The Carcinogenicity of Compounds Related to2-Acetylaminofluorene

III. Aminobiphenyl and Benzidine Derivatives*^

E. C. MILLER,R. B. SANDIN,J. A. MILLER,ANDH. P. RUSCH

(McArdle Memorial Laboratory, Medical School, University of Wisconsin, Madison 6, Wisconsin, andDepartment of Chemistry, University of Alberta, Edmonton, Alberta, Canada)

An interesting feature of the carcinogenicactivity of 2-acetylaminofluorene (AAF) is thewide range of tissues of the rat in which thiscompound can induce neoplasia (for references,see 21, 26, 28). Studies on the carcinogenicitiesof various derivatives and structural analogsof AAF have shown that they differ fromAAF and from each other not only in theirover-all potencies but in their tissue specificitiesas well. Thus, it appears from our earlier studies(22, 26) that a méthylènebridge in the 9 positionis required for significant activity toward theliver, while certain other bridges, for instance—¿�S—and —¿�CH= CH—,can be substituted without loss of potency for the ear duct, mammarygland, and epithelium of the small intestine. 4-Acetylaminobiphenyl, which has no bridge in thisposition, has greatly decreased activity toward theintestinal epithelium and the ear duct, but is asactive as AAF toward the mammary gland. Wai-pole and his associates (36) have likewise foundtumors at various sites, especially in the largeintestine, in rats given repeated subcutaneous injections of 4-aminobiphenyl after experimentalperiods of 500 days.

The high activity of 4-acetylaminobiphenyl forthe mammary gland and its structural simplicityprompted us to compare the carcinogenicities ofthe three isomerie acetylaminobiphenyls and todetermine the effects of various ring substituentson the activity of 4-acetylaminobiphenyl. The latter group included certain methyl, amino, fluoro,and hydroxy derivatives. The 2'- and 4'-fluoro

* This work was supported by grants-in-aid from the

National Cancer Institute, United States Public Health Service (No. C355), and the Alberta Branch of the CanadianCancer Society, and by an institutional grant from the Alexander and Margaret Stewart Trust Fund.

t Some of these data have been presented in preliminaryform (21, 25, 31).

Received for publication January 16, 1956.

derivatives were tested, since substitution offluorine in the 7 position of AAF considerably enhances its activity toward the liver but not forother tissues (26). Similarly, with the exception ofdyes substituted in both the 2 and 6 positions, thefluorinated derivatives of 4-dimethylaminoazo-benzene are as active or more active hepatic carcinogens than the parent dye (23, 24, 27). The 3-hydroxy derivatives of 4-aminobiphenyl and 4-acetylaminobiphenyl were studied as a direct testof the hypothesis of Walpole and associates (36)that these amines are active as a result of conversion in vivo to carcinogenic ortho hydroxy-amines.

Certain ring-methyl derivatives of N,N'-di-

acetylbenzidine were also assayed for carcinogenic activity. These compounds are of interestbecause of the definite, though weak, activity ofbenzidine (34) and also since they can be considered as derivatives of 4-acetylaminobiphenyl.

Since these studies were designed to obtaincomparative data on structure and carcinogenicactivity, the compounds were generally incorporated in the diet at equimolar levels and fed for astandard period. It is recognized that the relativeactivities of certain of the compounds might bedifferent under other regimens and that compounds which we found to be inactive might havesome carcinogenic activity under other conditions.

MATERIALS AND METHODSPreparation of compounds.'—TheAAF (22), 4-acetylamino

biphenyl (26), and 4-dimethylaminobiphenyl (22) were prepared as described previously. 2-Methyl-4-acetylaminobiphenyl(ni.p., 125°C.) and 2'-methyl-4-acetylaminobiphenyl (m.p.,148°-144°C.) were synthesized according to the procedures of

Melby, Brown, and Sandin (20). The acetanilide was a commercial sample (Eastman).

For the preparation of 8-hydroxy-4-aminobiphenyl (m.p.,185°-186°C.) and 3-hydroxy-4-acetylaminobiphenyl (m.p.,

1The assistance of Dr. R. Melby, Dr. F. Chubb, RobertCrawford, and Sidney Levine in the preparative work isgratefully acknowledged.

525

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526 Cancer Research

194°-195°C.), 4-nitrobiphenyl was heated with potassium

hydroxide and benzene as described by Colbert et al. (9); a50 per cent yield of 8-hydroxy-4-nitrobiphenyl (m.p., 102°-108°C.) was obtained. The nitro compound, dissolved in a

1:1 mixture of ethanol and ethyl acetate, was hydrogenatedat 50 p.s.i. in the presence of Raney nickel. The yield of3-hydroxy-4-aminobiphenyl (calculated for C«HnNO: C,77.8; H, 6.0; found: C, 78.2; H, 6.0) was 85 per cent. Theamine was acetylated in 90 per cent yield to 3-hydroxy-4-acetylaminobiphenyl (calculated for CnHiiNOj: C, 74.0;H, 5.7; found: C, 73.6; H, 5.7) which was crystallized fromalcohol or acetic acid.

3-Amino-4-acetylaminobiphenyl (m.p., 154.5°-155°C.; literature value, 155°C. [4]) was prepared by the hydrogénationat 50 p.s.i. of 8-nitro-4-acetylaminobiphenyl (m.p., 182°C.)

dissolved in ethyl acetate; Raney nickel was used as catalyst.The product was recrystallized from ethanol. S-Amino-4-dimethylaminobiphenyl (m.p., 48°-49°C.; b.p., 210°C. at

2.2 mm.) was similarly prepared by the hydrogénationof3-nitro-4-dimethylaminobiphenyl (m.p., 112°-113°C.) (3).

4'-Fluoro-4-acetylaminobiphenyl (m.p., 205°-206°C.)was

synthesized by the procedure of van Hove (85) and crystallizedfrom acetic acid. 2'-Fluoro-4-acetylaminobiphenyI (m.p.,157°-158°C.) was prepared from 2'-fluoro-4-acetylbiphenyl.

Twenty gm. were heated with 10 gm. hydroxylamine sulfate,10 gm. sodium hydroxide, 125 ml. water, and 50 ml. ethanoluntil a clear solution was obtained. On cooling, 21.5 gm.of crude oxime with a m.p. of 143°-147°C. was obtained.The over-all yield of oxime after recrystallization fromethanol was 89 per cent; the purified compound melted at 147°-148°C. and had the the correct analysis (calculated for

CiJIijNOF: C, 73.4; H. 5.3; found: C, 73.3; H, 5.5). Theoxime of 2'-fluoro-4-acetylbiphenyl (4.6 gm.) was dissolved in

60 ml. of ethyl ether, 4.5 gm. of phosphorus pentachloridewas added slowly, and the mixture was refluxed for 15 minutesand then cooled. The phosphorus pentachloride was decomposed by the cautious addition of water, the ether was evaporated, and the residue was refluxed with 6 N HC1 until a clearsolution was obtained. On cooling, the crystalline hydro-chloride of 2'-fluoro-4-aminobiphenyl was obtained in 90

per cent yield. The hydrochloride was acetylated (14) in 80per cent yield to 2'-fluoro-4-acetylaminobiphenyl (calculated

for CiiHisNOF: C, 73.4; H, 5.3; found: C, 73.2; H, 5.6), whichwas crystallized from acetic acid.

2-Acetylaminobiphenyl (m.p., 118.5°-120°C.) and 3-acetylaminobiphenyl (m.p., 148°C.) were obtained by the

acetylation (14) of the respective amines; these compoundswere crystallized from alcohol. The 2-aminobiphenyl was acommercial product (Eastman), while the 3-aminobiphenylwas prepared as previously described (13) except that the3-nitrobiphenyl was reduced with hydrogen over Raneynickel. 4-Fluorobiphenyl was synthesized by the procedure ofSchiemann and Roselius (32).

N,N'-Diacetylbenzidine (m.p., 322°-S23°C.), 2-methyl-N.N'-diacetylbenzidine (m.p., 308°-309°C.), 2,2'-dimethyl-N,N'-diacetylbenzidine (m.p., 273°-275°C.), and 3,3'-di-methyl-N,N'-diacetylbenzidine (m.p., 314°-315°C.) were pre

pared by the acetylation of the corresponding benzidines with»ceticanhydride in either benzene or acetic acid. Benzidineand its 3,3'-dimethyl derivative were commercial samples(Eastman). The 2-methyl- and 2,2'-dimethylbenzidine weresynthesized from 2-methylazobenzene (18) and S-nitrotoluene(33), respectively.

For the preparation of 4-acetylamino-p-terphenyl (m.p.,287°-289°C.) p-terphenyl was nitrated by the procedure of

Allen and Burness (1). The nitro compound was reduced withstannous chloride; the amine was then acetylated according

to Pummerer and Bittner (30), and the acetyl derivative wascrystallized from acetic acid.

3,4-Dimethylacetanilide was prepared by two differentprocedures. 8,4-Dimethylacetophenone was made fromfenchone according to Zaugg (88) or in 82 per cent yield fromo-xylene, acetyl chloride, and anhydrous aluminum chloride incarbon dioxide. In the ./irsi method the oxime of 3,4-dimethyl-acetophenone was converted by the Beckmann rearrangementto 3,4-dimethylaniline. Acetylation to 3,4-dimethykcetanilide(m.p., 99°C.) was carried out according to Fieser (14). Thesecond procedure involved the reaction of 3,4-dimethyIaceto-phenone with hydrazoic acid. To a solution of 21.4 gm. of8,4-dimethylacetophenone in benzene was added, with stirring,80 ml. of concentrated sulfuric acid; 123 ml. of a 6.4 per centsolution of hydrazoic acid in benzene was then added dropwiseover a 50-minute period with continuous stirring and whilethe temperature was maintained at 40°C. After the reaction

mixture was cooled, the sulfuric acid layer was separated,poured into 400 ml. of ice-water, and made alkaline with ammonium hydroxide. The precipitate was separated, washedwith water, and refluxed for 2 hours with concentrated hydrochloric acid. The reaction mixture was diluted with water,made alkaline, and extracted with ethyl ether. After theether was dried over anhydrous sodium sulfate, the ether wasevaporated, and the amine was distilled under reduced pressure (b.p., 120°C. at 18 mm.). The amine, which was obtained in 85 per cent yield, was acetylated (14) to 8,4-dimethyl-acetanilide (m.p., 99°C.).

All the compounds assayed for carcinogenicity were purifiedto constant melting point by crystallization from an appropriate solvent.

Method of assay.—Except where the amount ofcompound was insufficient, each compound wasfed to at least eight male and eight female albinorats;2 the number of animals in each group isindicated in the tables. The rats, weighing 190-210 gm. at the beginning of the experiments, werefed a stock grain diet (26) in which the compoundswere generally incorporated at a level of 1.62mM/kg of diet by the addition of 10 gm/kg of aglucose triturate prepared in a mortar. The exceptions are noted in the tables. In one experiment4-dimethylaminobiphenyl was fed at a level of5.32 mM/kg in a semi-purified diet composed of 12per cent crude casein, 2 per cent Vitab,3 4 per centsalt mixture, 77 per cent glucose monohydrate, 5per cent corn oil, and 2 mg/kg of riboflavin (including that in the crude casein). After 8 monthsthe feeding of the test compounds was generallydiscontinued, and the rats were maintained on thebasal grain diet for 2 months to allow small tumorsto develop. A few compounds were fed for longerperiods, and these feeding times and the times atwhich the experiments were terminated are givenin the tables.

After 4 months the rats were examined monthlyor bimonthly for external tumors. The autopsy for

*Holtzman Rat Company, Madison, Wis.'National Oil Products Company, Harrison, N.J.; the

manufacture of this rice bran extract has been discontinued.

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MILLERet al.—Carcinogenicity of Compounds Related to AAF. Ill 527

each rat included a gross examination of the earducts and mammary glands after the removal ofthe overlying tissue in each case, and of the liver,kidneys, heart, lungs, reproductive organs, spleen,thymus, and gastrointestinal tract. The entirelength of the small intestine was routinely passedbetween the thumb and forefinger, since many ofthe tumors are too small to be detected otherwise.Where the number of tumors available permitted,at least four, and usually more, gross tumors ofeach type from the rats fed each compound werestudied histologically after being fixed in Moss-man's acetic acid-formalin fixative (15), sectioned,

and stained with hematoxylin and eosin. All smallor questionable tumors and certain other tissueswere also examined histologically.

RESULTSIn general, the health and survival of the rats

was good up to the time of tumor formation, andthe number of rats which died without detectabletumors (negative deaths) before 8 months waslow except for the rats fed N.N'-diacetylbenzidine.

This compound caused a fatal glomerulonephritiswhich prevented adequate tests for carcinogenicity(16). In the tabulation of the data, rats bearingprimary tumors in more than one tissue werecounted under each classification. Multiple tumors of the ear duct, mammary gland, small intestinal epithelium, and liver occurred frequentlywhen the more active compounds were administered, but, to simplify the tables, no differentiation is made between animals with single ormultiple tumors derived from a given tissue. Inthe tables the incidences of tumors are given ascumulative totals at the times indicated.

As in our earlier experiments (19, 22, 26), AAF(Table 1, no. 1) induced liver tumors (malignanthepatomas and cholangiomas) in most of the malerats, adenocarcinomas of the mammary gland inmost of the female rats, ear duct tumors (carcinomas of the sebaceous glands and squamous-cellcarcinomas) in about 50 per cent of the animalsof both sexes, and adenocarcinomas of the epithelium of the small intestine in about 20 per centof the animals of either sex. 4-Acetylaminobi-phenyl (no. 2) was as active as AAF in the induction of adenocarcinomas of the mammary gland,but in the ear duct, small intestine, and liver, onlythree, two, and no tumors, respectively, werefound. 4-Dimethylaminobiphenyl (no. 3) had apotency similar to that of 4-acetylaminobiphenyland AAF as a mammary carcinogen for femalerats when fed under comparable conditions. Atthis level of compound (1.62 nnw/kg) the only

other tumor found was an adenocarcinoma of thesmall intestine in a male rat. In an earlier experiment (22) in which 4-dimethylaminobiphenyl wasfed at a higher level (5.32 mM/kg) in a semi-purified diet it induced a variety of tumors in malerats, including adenocarcinomas of the mammarygland, epidermoid carcinomas, and benign hepatomas. When the compound was retested underthese conditions, several tumors were found inmale rats, although in this case no liver tumors were detected. As in previous experiments(26) control rats fed the grain diet alone (Table2, no. 22) were tumor-free except for an occasional fibroma or fibroadenoma of the mammary gland.The incidence of benign tumors of the mammarygland was higher in this series than previously; apartial explanation may be that the control animalsin these experiments were kept for 13 months, whilepreviously they were killed after 10 months.

Substitution of a fluorine atom in the 4'-positionof 4-acetylaminobiphenyl yielded a compoundwith hepatocarcinogenic activity for the rat. Thelatent period was about twice as long as withAAF, but tumors, usually multiple, were found at10-12 months in the livers of each of the five malerats fed 4'-fluoro-4-acetylaminobiphenyl (Table 1,

no. 4) which survived more than 9 months. In onerat metastatic liver tumors were found in the kidney; in another there were métastasesto theomentum. On microscopic examination the tumors were classified as hepatomas and cholangiomas. Little nodularity or other pathology wasseen grossly in the uninvolved portions of thelivers. No liver tumors were found in the femalerats, but nine of the ten females developedadenocarcinomas of the mammary gland. Tumorsof the ear duct were detected in three of theseventeen rats fed the compound. The carcino-genicities of 4'-fluoro-4-acetylaminobiphenyl and4'-fluoro-4-aminobiphenyl have also been studied

by Morris and associates (28) and Hendry et al.(17), respectively. In contrast to the high incidence of tumors induced by 4'-fluoro-4-acetyl-

aminobiphenyl, no tumors were found in the ratsfed the isomerie compound 2'-fluoro-4-acetyl-aminobiphenyl (no. 5). 4-Fluorobiphenyl (no. 6)was likewise inactive.

No carcinogenic activity was observed for 3-hydroxy-4-aminobiphenyl (no. 7) or 3-hydroxy-4-acetylaminobiphenyl (no. 8); these compoundswere fed for 8 and 10 months, respectively, andthe surviving rats were killed 13 months after thebeginning of the experiment. Single fibroadenomasof the mammary gland, similar to those seen occasionally in the control rats on the grain diet,

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MILLER et al.—Carcínogenícüyof Compounds Related to AAF. Ill 531

were found in one of the female rats fed 3-hydroxy-4-acetylaminobiphenyl and in three fed 3-hydroxy-4-aminobiphenyl. Introduction of anamino group in the 3 position likewise led to aloss of activity. Except for one fibroadenoma ofthe mammary gland and an angiomatous polypof the uterus, no tumors were found in rats fedeither 3-amino-4-acetylaminobiphenyl (no. 9) or3-amino-4-dimethylaminobiphenyl (no. 10).

Introduction of a methyl group ortho to the bi-phenyl linkage of 4-acetylaminobiphenyl also destroyed its carcinogenic activity. 2-Methyl-4-acetylaminobiphenyl (no. 11) and 2'-methyl-4-

acetylaminobiphenyl (no. 12) both failed to induce tumors in these experiments. Walpole et al.(37) were similarly unable to induce tumors with2-methyl-4-aminobiphenyl.

The isomerie 2- and 3-acetylaminobiphenylshad little, if any, carcinogenicity. One male rat fed2-acetylaminobiphenyl (no. 13) developed anadenocarcinoma of the mammary gland withmétastasesto the lung, and one female rat had aleiomyosarcoma in the uterus. Adenocarcinomas ofthe mammary gland were found in two female ratsand a fibrosarcoma in one female rat fed 3-acetyl-aminobiphenyl (no. 14).

Like benzidine (34), N,N'-diacetylbenzidine

(Table 2, no. 15) has some carcinogenic activity.In the experiment listed in Table 2, two of themale rats developed squamous-cell carcinomasof the ear duct at 5 and 7 months, and in otherexperiments similar tumors were seen at 4-7months in four of twenty male rats fed N,N'-

diacetylbenzidine. However, its potency is difficultto assess, since rats fed the compound developeda fatal chronic glomerulonephritis at or before thetime that tumors would be expected (16). Afteronly 3-4 weeks on the diet, rats of either sex hada considerable proteinuria. The proteinuria progressed rapidly in the females so that at 2 monthsthey excreted as much as 0.1 gm. of protein perday; the severely anemic and edematous rats diedat 3-6 months. The male rats developed proteinuria more gradually, manifested a mild ne-phrotic syndrome only after 5 months, and died at5-8 months. Pathological study revealed a pro-liferative glomerulonephritis which affected nearlyall the glomeruli of each kidney. No gross evidenceof this lesion was seen in the rats fed the substituted diacetylbenzidines or any of the othercompounds studied.

Introduction of a methyl group in the 2 positionof N,N'-diacetylbenzidine gave a compound with

moderate carcinogenic activity (no. 16). Adenocarcinomas of the mammary gland were found inseven of the eight female rats; squamous-cell car

cinomas of the ear duct and adenocarcinomas ofthe small intestinal epithelium were detected in nineand five, respectively, of the seventeen rats fed thecompound. On the other hand, 2,2'-dimethyl-N.N'-diacetylbenzidine (no. 17) was inactive in ourtests. 3,8'-Dimethyl-N,N'-diacetylbenzidine (no.

18) had weak carcinogenic activity; adenocarcinomas of the mammary gland were found in two ofthe nine female rats and tumors of the ear ductin four of the eighteen rats fed the compound.

4-Acetylamino-p-terphenyl (no. 19) and 3,4-dimethylacetanilide (no. 20) appear to be veryweak carcinogens. One rat fed 4-acetylamino-p-terphenyl developed an adenocarcinoma of themammary gland and another an adenocarcinomain the small intestine. Of the 37 male rats fed 3,4-dimethylacetanilide one developed a cholan-gioma, while two of 38 female rats had adenocarcinomas of the mammary gland and one asquamous-cell carcinoma of the ear duct. Therelatively extensive tests with 3,4-dimethyl-acetanilide were made as a result of the suggestionfrom the first experiment that it might be ahepatic carcinogen. This possibility was not borneout when it was retested at higher levels. Asanticipated from earlier studies on aniline (10, 11)and acetanilide (6, 29), acetanilide (no. 21) wasinactive when fed at a level of 8.1 mM/kg of diet.Ekman and Strombeck (12) have reported metaplasia in the bladders of rats fed aniline; no grosschanges were seen in the bladders of our rats, butmicroscopic examinations were not made.

The histological characteristics of the tumors ofthe mammary gland, ear duct, small intestine,and liver were similar regardless of the compoundfed. These tumors have been described previously (22, 26).

DISCUSSIONIn general the carcinogenic activities of these

biphenyl and benzidine derivatives fall into thegeneral pattern seen previously (26). Thus, thecompounds of this group with carcinogenic activity usually induced primarily adenocarcinomas ofthe mammary gland ; the requirement for activityat this site was earlier found to be less specificunder our conditions than for tumors of the earduct, small intestinal epithelium, or liver, and toinvolve a biphenyl linkage. Several compoundsalso induced tumors of the ear duct and small intestine, but the only compound of this series withsignificant carcinogenic activity for the liver was4'-fluoro-4-acetylaminobiphenyl. This compoundlacks the méthylènebridge which is generally associated with hepatocarcinogenic activity in thisclass of compounds; however, the fluorine sub-

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532 Cancer Research

stituent para to the biphenyl linkage has beenshown previously to augment carcinogenic activity for the liver in the case of AAF. Two other reports have appeared recently on the carcinogenicactivity of 4'-fluoro-4-aminobiphenyl and its N-

acetyl derivative. Morris (28) found a high incidence of hepatomas and mammary tumors inBuffalo strain female rats given 4'-fluoro-4-

acetylaminobiphenyl in the diet. Hendry et al. (17)administered 4'-fluoro-4-aminobiphenyl by daily

subcutaneous injections and found liver tumors in22 of 24 male Wistar rats as well as large numbersof tumors in the kidneys and intestine. The survival time of these animals ranged from 381 to626 days, but the daily dose was only about one-half that administered in our experiments.

The mechanism by which a 4'-fluoro substituentenhances the carcinogenic activity of 4-amino-biphenyl and its N-acetyl derivative is not known.However, as also suggested by Hendry et al. (17),it seems likely that it acts, at least in part, by preventing hydroxylation in this position so thatmore of the administered compound is availablefor reactions leading to carcinogenesis. Hendryand associates have further indicated that interference with hydroxylation in the 4' position might

favor the formation of the ortho hydroxyaminoderivative, which this group of investigators haspostulated to be the effective carcinogen (36).Similar suggestions concerning the central role ofortho hydroxyamino derivatives in the carcino-genicity of various aromatic amines have beenmade by others (2, 5, 8), but only in the case of/3-naphthylamine (5) and benzidine (2) have directsupporting data been presented. The recent finding (7) that, following the ingestion of benzidineor 4-aminobiphenyl, small amounts of theirortho hydroxy derivatives are excreted in theurine of the dog is of interest. However, the relationship of these metabolites to the action of theseamines as bladder carcinogens remains to bedemonstrated. In the case of the aminobiphenylsour failure to induce any malignant tumors witheither 3-hydroxy-4-aminobiphenyl or its N-acetylderivative under conditions suitable for the induction of high incidences of tumors with 4-acetyl-aminobiphenyl, its 4'-fluoro derivative, or 4-

dimethylaminobiphenyl argues against a central role in carcinogenesis for the ortho hydroxyamino derivatives. Possible differences in thedegrees of absorption of various compounds always introduce some uncertainty in assigning relative biological activities to the compounds of aseries. In this case, however, the 3-hydroxy compounds presumably were available for direct action on the intestine. Although the epithelium of

the small intestine of our rats has been shown tobe quite susceptible to carcinogenesis by AAF andrelated compounds, no intestinal tumors werefound in the rats given the ortho hydroxy amines.In this connection, it should be noted that Wai-pole et al. (36) and Clayson (8) also suggested thatthe carcinogenic action of the aminoazo dyes wasdue to their conversion to ortho hydroxyaminoderivatives. Again, a direct test of this hypothesisby feeding 3-hydroxy-4-dimethylaminoazobenzenegave negative results; no tumors were induced inrats fed the compound as 0.064 per cent of thediet for 8 months.4 Other aspects of this problemhave been considered in a recent review (21).

Introduction of methyl groups in the rings of 4-aminobiphenyl or benzidine results in compoundsof widely different activities. Certain derivatives,such as 2-methyl-4-aminobiphenyl (37), 2- or 2'-methyl-4-acetylaminobiphenyl, or 2,2'-dimethyI-N,N'-diacetylbenzidine have shown no activity.2-Methyl-N,N'-diacetylbenzidine has moderatecarcinogenic activity. Derivatives of 4-aminobiphenyl with a methyl group in the 3 positionhave proved more active than 4-aminobiphenyl intests by Walpole and associates (36, 37), while3,3'-dimethyl-N,N'-diacetylbenzidine was considerably less active than 2-methyl-N,N'-diacetyl-

benzidine in our tests. The explanation for theseeffects of methyl substituents is not clear. Walpole et al. (36), extrapolating from data on themetabolism of aniline and o-toluidine by dogs,have suggested that the 3-methyl group in 4-aminobiphenyl promotes the formation of theortho hydroxyamino derivative in vivo. The lackof pertinent metabolic data for these compoundsin the rat and the inactivity of 3-hydroxy-4-aminobiphenyl and its N-acetyl derivative greatlyweaken this argument. In the tests by Walpoleand associates (36, 37) the compounds wereusually administered by subcutaneous injection,but 3,2'-dimethyl-4-aminobiphenyl was found to

be equally effective by either the oral or subcutaneous route. At the opposite extreme one mightsuggest that in the rat hydroxylation of 4-aminobiphenyl in the 3(5) position is a major pathwayof metabolism and that this hypothetical metabolite is noncarcinogenic; if so, blocking one of the3(5) positions with a methyl group might permitmore of the metabolism to proceed via carcinogenic metabolites. The high activity of 3,2'-dimethyl-4-aminobiphenyl (36) appears to ruleout maintenance of planarity between the benzene rings (22, 31) as a major structural requirement for carcinogenic activity, although it maystill play some role.

«J.A. Miller and E. C. Miller, unpublished data.

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MiLLEB et al.—Carcinogenicity of Compounds Related to AAF. Ill 533

SUMMARY1. The carcinogenic activities for the rat of 4-

acetylaminobiphenyl, N,N'-diacetylbenzidine, and

eighteen related compounds have been investigated. The compounds were administered orallyfor 8-10 months.

2. Under these conditions 4-acetylaminobi-phenyl induced a high incidence of adenocarcino-mas of the mammary gland in female rats andsmall numbers of adenocarcinomas of the smallintestine and squamous-cell carcinomas andsebaceous gland carcinomas of the ear duct. Inaddition to these tumors, 4'-fluoro-4-acetylamino-

biphenyl induced a high incidence of liver tumorsin male rats at 10-12 months.

3. 3-Hydroxy-4-aminobiphenyl and its N-acetyl derivative were noncarcinogenic. Thesecompounds do not appear to be the effective carcinogen when 4-aminobiphenyl or its N-acetylderivative is administered.

4. The following compounds had little or nocarcinogenic activity under our conditions: 2-methyl-, 2'-methyl-, 2'-fluoro-, and 3-amino-4-acetylaminobiphenyl ; 3-amino-4-dimethylamino-biphenyl; 2- and 3-acetylaminobiphenyl; 4-fluoro-biphenyl; 4-acetylamino-p-terphenyl; acetanilide;and 3,4-dimethylacetanilide.

5. Rats fed N,N'-diacetylbenzidine developed a

fatal glomerulonephritis which prevented adequate tests for carcinogenic activity. 2-Methyl-N,N'-diacetylbenzidine had moderate carcinogenic activity similar to that of 4-acetylamino-biphenyl; 3,3'-dimethyl-N,N'-diacetylbenzidinehad weak activity; and 2,2'-dimethyl-N,N'-dia-cetylbenzidine was inactive.

6. The syntheses of the following new compounds are described: 3-hydroxy-4-aminobi-phenyl, 3-hydroxy-4-acetylaminobiphenyl, and2'-fluoro-4-acetylammobiphenyl.

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1956;16:525-534. Cancer Res   E. C. Miller, R. B. Sandin, J. A. Miller, et al.   Derivatives2-Acetylaminofluorene: III. Aminobiphenyl and Benzidine The Carcinogenicity of Compounds Related to

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