade Iâ€”Il(2) ; diffusefibroushyperplasia(1)

Lobular carcinoma, Grade 0â€”I(2) ; fibroadenomatous hyperplasia (1); 7-gmmassâ€”undetermined (1)

Lobular carcinoma, Grade 0-IAdenocarcinoma, Grade II, cortexTubular carcinoma, Grade II, cortexAdenomatous hyperplasia, some extension

of proliferated breast lobules into skele

tal muscle, glandular tissue does notseem malignant

Focal lymphocyte-monocyte collectionDiffuse fibroadenomatous hyperplasia

Lobular carcinoma, Grade 0â€”11Adenocarcinoma, Grade IILobular carcinoma, Grade 0â€”Il,mucinous

carcinoma, Grade 0â€”I(1)Portal hemopoietic tissue compatible with

leukemiaLobular carcinoma, Grade 0â€”Il

Adenocarcinoma, Grade II, tubular type,invasive

Dermoid cyst with precancerous changeRenal carcinoma, nephron type, Grade 0â€”ILobular carcinoma, Grade 0â€”Ill (6);

marked fibroadenomatous hyperplasia(1)

Lobular carcinoma, Grade 0â€”I(1); diffusefibroadenomatous hyperplasia (1)

Diffuse hyperplasia (1); diffuse hyperplasia, benignâ€”the hyperplasia is asextensive as a neoplasm but entirely inactive lobules (1)

Breast (18)â€•

Breast (1)

Kidney (1)

Breast (1)Breast (2)

Breast(1)Breast (1)

Breast(1)Breast (8)

Kidney (1)

Stomach (2)

Breast (3)

Breast (4)

Breast (1)

Kidney (1)Kidney (1)Breast (1)

Liver (1)Breast (1)

Breast (6)Kidney (1)Breast (5)

Liver (1)

Breast (4)

Kidney (1)

Ovary (1)

Kidney (1)Breast (7)

Breast (2)

Breast (2)

6-Methylcyclopentaanthracene3-Ethyl-6-methylcyclopentaan

thracene

Dinitrosopiperazine

1-(2-Chloroethyl)-3-cyclohexyl-1-nitrosourea

Octafluorobenzidine

2-Anthramine

Dimethyl-p-styrylaniline

4'-Fluoro-4-biphenylamine

7-Chloro-2-fiuorenylacetamideTrimethylacetanilide4,4'-Oxydianiline2-Chrysenamine

Tetrachioro - 2,7 - diacetamidofluorene

2 ,7-Fluorenediamine3-Methyl-2-naphthylamine. HC1

N-Hydroxy-N-2-fluorenylacetamide

4,4'-SulfonyldianilineAcid Blue 9N-6- (3,4-Benzocoumarinyl)aceta

mide

16

b9

20

17

b318

20

18

15

24

18211820

20

3218

15

191920

20

b9

Diphenylhydantoin

Aminotriazole



Ovary (1)

Breast (1)Liver (1)

Breast (2)Lung (1)

D. P. Griswold, ,Jr., A. E. Casey, E. K. Weisburger, J. H. Weisburger, and F. M. Schabel, Jr.

TABLE 2â€”Continued

Description'SDiseased tissue@'

Possibly early granulosa cell tumors (3)in situ

I)iffusehyperplasia,moderateExt ramedullary hemopoiesis , portal

spaces; possible leukemia or leukemoidhyperplasia, portal spaces

Lobular carcinoma, Grade 0â€”IlAdenocarcinoma bronchus, (rade 0, in

situ (adenoma malignum)Lobular carcinoma, (rade IFibroadenomatous hyperplasiaTubular carcinomaLobular carcinoma, Grade 0-IBasal hyperactivity compatible with pre

cancerous change

Agent@. vors

Thioacetaniide@ 18

Ethionine@ 31

l)ieklrin 34

I )in iI rochloronaphthalene@ 19

p-Ureidohenzenearsonic acid 19Sesame oil control 84

Breast (1)Breast (1)Kidney (1)Lung (1)Bronchus (1)

a Numbers in parentheses indicate the number of rats affected.b Ilyperplasia of the breast, not considered precancerous, was noted with auramine 0 (2 rats), acid

blue 9 (1), 2,t@.'-azonaphthalene (2), and tolylazo-o-toluidine (1).C One group of 4, each with 20 rats (see text).

d Three rats with breast lesions died or were sacrificed before the end of the 6-month period.

tends to yield carcinomas and fibroadenomas spontaneously,rendering the interpretation of the data more difficult.

Carcinoma of the breast was found following the administration of 14 compounds. Of 89 control animals fed sesame oil, histopathologic change was seen in breast tissue of only 1 rat; thischange was limited to fibroadenomatous hyperplasia. The highestincidences of breast cancer were seen in animals fed DMBA(75% +) ; 2-anthramine (30%) ; N-6-(3 ,4-benzocoumarinyl)-acetamide (30%) ; 3-methvl-2-naphthylamine hydrochloride(28%); N-hydroxv-N-2-fluorenylacetamide (20%); and 2,7-fluorenediamine (19%).

Palpable nodules during the experiment which persisted forvarying periods and then regressed were also noted with the following compounds : 1-(2-chloroethyl)-3-cyclohexyl-1 -nitrosourea,1 ; 4'-fluoro-4-biphenylamine, 1; N-(7-chloro-2-fluorenyl)acetamide, 2; 3-methyl-2-naphthylamine hydrochloride, 1; N-6-(3 ,4-benzocoumarinyl)acetamide, 2; ethionine, 1; and dieldrin, 3small.

Tissue changes coml)atible with leukemia were observed in ratswhich were fed 3-methvl-2-naphthylamine .HC1 and dieldrin,and rr@ancerous changes were found in the ovary of 1 rat fed4 ,4'-sulfonvldianiline. An ovary of a rat fed thioacetamide hadPossible early granulosa-cell tumors; this tumor type was reported to have occurred spontaneously in an untreated SpragueDawley rat (35). Carcinoma of the lung was found in 1 controlrat and 1 fed dinitrochloronaphthalene. Davis reported 1 spontaneous lung carcinoma in Sprague-Dawley rats (8).

Also of interest were the observations of carcinoma of the kidney in control as well as test animals. Thompson et al (35) reported 1 spontaneous nel)hroblastoma in a female SpragueDawley rat killed at 8 months of age, while Davis (8), in hisextensive studies, reported none. Bullock and Curtis (3) havereported 6 embryonal carcinomas, 1 carcinoma and 1 sarcoma,all spontaneous tumors of the rat kidney. Rosen et al. (27) reported no renal tumors in 41 Sprague-Dawley rats, but a high

percentage of renal tumors in irradiated rats. In addition to thetubular carcinoma found in the kidney of 1 of 89 control rats,

renal tumors were found in 1 of the 20 rats each which were fed

the MTD of cyclopentaphenanthrene, 2-anthramine, trimethylacetanilide (1 of 40 rats), 4 ,4'-oxydianiline, 3-methyl-2-naphthylamine .HC1, N-hydroxy-N-2-fluorenylacetamide, and ColourIndex Acid Blue 9. The cause-effect relationship in this seriesremains to be established.

Discussion

One of the aims of this work was to determine whether thisrapid tumor induction system could be reliably used to detect

inherent carcinogenicity of a variety of chemicals. It appears

that compounds of the polynuclear aromatic type, aromaticamines, certain coumarins, and possibly polynitro halogenatedpolynuclear aromatic hydrocarbons, would respond in a positivemanner and induce mammary cancer rapidly in the young female

Sprague-Dawley rats. Additional criteria such as multiple ratherthan single dosing, and perhaps extension of the experimentalperiod, might enhance sensitivity of this test. Germ-free rats,have reportedly a more uniform response (25). However, it is

also clear that the potent carcinogens of the nitrosamine typeand fairly polar small molecules such as ethionine may not be

detected by this technic. Likewise, the series of dyestuffs examined, some of which have been shown carcinogenic in other moreprolonged tests, were negative.

It can be concluded that to be carcinogenic in this system acompound will need to possess some selective ability to concentrate in the mammary gland and remain therein for a sufficientperiod of time to initiate the carcinogenic process. Those typesof compounds which responded positively probably have thisproperty. In addition, the development of mammary gland cancer involves particij)ation of hormonal factors. The regressionsnoted, even with DMBA, may have resulted from a secondaryalteration in hormonal stimulus.

624 CANCEH RESEARCH VOL. 26



Tests for Chemical Carcinogens

Mammary carcinoma in female Sprague-Dawley rats forms auseful tool not only to study hormonal factors required for thedevelopment of the tumors (6), but also to assess their responseto chemotherapy. The present work makes available a number ofnonhydrocarbons which with some additional effort to optimizeconditions may be suited to induce these cancers reliably. Theypossess advantages such as more rapid elimination and possiblylesser biologic and biochemical effects (damage, enzyme induetion) in other tissues.

Acknowledgments

The authors wish to gratefully acknowledge the assistance ofMr. Charles Grccii, Mrs. Joan Belzer, Mr. Jack Moore, Mr. J. R..Thomson, and Mr. Frank Hogg. We are greatly indebted to Dr.Charles Huggins, University of Chicago, for invaluable discussions.

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APRIL 1966 625



1966;26:619-625. Cancer Res D. P. Griswold, Jr., A. E. Casey, E. K. Weisburger, et al. Sprague-Dawley RatsCoumarins, and Miscellaneous Chemicals in FemaleHydrocarbons, Nitrosamines, Aromatic Amines, Dyes, On the Carcinogenicity of a Single Intragastric Dose of

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