Structure-Activity Studies of the Carcinogenicities in the Mouse and Rat of Some Naturally Occurring and Synthetic Alkenylbenzen

[CANCER RESEARCH 43, 11 24-1134, March 1983] 0008-5472/83/0043-OOOOS02.00 Structure-Activity Studies of the Carcinogenicities in the Mouse and Rat of Some Naturally Occurring and Synthetic Alkenylbenzene Derivatives Related to Safrole and EstragÃ³le1

Elizabeth C. Miller, Anne B. Swanson,2 David H. Phillips,3 T. Lloyd Fletcher, Amy Liem, and James A. Miller

McArdle Laboratory for Cancer Research, University of Wisconsin Medical School, Madison, Wisconsin 53706 [E. C. M., A. B. S., D. H. P., A. L., J. A. M.], and The Fred Hutchinson Cancer Research Center [T. L. FJ Seattle, Washington 98104

ysafrole, and 1'-hydroxyestragole, when administered topically ABSTRACT to female CD-1 mice at relatively high doses, initiated benign Twenty-three naturally occurring and synthetic alkenylben- skin tumors that could be promoted with croton oil. zene derivatives structurally related to the hepatocarcinogen safrole (1-allyl-3,4-methylenedioxybenzene) were assayed for INTRODUCTION their hepatocarcinogenicity in mice. Some of these compounds (safrole, estragÃ³le, eugenol, anethole, methyleugenol, myristi- Safrole (1-allyl-3,4-methylenedioxybenzene), a major con cin, elemicin, and dill and parsley apiols) may be ingested in stituent of oil of sassafras and a component of certain other very small amounts by humans as natural components of essential oils4 (9, 11, 12, 22), has induced hepatic tumors certain spices, essential oils, or vegetables. EstragÃ³le (1-allyl- when fed as 0.5 to 1% of the diet of rats or mice for long 4-methoxybenzene) and its proximate carcinogenic metabolite periods and on administration to male CD-1 mice during the 1'-hydroxyestragole, previously shown to induce hepatic tu preweaning period only (1, 7, 16-18, 54). Hepatic tumors also mors when administered to male CD-1 mice only during the developed in male C57BL/6 x C3H F, (hereafter called preweaning period, also induced hepatic tumors on administra B6C3F,) mice that had been nursed by safrole-treated mothers tion for 12 months in the diet of female CD-1 mice. Eugenol (1- (52); female mice born to mothers given safrole during preg allyl-4-hydroxy-3-methoxybenzene) and anethole -(frans-4- nancy developed a low incidence of renal carcinomas but no methoxy-1-propenylbenzene) were inactive in this assay; they hepatic tumors (52). EstragÃ³le (1-allyl-4-methoxybenzene), a were also inactive when administered i.p. during the prewean major constituent of the oils of tarragon (estragon) and sweet ing period at total doses of up to 9.45 /imol/mouse to male basil (9,11,12, 22), had hepatocarcinogenic activity similar to CD-1 or C57BL/6 x C3H F, (hereafter called B6C3F,) mice. that of safrole in mice given injections of the compound prior Methyleugenol (1-allyl-3,4-dimethoxybenzene) and its 1'-hy- to weaning (7). Prior to the current study, estragÃ³le had not droxy metabolite had activities similar to those of estragÃ³le and been assayed in other systems. In addition to safrole and its 1'-hydroxy metabolite for the induction of hepatic tumors in estragÃ³le, a number of other allylic and propenylic benzene male B6C3Ft mice treated prior to weaning; 1-allyl-1 '-hydroxy- derivatives (Chart 1) with ring-methoxy and/or ring-methyle- 4-methoxynaphthalene was somewhat less active. At the levels nedioxy substituents occur, usually at quite low levels, in tested, myristicin (1-allyl-5-methoxy-3,4-methylenedioxyben- various foods, including spices, herbs, and vegetables (4, 9, zene), elemicin (1-a!lyl-3,4,5-trimethoxybenzene) and its 1'- 11, 12, 22, 23, 45). Of these, isosafrole (3,4-methylenedioxy- hydroxy metabolite, dill apiol (1-allyl-2,3-dimethoxy-4,5-meth- 1-propenylbenzene) showed weak hepatocarcinogenic activity ylenedioxybenzene), parsley apiol (1-allyl-2,5-dimethoxy-3,4- for mice (16). /S-Asarone (c/s-1-propenyl-2,4,5-trimethoxyben- methylenedioxybenzene), 1'-hydroxyallylbenzene, 3'-hydrox- zene) is a major component of oil of calamus, a bitters flavoring yanethole, and benzyl and anisyl alcohols had no detectable agent (10, 22). The ingestion of a high level of either this oil or activity for the initiation of hepatic tumors on administration to ÃŸ-asarone induced leiomyosarcomas of the small intestine in male mice prior to weaning. The acetylenic derivative 1'-hy- rats (10, 20). droxy-2',3'-dehydroestragole was much more active than Both safrole and estragÃ³le are metabolized by rat and mouse either 1'-hydroxysafrole or 1'-hydroxyestragole when admin liver microsomes to their 1'-hydroxy derivatives (50), which are istered to preweanling mice. The 2',3'-oxides of safrole, estra excreted as conjugates in the urine of safrole- or estragole- gÃ³le,eugenol, and 1'-hydroxysafrole, which are metabolites of treated animals (2, 7). The 1'-hydroxy metabolites are stronger these alkenylbenzenes, had little or no activity in this test. The hepatocarcinogens than are the parent compounds (1, 7). Low 2',3'-oxides of safrole and estragÃ³le and their 1'-hydroxy de levels of Mannich bases (/8-aminoketones of the general struc rivatives likewise had little or no activity for the induction of ture Râ€”COâ€”CH2â€”CH2â€”NR,R2)areexcreted in the urine of lung adenomas in female A/J mice or for the induction of safrole-treated rats (35), and this suggests the formation in tumors on repetitive injections s.c. in male Fischer rats. How vivo of the electrophilic derivative 1'-oxosafrole (53). Epoxi- ever, the 2',3'-oxides of safrole, estragÃ³le, eugenol, 1'-hydrox- dation of the allylic side chains of safrole, estragÃ³le, and their 1'-hydroxy derivatives occurs with NADPH-fortified hepatic ' Supported by Grants CA-07175, CA-22484, and CA-09020 of the National Cancer Institute, United States Department of Health and Human Services. microsomes (50, 53), and these electrophilic metabolites are 2Present address: Department of Physical Science, Edgewood College, Mad also apparently formed in vivo (42, 48). Activation through a ison, Wis 53711. 3'-phosphoadenosine 5'-phosphosulfate-mediated reaction 3 Present address: Chester Beatty Research Institute, Institute of Cancer Research, Royal Cancer Hospital, Fulham Road, London, SW3 6JB, United Kingdom. ' Essential oils are the volatile oils obtained from plant materials by steam Received September 21, 1982; accepted December 7, 1982. distillation, solvent extraction, or expression.

1124 CANCER RESEARCH VOL. 43

and 1'-hydroxyestragole, were synthesized and characterized as noted in the references cited. Elemicin (1 -allyl-3.4,5-trimethoxybenzene) was prepared as described previously (25). Methyleugenol (l-allyl-3,4-dimethoxybenzene) was prepared from eugenol by a methylation pro cedure (24) used for similar phenols. The 1'-hydroxy derivatives of elemicin and methyleugenol were prepared as described recently (43, 44) by the procedure previously used for the preparation of 1'-hydrox ysafrole (2). These 1'-hydroxy derivatives were purified by dry-column chromatography on silica gel (60 to 200 mesh) developed with ether/ hexanes (75/25, v/v). The IR and nuclear magnetic resonance spectra and the elemental analyses of the preparations of elemicin, methyleu genol, and their 1'-hydroxy derivatives were entirely consistent with the expected structures. Dill apiol (1-allyl-2,3-dimethoxy-4,5-methyl- PROPENYLBENZENE enedioxybenzene) (6) was kindly prepared for us by Professor Dr. Chart 1. Basic structures found in the majority of the naturally occurring Franz Daliacker, Institut fÃ¼rOrganische Chemie, Technischen Hochs alkenylbenzenes. chule, Aachen, Germany, and myristicin (1-allyl-5-methoxy-3,4-methylenedioxybenzene) was generously made available by Drs. W. Gaietto, has been demonstrated for 1'-hydroxysafrole with hepatic cy- J. L. Welbourn, and R. L. Hall, McCormick and Co., Research and tosols (53). Studies on the structures of the hepatic DNA Development Laboratories, Inc., Hunt Valley, Md. All of the above adducts of mice given either 1'-hydroxysafrole or 1'-hydrox- compounds were assayed by high-performance liquid chromatography yestragole indicate that esters (probably the sulfuric acid es and, based on the absorbance at 254 or 280 nm of the material eluted ters) are the major electrophilic metabolites reacting with the from the column, were judged to have purities of at least 98%. hepatic DNA in vivo (36, 37). PhÃ©nobarbital and ethyl carbamate were obtained from J. T. Baker The 2',3'-oxides of safrole and estragÃ³le, the 1'-hydroxy Chemical Co., Phillipsburg, N. J., and Aldrich Chemical Co., respec derivatives of these oxides, and the 1'-acetoxy derivatives of tively. Trioctanoin was obtained from either Sigma Chemical Co. (St. Louis, Mo.) or Pfaltz and Bauer, Inc. (Stamford, Conn.); it was sterilized safrole and estragÃ³le (models for the synthetically unavailable by dry heat at 100Â° for 3 hr. Croton oil was purchased from Amend sulfuric acid esters) are all mutagenic for the missense mutants Drug and Chemical Co., New York, N. Y. Salmonella typhimurium TA1535 and TA100 without addition The trioctanoin solutions for injections or intubations were prepared of a metabolic activation system (7, 26, 49, 54). No mutagen- at room temperature and were stored at -20Â°. Fresh solutions were icity was detected for the corresponding 1'-oxo derivatives at prepared at 2- to 4-week intervals. Disposable plastic gloves were levels at which their toxicities permitted the assays to be carried used in the handling of the alkenylbenzenes and their derivatives. out (36, 54). 1'-Acetoxysafrole was hepatocarcinogenic after administration to preweanling mice and had weak carcinogenic Animals activity at sites of application (e.g., the forestomach and s.c. injection site) (1 ). 1'-Oxosafrole showed no carcinogenic activ Induction of Hepatic Tumors by Administration Prior to Weaning. CD-1 breeding stock were obtained from the Charles River Breeding ity under similar conditions (54). Laboratories, Inc. (Wilmington, Mass.) and female C57BL/6J and male The studies reported here were carried out to further explore the carcinogenicities of safrole and estragÃ³le and their 1'- C3H/HeJ mice were obtained from The Jackson Laboratory (Bar Harbor, Maine). These mice were bred in our laboratory, and the young hydroxy derivatives and to assess the carcinogenic activities were given injections according to the schedules given in the tables. for mouse liver of a number of other naturally occurring alken- For most experiments, the injections were given on Days 1,8, 15, and ylbenzenes and some of their 1'- or 3'-hydroxy metabolites.5 22; Day 1 was the first 24 hr after birth. The average body weights of In addition, the carcinogenicities of some metabolites of safrole mice of both sexes were 1.4 g (Day 1), 3.5 g (Day 8), 7 g (Day 15), and and estragÃ³le were further examined. Particular attention was 13 g (Day 22). The CD-1 litters were generally reduced to 12 young, placed on the 2',3'-oxides, which had previously received little and the B6C3F, litters were reduced to no more than 10 by killing study for carcinogenic activity. Several synthetic compounds some or all of the female young. All of the breeding stock were maintained in plastic cages on which were related to the alkenylbenzenes and their metabo hardwood bedding (Betta-Chip; Northeastern Products Corp., War- lites were also tested. rensburg, N. Y.). In general, the experimental mice were also housed in plastic cages in groups of 4 to 5 from weaning until termination of MATERIALS AND METHODS the experiments. In 2 experiments, the mice were housed in screen- bottomed cages (4 to 5/cage) for the first 8 months and were then Chemicals transferred to plastic cages. All of the breeding stock were fed Wayne Breeder Blox pellets (Allied Anethole (frans-4-methoxy-1-propenylbenzene), estragÃ³le, safrole, Mills, Inc., Chicago, III.). These pellets were also used for most of the and eugenol (1-allyl-4-hydroxy-3-methoxybenzene), benzyl alcohol, experimental mice; for one experiment, a pelleted purified diet prepared and anisyl alcohol (p-methoxybenzyl alcohol) were obtained from by Teklad Test Diets (Madison, Wis.) was used. This diet contained Aldrich Chemical Co., Inc. (Milwaukee, Wis.). Parsley apiol (1-allyl-2,5- (per kg) vitamin-free casein, 200 g; DL-methionine, 3 g; corn starch, dimethoxy-3,4-methylenedioxybenzene) was obtained from Saber Lab 250 g; sucrose, 421 g; corn oil, 60 g; cellulose, 20 g; mineral mix oratories, Inc. (Morton Grove, III.). 1'-Hydroxysafrole (2), 1'-hydrox- (Teklad No. 76514), 35 g; calcium carbonate, 0.81 g; vitamin mix yestragole (7), 3'-hydroxyanethole (49), l-allyl-1'-hydroxy-4-methox- (Teklad No. 40060), 10 g. All animals were fed ad libitum, and the diet ynaphthalene (7), 1'-hydroxyallylbenzene (7), 1'-hydroxy-2',3'-dehy- used for each experiment is specified in the tables. droestragole (36), 1'-acetoxysafrole (2), and the 2',3'-oxides (49, 53) The i.p. injections into the preweanling mice were made with either of safrole, estragÃ³le, eugenol, 1'-hydroxysafrole, 1'-acetoxysafrole, a 100-fil Hamilton syringe fitted with 27-gauge needle or with 0.5-ml insulin syringes graduated to 0.01 ml and fitted with an integral 27- 5 Some of these data have been presented in a preliminary form (28, 29). gauge needle. In one experiment, the mice were given the trioctanoin

MARCH 1983 1125

Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 1983 American Association for Cancer Research. E. C. Miller et al. solutions P.O. through a No. 24 stainless steel feeding tube (Perfektum; formalin, sectioned at 5 to 6 Â¡im,and stained with hematoxylin and Popper and Sons, Inc., New Hyde Park, N. Y.). eosin. We are indebted to Dr. Henry C. Pilot of the McArdle Laboratory Induction of Tumors by Administration for 12 Months in the Diet for the examination of these sections and for the histological diagnoses. of CD-1 Mice. Based on earlier studies with safrole and 1'-hydroxy- Statistical Evaluations. Comparisons of tumor incidences were safrole(1, 54), estragÃ³le, 1'-hydroxyestragole, eugenol, and anethole made by Fisher's exact test (Ref. 3, p. 195). Differences in tumor were assayed for their carcinogenicity on administration in a grain diet multiplicity were examined by the Mann-Whitney (Wilcoxon) test (Ref. (38) to female CD-1 mice. These mice were obtained directly from the 15, p. 68). Charles River Breeding Laboratories and were started on the diets at 5 weeks of age; they were maintained in ventilated cages. Solutions of RESULTS the chemicals in redistilled acetone were added to the grain diet in a hood. As soon as the acetone had evaporated, the diets were mixed in Induction of Hepatic Tumors by Allylbenzene and Propenyl- closed plastic bags, stored in closed cans in a refrigerator, and added benzene Derivatives Related to Safrole and EstragÃ³le and to the feeders 3 times/week. Because of their volatility at room tem Their 1'-Hydroxy Derivatives perature, the amounts of the alkenylbenzene derivatives actually ob tained by the mice were somewhat lower than those indicated in the Compounds Administered Prior To Weaning to CD-1 or tables. The loss of the compounds from diets stored in closed cans in the refrigerator for up to 2 weeks was generally less than 10%; the B6C3F, Mice. Administration of a variety of chemicals only loss on standing in open containers at room temperature was of the during the preweaning period to male mice derived from certain order of 5%/day. stocks or strains of mice (e.g., C3H, CBA, CD-1 ) has proved to Since earlier studies had shown that CD-1 mice do not tolerate be a sensitive assay for the induction of hepatic tumors (1, 7, administration of safrole or 1'-hydroxysafrole as 0.25% or more of the 8, 27, 39). With this assay, the structures of safrole and diet unless they are first adapted by administration of lower doses (54), estragÃ³le appear to be relatively favorable among the allyl- and all of the mice were fed diets for 2 or 3 ten-day periods with progres propenylbenzene compounds studied for the induction of he sively increasing levels of the compounds. The details are given in patic tumors (Tables 1 to 3). With CD-1 mice and administration Table 4. Some of the mice also received 0.05% phÃ©nobarbital in their by either gastric tube or i.p. injection, neither anethole nor drinking water for the entire experiment. Induction of Skin Tumors by Topical Administration of 2',3'-Ox- eugenol showed hepatocarcinogenic activity at doses at which ides. Female CD-1 mice from the Charles River Breeding Laboratories 61 to 73% of the male mice treated with safrole or estragÃ³le were used at 7 to 8 weeks of age. After being shaved, the mice were developed hepatic tumors and for which the average multiplic treated topically 4 days/week for 6 weeks with 0.15 ml of redistilled ities of tumors per liver were 1.7 to 3.5 by about 1 year. The acetone containing 11.2 /Â¿molofthe 2',3'-oxides of safrole, estragÃ³le, uninjected and solvent-treated control male mice had inci eugenol, 1'-hydroxysafrole, or 1'-hydroxyestragole. Starting 1 week dences of 15 to 26% and average multiplicities of only 0.2 to after the last dose of epoxide, all of the mice were treated topically 0.6 tumor/liver (Table 1). In agreement with previous studies twice weekly with 0.15 ml of 0.6% croton oil in redistilled acetone. The which used this assay (1, 39), solvent-treated female mice had mice were reshaved as necessary, and papillomas were enumerated at a much lower multiplicity of hepatomas (0.02/mouse) than did biweekly intervals. The experiment was terminated at 40 weeks. These the males. Safrole had only marginal activity in the female mice, mice were maintained in groups of 5 in screen-bottomed cages. while estragÃ³le, anethole, and eugenol showed no activity. For Induction of Lung Adenomas in Female A/J Mice. Groups of 25 female 8-week-old A/J mice from The Jackson Laboratory were given these and subsequent data in this paper, the calculations of tumor multiplicities, as well as the incidences, are based on all injections twice weekly for 12 weeks of 0.5 or 1 /imol of alkenylbenzene derivative per 0.005 ml sterile trioctanoin per g of body weight. The of the animals considered to be at risk (e.g., those autopsied mice were housed in groups of 5 in screen-bottomed cages. The mice at 11 to 14 months in Experiment 1). were killed 8 months after the first injection, and the lungs were fixed Since, with the outbred CD-1 stock, the incidence by 15 in neutral 10% formalin. The adenomas 2:1 mm in diameter on the months of hepatic tumors in male mice of the treated groups surface of the lungs were enumerated. rarely surpassed 75 to 80% (Refs. 1 and 54; Table 1), B6C3F, Induction of Tumors by Repetitive s.c. Injections in Fischer Rats. mice were used in subsequent studies. As expected from their Groups of 20 male Fischer rats from the Charles River Breeding genetic uniformity, the development of hepatic tumors in these Laboratories were given s.c. injections twice weekly, starting at 5 mice was more uniform than for the CD-1 stock (Tables 2 and weeks of age, of 0.2 ml of sterile trioctanoin containing 30 to 100 fimol 3). 1-Allyl-1'-hydroxy-4-methoxynaphthalene was somewhat of compound. Each rat was given 20 injections, and all of the injections less active than was 1'-hydroxyestragole or 1'-hydroxysafrole, were made at approximately the same s.c. site in the right hind leg. and 1'-hydroxyallylbenzene had no evident activity as an he The rats were housed singly in screen-bottomed cages. General Care and Autopsies. All of the animals were weighed patic carcinogen. Benzyl and anisyl alcohols were also inactive. The synthetic derivative 1-allyl-1 '-hydroxy-4-methoxynaphtha- initially and at least monthly throughout the experiments. Unless other wise indicated, all of the animals received Wayne Breeder Blox ad lene was of interest because its acetic acid ester was more libitum. The animals were kept in rooms without windows on a 12-hr- electrophilic than 1'-acetoxyestragole toward inosine and was light-12-hr-dark cycle; the rooms were maintained at 22-25Â°F. approximately equal in activity to 1'-acetoxyestragole as a Autopsies were performed on all animals found dead or seriously ill direct mutagen for S. typhimurium TA100 (7). during the experiments and, at the termination, on all of the surviving Table 3 details results obtained on comparison of a number animals. Major attention was given to the target organ for each exper of naturally occurring alkenylbenzenes. At a total dose of 4.75 iment (liver for mice given injections prior to weaning, lung for A/J /imol/mouse in B6C3Fi mice, methyleugenol (Group 7) had an mice, and skin for those treated topically) but, in addition, a gross activity similar to that of estragÃ³le (Group 1) for the induction examination was made of the organs of the pleural and peritoneal cavity and of the s.c. tissue of each animal. Representative liver tumors of hepatic tumors, but elemicin, myristicin, anethole, and dill from each animal, approximately one-third of the skin tumors from the and parsley apiols (Groups 2 to 6) had no detectable activity. mice treated topically and of the lung adenomas from the A/J mice, A negative result was also observed on assay of myristicin and all other tumors or grossly abnormal tissues were fixed in neutral (total dose, 3.75 Â¡umol)inthe previous experiment (Table 2).

1126 CANCER RESEARCH VOL. 43

Table 1 Development of hepatomas in CD-7 mice given various alkenylbenzenes or their derivatives during the first 4 or 5 weeks after birth The test chemicals were administered to CD-1 mice in sterile Irioctanoin. For Experiment 1, the indicated doses (based on body weight) were administered by stomach tube twice weekly for 10 times starting on Day 4; 0.01 ml of trioctanoin was administered per g of body weight. For Experiment 2, male mice received the chemicals by i.p. injections; the total doses are given below. The fractions of the dose administered on Days 1, 8,15, and 22 were in the ratio of 1:2:4:8, respectively. These doses were injected in 0.025, 0.05, 0.05, and 0.1 ml on Days 1,8, 15, and 22, respectively. The mice from Experiment 1 were weaned at 35 days (after the last intubation); those from Experiment 2 were weaned at 22 days. All mice were fed Wayne Breeder Blox pellets. Some male mice from Groups 1 and 2 of Experiment 1 died with hepatic tumors as early as 11 months, but the experiment was not terminated until 14 months, to permit a longer observation time for the eugenol- and anethole-treated mice. Experiment 2 was terminated at 12 months.

of Of of no. of mice with mice mice ex hepatomas/ lung ade mousea3.0e0.2d3.5e0.10.500.30.020.60.040.60.021.9e1.7e0.60.52.7e0.31.0'0.50.60.50.50.2No.nomas"337173222202743255255612 QroupCompound1 (/imol)Experiment2.5/g weaned11-14 amined494849445152515659505947484645424644514148494246Hepatoma-bearing(%)61e1373e925018231424267e65'243365e1455d3940312615Av.

atP.O.P.O.P.O.P.O.P.O.P.O.P.O.P.O.P.O.P.O.P.O.P.O.MFMFMFMFMFMFExperiment: mice autopsied mos.615355495955566167556552Ã•2mo."555252535252565256535950No. Safrole2 X102.5/g X102.5/g

EstragÃ³le3 X102.5/g x102.5/g

Eugenol4 x102.5/g x102.5/g

Anethole5 x102.5/g x105/g

Anethole6 x105/g x 10RouteSex)

(Trioctanoin)Dose

at1 2: mice killed Safrole2 EstragÃ³le3 Eugenol4 Anethole5 '-Hydroxysafrole61 2',3'-oxide7 Safrole 2',3'-oxide8 1'-Hydroxysafrole

2',3'-oxide9 1'-Acetoxysafrole

2',3'-oxide10 EstragÃ³le 2',3'-oxide1 Eugenol only)121 {Trioctanoin (Not injected)9.459.459.459.454.729.459.459.459.459.45i.p.i.p.i.p.Â¡.p.i.p.i.p.i.p.i.p.i.p.i.p.i.p.MMMMMMMMMMMMNo. Hepatic tumors 22 mm in diameter are tabulated. 6 In most cases, each mouse had only one grossly visible lung adenoma (al mm in diameter). 0 Statistically different (p < 0.001) from the value for the males of Group 6 or of Group 11 for Experiments 1 and 2, respectively. 0 Statistically different (p < 0.05) from the value for the female mice of Group 6 or the male mice of Group 11 for Experiments 1 and 2. respectively. e In addition, for Experiment 2. one mouse in each of Groups 1 and 2 developed generalized lymphoma. and one mouse in each of Groups 2 and 9 developed a reticulum cell sarcoma. ' Statistically different (p < 0.01 ) from the value for the mice of Group 11.

1'-Hydroxyelemicin, 1'-hydroxymethyleugenol, and 3'-hy- treated with this compound and examined by laparotomy at 13 droxyanethole, metabolites of the corresponding parent alken months had hepatic tumors with an average multiplicity of 10, ylbenzenes (42-44, 50), were also included in the experiment even though the maximum dose that could be administered shown in Table 3. 1'-Hydroxymethyleugenol (Group 13) was a was 1.9 Â¿imol/mouse. Because of the extensive tumors, only relatively strong carcinogen in this system; because of its 6 of these mice survived to 18 months. The reason for the lack toxicity for 1- and 8-day-old mice, the total dose of 1'-hydrox of a dose response in tumor formation on administration of 1'- ymethyleugenol was only 2.85 /imol/mouse. Neither 1'-hy- hydroxyestragole at doses of 1.9 to 4.65 jumol/mouse (Groups droxyelemicin nor 3'-hydroxyanethole (Groups 11 and 12), 8 to 10) is not evident, although it is possible that the enzyme(s) administered at levels of 4.75 jimol/mouse, showed activity as responsible for its metabolism to the ultimate carcinogenic form an hepatic carcinogen in this assay; thus, the inactivity of their are limiting factors. In any case, this result made quantitative parent compounds cannot be ascribed to a relative inability to comparisons of the relative activities of the various compounds be converted to side-chain hydroxy derivatives. uncertain. The synthetic acetylenic derivative 1'-hydroxy-2',3'-dehy- For the data in Table 3, similar conclusions with regard to droestragole proved to be the most active of the compounds the hepatocarcinogenicities of the compounds were obtained studied (Table 3, Group 14). This compound was available as on examination of the livers of approximately 20 mice/group an intermediate in the synthesis of [2',3'-3H]-1'-hydroxyestra- at 13 months and on autopsy of groups of 40 to 60 mice gole (36). It is closely related in structure to the carcinogenic (including those examined by laparotomy) at 13 to 18 months. diaryl acetylenic carbamates (13, 14, 40). All of the mice Thus, the average numbers of hepatic tumors per mouse were

MARCH 1983 1127

Table 2 Development of hepatomas in male B6C3F, mice which received injections of alkenylbenzene derivatives prior to weaning The test chemicals were dissolved in sterile trioctanoin and injected i.p. on Days 1, 8, 15. and 22. The total doses are indicated below; the amounts injected per dose were in the ratio of 1:2:4:8. The volumes of trioctanoin per mouse were 25, 50, 50, and 100 /Â»I,respectively, for these injections. The mice were weaned at 4 weeks of age. Both the mothers and the treated animals were fed Wayne Breeder Blox pellets throughout the experiment. All surviving mice were killed at 12 months for enumeration of hepatomas. mos.Compound1 Mice killed at 12

ofmiceweaned353036353534333335No.ofmiceexamined272634323032333235Hepatoma-bearingmice(%)93C92C65C131016211515Av.no. ofhepa-tomas/mouse82.7C2.7Â°1.1C0.10.10.20.20.20.1 dose(/imol)1.87"3.753.753.753.753.753.75No. '-Hydroxyestragole1 '-Hydroxysafrole1 -Allyl-1'-hydroxy-4-methoxynaphthalene1

'-HydroxyallylbenzeneBenzyl alcoholAnisyl alcoholMyristicin(Trioctanoin)UninjectedTotal

Nodules >2 mm in diameter. " 1'-Hydroxyestragole was administered at one-half of the level used for the other compounds, in view of its greater toxicity for Day 1 mice. c Statistically different from the value for the trioctanoin-treated controls (p < 0.001 ).

1.2 and 1.5 for the estragÃ³le- and methyleugenol-treated mice, an average life span of only 13.6 months after initiation of respectively, at 13 months; the values were 2.4 and 3.2, treatment, as compared to survivals in excess of 18 months for respectively, for the mice examined at autopsy at 13 to 18 each of the other groups. This shorter survival period probably months. The solvent-treated and uninjected controls showed accounted for the relatively low multiplicity of hepatomas in the average multiplicities of 0.1 to 0.2 and 0.5 tumor/liver on 1'-hydroxyestragole-fed mice. Angiosarcomas were observed laparotomy at 13 months and autopsy, respectively. Most of in mice that were fed 1'-hydroxysafrole and, at low incidence, the mice that did not develop tumors survived to 18 months in mice that were fed safrole in earlier experiments (1, 54). It is (note the number of mice that had no gross liver tumors when not evident why the incidence of angiosarcomas was relatively they were killed at 18 months (Table 3)). The tumors observed high for the mice which were fed 0.5% safrole in the first on laparotomy at 13 months rarely exceeded 1 cm in diameter experiment in Table 4, while none of these tumors were ob and were easily enumerated; however, occasional tumors on served in the similarly treated safrole-fed mice in Experiment the small lobes or on the underside of the lobes, which were 2. found at autopsy, were missed on examination by laparotomy. Histology of the Hepatic Tumors. With the exception of a By 18 months, the tumors were frequently 1 to 2 cm or more few tumors which were diagnosed as hepatic angiosarcomas in diameter and the accuracy of enumeration of the tumors was or hemangioendotheliosarcomas, all of the liver tumors induced lower, since large tumors may have been formed from coalition by injection of the allylbenzene derivatives prior to weaning or of 2 or more tumors. by administration of these compounds in the diets of adult mice Compounds Administered in the Diet of Female CD-1 Mice. were diagnosed as hepatomas types A or B or mixed types A As previously demonstrated for mice that were fed safrole or and B (7, 21 ). In the case of the mice that were treated prior to its 1'-hydroxy derivative (54), at least 50% of female CD-1 weaning, the tumors developed in livers that were otherwise mice which were fed 0.23 or 0.46% of estragÃ³le (15.5 or 31 histologically normal. On the other hand, in addition to hepa mmol/kg) or 0.25% of 1'-hydroxyestragole in the diet for 12 tomas, the mice which were fed estragÃ³le, safrole, or 1'-hy months developed hepatic tumors by 18 months (Table 4). droxyestragole for long periods had finely nodular livers and Under the same conditions, administration of 0.5% of eugenol occasional larger nodules that were not well demarcated from or 0.46% of anethole (31 mmol/kg of diet) did not result in the surrounding liver. Much of this nodularity gradually re hepatic tumors, whether or not the mice received 0.05% of gressed after administration of the alkenylbenzene derivatives phÃ©nobarbital in their drinking water throughout the entire 20- was terminated at 12 months. Histologically, these livers month experiment. None of the female mice which were fed showed various degrees of chronic inflammation, portal fibro- the control diet for 18 or 20 months developed an hepatic sis, bile duct proliferation, and various numbers of ceroid-laden tumor, and only 2 of 30 mice that were fed the control diet and histiocytes and focal areas of atypical cellular hyperplasia and also given phÃ©nobarbital developed hepatic tumors by 20 megalocytosis. months. In addition to the hepatic tumors, some of the mice which Induction of Tumors by the 2',3'-Oxides of Allylbenzene were fed the test compounds also developed angiosarcomas Derivatives in the s.c. tissue between the shoulder blades. The angiosar comas were observed as early as 9 months in mice that were Hepatic Tumors in Male CD-1 Mice Treated Prior To Wean fed 1'-hydroxyestragole. The relatively short latent period and ing. When administered to male CD-1 mice prior to weaning, the high incidence of angiosarcomas in this group resulted in 1'-hydroxysafrole 2',3'-oxide caused a significant increase in

1128 CANCER RESEARCH VOL. 43

Table 3 Development of hepatomas in male B6C3F, mice which received injections of alkenylbenzenes or their derivatives prior to weaning The test chemicals were dissolved in sterile trioctanoin and injected i.p. on Days 1,8, 15, and 22. The total doses are indicated in the table; the /Â«noiinjected/ mouse/dose were in the ratio 1:2:4:12 for Days 1,8, 15, and 22, respectively; the volumes of trioctanoin per mouse were 25, 50, 25, and 75 Â¡Â¡Â¡,respectively,for these injections. The mice in Groups 8 and 14 were an exception. Since over 50% of the mice in each of these groups died within 1 week after injection of the standard amount on Day 1, the doses were injected in the ratios of 0.6:2:4:12 on Days 1,8, 15, and 22, respectively, and the volumes of trioctanoin per mouse were 15, 50, 25, and 75 pi, respectively. The mice were weaned at 4 weeks of age. The mothers were fed the purified diet from the date on which they gave birth until the mice were weaned; the young were maintained on the purified diet from weaning until the termination of the experiment. The 13-month data were obtained by examination of the livers on laparotomy, except for those mice that were killed at this time with extensive hepatic tumors. The surviving mice were killed at 18 months. 13-18Data Data for mice dead or killed at mos.No. obtained at 13 ofmicekilled with ofTotal of No. of Hepatoma- Av. no. of No. no.ofhepatomas/mouse82.4b0.40.40.40.40.23.2"5.8"5.1"5.6"0.60.33.5"9.460.50.5No.out hepato ex-Group8mos.5C28"28"30'39g44"1f1'0oy36*42'3m0"3254Â°'TWOdose mice mice ex- bearing hepatomas/ mice mice(%)63"3431322320<98"100"98"411993"97"4128Av.mas at 1 amined1 Compound (/imol) weaned amined mice (%) mouse3

412 EstragÃ³le 4.75 47 23 56 1.2 443 Elemicin 4.75 46 19 21 0.3 454 Myristicin 4.75 45 20 10 0.2 475 Anethole 4.75 50 14 7 0.1 526 Dillapiol 4.75 55 18 6 0.1 557 Parsley apiol 4.75 55 21 00 588 Methyleugenol 4.75 59 23 70 1.5 469 1'-Hydroxyestragole 4.65 49 21 100 4.8 4010 1'-Hydroxyestragole 2.85 44 17 88 3.5 6011 1'-Hydroxyestragole 1.90 63 20 95 4.0 6312 1'-Hydroxyelemicin 4.75 65 20 5 0.1 5313 3'-Hydroxyanethole 4.75 54 23 13 0.3 4414 1'-Hydroxymethyleugenol 2.85 44 22 68 2.3 30dehydroestragole151'-Hydroxy-2',3'- 1.86 33 17 100 10

5816 (Trioctanoin only) 60 20 5 0.1 82a Uninjected 82 26 12 0.2 diameter.bNodules s2 mm in 5.cStatistically different (p < 0.001 ) from the value for Group 1 Onespleen.One mouse had multiple hemangioendotheliosarcomas in the liver and adenoma.8mouse had a hemangiosarcoma in the liver, and 2 mice each had a lung Two mice each had a lung adenoma.mos.Hepatoma-bearing liver.9 mice each had a papillary adenoma of the Harderian gland, and one had a hemangioendotheliosarcoma in the adenoma.hOne mouse had a lung liver.'One mouse had a lung adenoma, and one had an angiosarcoma in the lymphomas.;One mouse had a fibrosarcoma (s.c.), and one had Oneneurofibroma.One mouse had a s.c. fibroma.'mouse each had a lung adenoma, papillary adenoma of the Harderian gland, and dermal adenomas.mOne mouse had an angiosarcoma in the liver, and 2 had lung spleen."One mouse had a lung adenoma, and one had a hemangioendotheliosarcoma in the tumors.Â°One mouse had a lung adenoma; 13 mice were killed at 13 months because of extensive hepatic Two mice each had a lung adenoma, and 2 mice had angiosarcomas (one in the spleen and the other in the lip).

both the incidence of hepatic tumor-bearing mice and hepatic of 1.1, and safrole 2',3'-oxide, estragÃ³le 2',3'-oxide, and eu tumor multiplicity (1.0 tumor/mouse) as compared to those for genol 2',3'-oxide induced skin tumors in 33 to 40% of the mice mice treated only with the solvent (trioctanoin) or not treated with average multiplicities of 0.6 to 0.9. The incidence for the (Table 1, Experiment 2). 1'-Hydroxysafrole 2',3'-oxide was, control group treated only with croton oil was 7%. however, less active than safrole, estragÃ³le, or 1'-hydroxysaf- Induction of Lung Adenomas in A/J Mice. At high doses role in this assay. The incidences of tumor-bearing mice and (1 fimol/g body weight for 24 doses), 1'-hydroxysafrole 2',3'- the average number of tumors/liver for mice treated with oxide and 1'-hydroxyestragole 2',3'-oxide appeared to have safrole 2',3'-oxide, 1'-acetoxysafrole 2',3'-oxide, estragÃ³le significant, but very low, activity for the induction of lung 2',3'-oxide, or eugenol 2',3'-oxide were not significantly differ adenomas in A/J mice (Table 6). The average multiplicities of ent from those for the mice treated only with the solvent or not adenomas per mouse were 0.7 and 0.8 for mice treated with treated at all. these 2 compounds, as compared to 0.04 and 0.1 adenoma Induction of Benign Skin Tumors in Adult CD-1 Mice. As per mouse for the 2 negative control groups. Safrole, estragÃ³le, observed previously (54), CD-1 mice treated topically with 1'- anethole, 1'-hydroxysafrole, 1'-hydroxyestragole, 3'-hydroxy- hydroxysafrole 2',3'-oxide and subsequently treated repeti anethole, safrole 2',3'-oxide, and estragole-2',3'-oxide showed tively with croton oil developed significantly more skin tumors no activity in this assay. (papillomas and keratoacanthomas) at the treatment site than Tumor Induction by Repetitive s.c. Injection in Fischer did mice that received only the croton oil treatments. The Rats. Repetitive s.c. injections of large amounts (total dose, 2 higher dose used in this experiment (45 /Â¿mol/week for 6 mmol/rat) of the 2',3'-oxides of safrole, estragÃ³le, eugenol, 1'- weeks as compared to 15 jumol/week for 6 weeks in the earlier hydroxysafrole, and 1'-hydroxyestragole into adult Fischer rats experiment) resulted in a much higher incidence (82% at 40 was an inefficient procedure for the induction of tumors (Table weeks) and average multiplicity (2.7 tumors/mouse) (Table 5). 7). Four of the 20 rats treated with 1'-hydroxysafrole 2',3'- At the same high dose, 1'-hydroxyestragole 2',3'-oxide caused oxide developed sarcomas at the injection site; no more than 44% of the mice to develop tumors with an average multiplicity 2 of the rats treated with any other epoxide developed sarco-

MARCH 1983 1129

Table 4 Development of hepatomas in female CD-I mice fed safrole, estragÃ³le, eugenol, or anethole in the diet for 12 months Groups of female CD-1 mice, approximately 8 weeks old and with average initial weights of 24 and 21 g for Experiments 1 and 2, respectively, were fed the indicated compounds in a grain diet (38). (See the comment in "Materials and Methods" on the loss by volatilization of these compounds from the diet.) For Experiment 1, the dietary levels were 25 and 50% of the final levels for the first and second 10-day periods, respectively. In Experiment 2, the compounds were fed at IS, 30, and 67% of the final levels for the first 3 successive 10-day periods. After 12 months, the mice were fed the grain diet without any supplement. Where indicated, phÃ©nobarbital was administered as 0.05% of the drinking water from the beginning of the experiment until its termination. Experiments 1 and 2 were terminated at 20 and 18 months, respectively. at:10mice alive at:1 gain (g)/mouse miceNo. of of no. of time mice mice hepato-mas/mouse82.1C2.3C1.4C1.8C1.2C01.8Â°0000.0300.13Av.on experi withan-giosar-comas6716043000000000No.withother Pheno-Compound ment(mos.)19.118.819.819.413.618.018.017.5No.tu ofmice834 barbitalExperimentand % in diet mos.4749484943503029253029303020mos.2526353403928*29*24'26'26^30*29/Wtmo.1.20.92.20.91.53.00.55.16.0-4.3-3.64.95.54mos.3.02.52.42.41.94.83.78.67.9-0.8-1.19.98.58mos.4.13.43.83.33.38.13.811100.30.21311Hepatoma-bearing mors5d1e7'891*7'1*4'02m1"1Â°2Â° 10.25% (72)c39 safrole0.50% (80)c27 safrole0.23% (56)c35(71)c24 estragÃ³le0.46% estragÃ³le0.25% 1'-hydroxy (56)Â°021 estragole(None)Experiment

20.50% (70)c0001 safrole0.50% eugenol0.50% +0.46% eugenol anethole0.46% +(None)(None)anethole (3)02

+No.Start50505050505030303030303030of (7)Av. 3 Numbers in parentheses, percentage. Percentage of hepatoma incidence and average multiplicity are based on the 10-month survival. 6 Located s.c. in the interscapular region, except that one mouse, fed 0.46% estragÃ³le and one fed 0.5% safrole had angiosarcomas s.c. on the hind leg and in the abdominal cavity, respectively. c Statistically different (p < 0.001) from the value for the untreated control group in the same experiment. " One osteosarcoma (s.c.), 2 lymphomas, and 2 lung adenomas. * One papillary adenoma of the Harderian gland. ' Six lymphomas and one lung adenoma. 9 Two lung adenomas, 5 lymphomas, and one sarcoma (abdominal cavity). " One lung adenoma. All of the mice in this group developed opacity of the lens within 11 months. ' Four lymphomas, 2 pulmonary adenomas, and one carcinoma of the Harderian gland. ' Number of mice alive at 18 months. * One pulmonary adenoma. ' One pulmonary adenoma, 2 thymic lymphomas. and one adenoacanthoma (mammary gland). m Two lymphomas. 17One lymphoma. Â°One pulmonary adenoma. " One pulmonary adenoma and one hemangioendotheliosarcoma (liver).

Table 5 mas. Injection of 1'-acetoxysafrole, which caused the devel Formation of benign skin tumors in female CD-1 mice treated topically with opment of sarcomas in 11 of 36 Charles River CD random-bred alkenylbenzene epoxides and then treated repetitively with croton oil rats in earlier experiments (1 ), gave rise to injection site sar Groups of 40 female CD-1 mice, about 8 weeks old and averaging 30 g, were shaved and treated topically 4 days/week for 6 weeks; each application con comas in only 4 of 20 Fischer rats, even though the dose (0.6 sisted of 0.15 ml of redistilled acetone which contained 11.2 umol of compound. mmol/rat) was approximately twice that used earlier. Three Starting 1 week after the last dose of epoxide, all of the mice were treated sarcomas occurred at the injection site of 1'-hydroxyestragole. topically twice weekly with 0.15 ml of 0.6% of croton oil in redistilled acetone. The mice were reshaved as necessary. Each group contained 38 to 40 mice at Hepatic carcinomas developed in 11 of 20 rats given s.c. the termination of the experiment at 40 weeks. The tumors were diagnosed as injections of 1'-hydroxysafrole with a total dose of 2 mmol/rat. epidermal papillomas and keratoacanthomas. The mice were fed Wayne Breeder Neither 1'-hydroxyestragole (which was tolerated at only one- Blox pellets. half of the dose used for 1'-hydroxysafrole) nor any of the wkCompoundSafrole 30 wk% epoxides tested showed significant carcinogenicity in the liver. no. of with no. of These latter 3 assays, which utilized topical applications to skin tu tumors/ skin tu tumors/ mors21 mouse0.4 mors36a mouse0.7a mouse skin, induction of lung adenomas in A/J mice, and 2',3'-oxide repetitive s.c. injections into adult rats, were used as ap 33a" 0.6a EstragÃ³le 2',3'-oxide 10 0.2 proaches for the detection of the carcinogenicity of electro- 40a82a44a- 0.9a Eugenol 2',3'-oxide 2553253Av.0.4 philic derivatives which, possibly because they are readily 1'-Hydroxysafrole 1.30.4 2.7a1.1a 2'.3'-oxide destroyed by reaction with nucleophilic constituents in vivo, 1'-Hydroxyestragole c7Av. may not be efficient for hepatic tumor induction. 2',3'-oxide (Acetone only)%with 0.0340 0.1 8 Statistically different (p < 0.005) from the value for the acetone-treated DISCUSSION controls. b In addition, one mouse had a fibromyosarcoma of the skin in the treatment The data presented here have further explored the structural area. c One of these mice also had an epidermal carcinoma in the treatment area. specificity of the hepatocarcinogenic activities of both naturally

1130 CANCER RESEARCH VOL. 43

Table 6 gÃ³le (1, 2, 7). 1'-Hydroxysafrole is a substrate for hepatic Development of lung adenomas in female A/J mice treated with safrole, sulfotransferase(s) in rat and mouse liver (53), and preliminary estragÃ³le, anethole, or certain of their side-chain hydroxy and epoxy derivatives data strongly suggest that the hepatocarcinogenicity of 1'- Starting at 8 weeks of age, groups of 25 female A/J mice were given injections of the indicated compounds twice weekly for 12 weeks; doses of 0.5 or 1 /imol/ hydroxysafrole in the mouse is dependent on the formation of g of body weight were administered i.p. in 5 fil of sterile trioctanoin per g of body its sulfuric acid ester.6 Thus, the abilities of the carcinogenic weight. The mice were killed 8 months after the first injection for enumeration of and noncarcinogenic 1'-hydroxy derivatives to serve as sub lung adenomas. The mice were fed Wayne Breeder Blox pellets. strates for hepatic sulfotransferase(s) may differ. In addition, mos.CompoundSafroleEstragÃ³leAnethole1 Mice examined at 8 the rates of ring oxidation and/or metabolism of the methoxy with no. of or methylenedioxy substituents of the various compounds with (/jmol/g of lung ade adenomas/ the formation of phenols, which may be readily conjugated and bodywt)1 nomas56181023610628452647100134Av.mouse0.050.060.240.100.230.060.110.060.330.65b0.320.79b7.2d0.130.04 excreted, also could be important factors. Similar considera X241 X241 tions may be important in understanding the very high activity X240.5 of 1'-hydroxy-2',3'-dehydroestragole as a hepatocarcinogen '-Hydroxysafrole1 X24a0.5 in the mouse. From data on the 1'-acetoxy derivatives, the '-Hydroxyestragole3'-HydroxyanetholeSafroleX24a1 X241 higher carcinogenicity of this acetylenic derivative, as com 2',3'-oxideEstragÃ³le x241 pared to that of 1'-hydroxyestragole, does not appear to be 2',3'-oxide1 x240.5 '-Hydroxysafrole2',3'-oxide1 explicable on the basis of a higher electrophilic reactivity of its X241 esters.7 Metabolic analyses of these various factors are in '-Hydroxysafrole2',3'-oxide1 X240.5 progress. '-Hydroxyestragole2'.3'-oxide1 X241 In addition to being assayed by administration to preweanling mice, eugenol and anethole were also assayed for their carci '-Hydroxyestragole2',3'-oxideEthyl X245.6 nogenic activity by administration for 12 months in the diet of carbamate(Trioctanoin X 1No.19181721221619171820191910e2425% adult female mice. Neither compound showed any activity in only)UninjectedDose either assay at levels higher than those required for the hepa tocarcinogenicity of safrole and estragÃ³le. These negative re A single i.p. dose of 1 fimol/g of body weight of 1'-hydroxyestragole caused the death of all of the mice within 1 week; 1'-hydroxysafrole at this dose caused sults for eugenol are consistent with the essentially negative about a 25% mortality. Accordingly, one-half of this dose was used. findings reported for eugenol in the studies conducted by the Statistically different (p < 0.01 ) from the value for the mice treated only with National Toxicology Program (32), in which eugenol was fed at trioctanoin. 0 Only 10 mice received injections of ethyl carbamate. similar doses but for longer times to both mice and rats. The a Statistically different (p < 0.001) from the value for the mice treated only report on these tests indicated that only "equivocal" evidence with trioctanoin. was obtained for an increased liver tumor incidence on feeding 0.3% or 0.6% of eugenol for up to 2 years to B6C3F, mice. occurring and synthetic alkenylbenzene derivatives. Methyleu- Anethole is a side chain isomer of the carcinogen estragÃ³le genol appears to be as active a carcinogen in the mouse liver (Chart 2). No other data on the carcinogenicity of anethole as safrole and estragÃ³le. In addition to its natural occurrence appear to have been reported; significantly higher levels of in many essential oils (22, 24), methyleugenol is one of several administration in the diet to mice are probably not feasible, alkenylbenzenes (eugenol, cis- and frans-methylisoeugenol, since in our experiment, administration of anethole as 0.46% elemicin, and irans-isoelemicin) that were found at levels of 4 of the diet adversely affected weight gain but not the survival to 40 ppb in the juice of oranges from trees sprayed with of the mice. The data on eugenol and anethole in Tables 1, 3, abscission agents before the mechanical harvesting of the fruit and 4 are of importance in view of the use of these compounds (30). These alkenylbenzenes were present in the essential oil as major spice-flavor food additives (31, 33, 34). Although the 2',3'-oxides of safrole, estragÃ³le, and their 1'- in the juice and were not detected in untreated oranges. In addition, methyleugenol has been used as an effective lure in hydroxy derivatives are moderately active mutagens for S. the agricultural control of the male oriental fruit fly (Dacus typhimurium TA1535 and TA100 (36, 49, 54), their weak dorsalis Hendel) (41, 46, 47) and as a surgical anesthetic in activities as initiators of papillomas on the skin of mice and rodents (5). their weak to negligible activities in the induction of tumors on The inactivities of dill and parsley apiols, myristicin, elemicin, administration to preweanling mice, on administration to A/J and eugenol as compared to the hepatocarcinogenic activities mice, and on s.c. injection into Fischer rats provide little evi of safrole, estragÃ³le, and methyleugenol for male mice treated dence for their importance as carcinogenic metabolites in vivo. prior to weaning show that the number and positions of meth- These results are consistent with the observations that the major DNA adducts in the livers of mice treated with either 1'- oxy and methylenedioxy substituents on the benzene ring have hydroxyestragole or 1'-hydroxysafrole appear to be derived a major effect in determining the carcinogenic activities of allylbenzene derivatives. The apiols, myristicin, and elemicin from esters of these proximate carcinogens (36, 37). In high- are each substituted in the 3, 4, and 5 positions by methoxy performance liquid chromatography systems, these adducts and/or methylenedioxy groups (Chart 2). The stereochemical cochromatograph with and thus appear to be identical to ad hindrance produced by such substitutions may alter the metab ducts formed in vitro from reactions of the synthetic electro philic acetic acid esters of these 1'-hydroxy metabolites with olism of these alkenylbenzenes. As is evident from the inactivity of 1'-hydroxyelemicin, the lack of activity of elemicin, at least, deoxyguanosine and deoxyadenosine. The results are also is apparently not due to an inability to be hydroxylated at the consistent with preliminary data which implicate sulfotransfer- 1' position in vivo', 1'-hydroxylation appears to be of major 6 E. W. Boberg, J. A. Miller, and E. C. Miller, unpublished results. importance in the carcinogenic activities of safrole and estra ' D. H. Phillips, J. A. Miller, and E. C. Miller, unpublished results.

MARCH 1983 1131

Table 7 Tumors in male Fischer rats given repeated s.c. injections of various derivatives of a/kenylbenzenes Groups of 20 male Fischer rats were given s.c. injections twice weekly for a total of 20 injections of the indicated compounds. All injections were in the right hind leg at approximately the same site; the solutions were dissolved in sterile trioctanoin (0.2 ml/injection). The surviving rats were killed at 24 months: 16 to 19 rats in each group survived for at least 20 months. The rats were fed Wayne Breeder Blox pellets. of rats withTumors

dose (injection car sites81 at other Compound1 (mmol/rat)0.6"Sarcomassite)4CHepaticcinomas1No. '-AcetoxysafroleTotal s.c. sarcoma 1 renal cell carcinoma 1 fibroadenoma (mammary gland)

1'-Hydroxysafrole 2 s.c. fibromas 1 hemangioendotheliosarcoma (s.c.)

1'-Hydroxyestragole 1 s.c. fibroliposarcoma 1 hemangioendotheliosarcoma (spleen) 1 lymphoma (spleen, liver, lymph nodes) 1 papillary mesothelioma (mesentery)

Safrole2EstragÃ³le 2'.3'-oxide 01

2Eugenol 2'.3'-oxide 0 1 basal cell carcinoma (lip) (duodenum)2 1 adenocarcinoma

21 2',3'-oxide 01 keratoacanthoma (skin) 1 sebaceous gland carcinoma (skin; near injection site) fibromyosarcoma4C 1

'-Hydroxysafrole 2 02 sarcomas (s.c.) 2',3'-oxide1 (s.c.)o' 1 fibroma

'-Hydroxyestragole 2 1 2 fibroma (s.c.) 2',3'-oxide(Trioctanoin 1 epidermal carcinoma 1 gastric adenocarcinoma 1 sarcoma (s.c.) (pancreas)0 1 islet cell adenoma

only)1 01 leiomyosarcoma (abdominal cavity) 1 fibrosarcoma (s.c.) 1 pulmonary adenoma In addition to the tumors listed, the majority of the rats that survived for at least 18 months also had unilateral or bilaterial interstitial cell tumors of the testes. 6 Only 0.03 mmol was given in each injection, since larger amounts caused severe ulcÃ©rationat the injection site. c Statistically different (p < 0.05) from the value for the rats treated only with trioctanoin. d Of the 11 rats with hepatic carcinomas, 7 had hepatocellular carcinomas, one had a cholangiocellular carcinoma, and 3 had mucinous adenocarcinomas of biliary origin. In addition, one rat had a cholangioma. Statistically different (p < 0.001) from the value for the rats treated only with trioctanoin. 8 Only 0.05 mmol was given in each injection. Doses of 0.1 mmol at twice weekly intervals caused weight loss and some deaths, as well as some ulcÃ©rationat the injection site. ' One rat developed an angiosarcoma at the injection site.

ase activity for 1'-hydroxysafrole as an important determinant induce hepatic tumors in mice are also carcinogenic for the in its hepatocarcinogenicity in mice.6 Direct tests of the sulfuric liver or other tissues of rats or mice with other protocols in acid esters of 1'-hydroxysafrole and 1'-hydroxyestragole are which few spontaneous tumors occur. It seems likely that male not possible at present, because the syntheses of these puta mice of the susceptible strains have a more favorable physio tive ultimate carcinogens have not been achieved. 1'-Acetox logical mechanism (hormonal?) for the promotion of "initiated" ysafrole, however, showed much more activity as a hepatocar- hepatic cells (whether spontaneous or due to an administered cinogen when administered to preweanling mice (1) than did chemical) than do male mice of other strains. 1'-hydroxysafrole 2',3'-oxide. About 30 alkenylbenzenes and a number of closely related Although administration of a variety of chemicals during the compounds have been found in the essential oils of various preweaning period to male mice derived from certain stocks or plants (4,9,11,12,19, 23, 45). For example, 13 monomeric strains of mice (e.g., C3H, CBA, and CD-1) has proved to be a and dimeric "phenylpropanoids" (e.g., myristicin and dehydro- sensitive assay for the induction of hepatic tumors (1, 8, 27, diisoeugenol) have been found in nutmeg (19). Of the 11 39), this assay has been criticized on the basis that male mice naturally occurring alkenylbenzenes that have been tested for of these stocks develop hepatic tumors spontaneously (i.e., carcinogenicity, 4 (safrole, estragÃ³le, methyleugenol, and iso- from unknown causes, endogenous or exogenous or both), safrole) are hepatocarcinogenic, and ÃŸ-asarone induces leio- especially after 1 year of age, and the incidences of these myosarcomas of the small intestine in male rats (10, 20). spontaneous tumors increase with the age of the mice (39). Isosafrole and /S-asarone appear to occur only rarely in essen However, as noted by Tomatis et al. (51 ), chemicals found to tial oils. Safrole, estragÃ³le, and methyleugenol occur more

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CARCINOGENIC IN ONE OR MORE TESTS of selected food additives by parenteral administration to infant Swiss mice. Toxicol. Appi. Pharmacol., Ã•6:321-334, 1970. OCHj 9. Furia, T. E., and Bellanca, N. (Eds.). Fenaroli's Handbook of Flavor Ingre dients, Ed. 2, Vol. 2, 928 pp. Cleveland: CRC Press. Inc.. 1975. 10. Gross. M. A., Jones, W. I., Cook, E. L., and Boone. C. C. Carcinogenicity of H2C-CH=CH2 oil of calamus. Proc. Am. Assoc. Cancer Res., 8. 24, 1967. SAFROLE METHYLEUGENOL 11. Guenther, E. The Essential Oils. Vol. 1, 427 pp., 1948; Vol. 3, 777 pp., (sassafras* (tarragon* (sweet bay, cloves, 1949; Vol. 4. 752 pp., 1950; Vol. 5. 507 pp., 1952; Vol. 6. 481 pp. New sweet basil, sweet basil* lemon grass) York: D. Van Nostrand Co.. 1948-1952. cinnamon)INACTIVEanise)PRESENT 12. Guenther, E., and Althausen, D. The Essential Oils, Vol. 2, 852 pp. New York: D. Van Nostrand Co., 1949. INOCHjH2C-CH=CH2ESTRAGÃ“LESTUDYOH 13. Harris, P. N., Gibson, W. R.. and Dillard, R. D. The oncogenicity of two 1,1- diaryl-2-propynyl N-cycloalkylcarbamates. Cancer Res., 30: 2952-2954, OCH, 1970. CN3Â°ÃŒÃ“TÂ°CH3 14. Harris. P. N., Gibson. W. R., and Dillard, R. D. The oncogenicity of six analogs of 1,1-diphenyl-2-propynyl N-cyclohexylcarbamate (1). Proc. Am. H2C-CH=CHj H2C-CH=CH2 Assoc. Cancer Res., 12: 26, 1971. H CHj 15. Hollander. M., and Wolfe. D. A. Nonparametric Statistical Methods, 503 pp. ANETHOLE EUGENOL ELEMICIN New York: John Wiley and Sons. Inc., 1973. (anise* fennel, (cloves* allspice, (nutmeg, elemi gum, 16. IARC Monographs on the Evaluation of Carcinogenic Risk of Chemicals to coriander) artichoke) sassafras) Man. Safrole, isosafrole and dihydrosafrole. Vol. 10, pp. 231-244. Lyon, France: International Agency for Research on Cancer. 1976. CHjO 17. InnÃ©s,J. R. M., Ulland, B. M.. Valerio, M. G., Petrucelli, L., Fishbein. L., Hart, E. R., Paliota. A. J., Bates, R. R., Falk, H. L., Gart. L. L.. Klein. M.. H2C-CHICH2 Mitchell, l., and Peter, J. Bioassay of pesticides and industrial chemicals for H2C-CH:CH2 tumorigenicity in mice: a preliminary note. J. Nati. Cancer Inst., 42: 1101- 1114, 1969. MYRISTICIN DILL APIOL PARSLEY APIOL (nutmeg*, mace* 18. loannides, C., Delaforge, M., and Parke. D. V. Safrole: its metabolism, (dill, indian dill) (parsley, fennel, Carcinogenicity and interactions with cytochrome P-450. Food Cosmet. black pepper, sassafras) Toxicol., 79. 657-666, 1981. carrots, parsnips) 19. Isogai, A., Murakoshi, S.. Suzuki, A., and Tamura. S. Structures of new Chart 2. Structures, occurrence in some representative spices and foods (4, dimeric phenylpropanoids from Myristica fragrans Houtt. Agrie. Biol. Chem., 37. 1479-1487, 1973. 9, 11, 12, 22, 23. 30, 45), and hepatocarcinogenic activity in the mouse of the naturally occurring alkenylbenzenes tested in the present study. *, oils with 20. Joint FAO/WHO Expert Committee of Food Additives. Toxicological Evalu ation of Certain Food Additives. WHO Tech. Rep. Ser. 669. pp. 33-44, relatively high concentrations. 1981. 21. Jones, G., and Butler, W. H. Morphology of spontaneous and induced widely as plant constituents. Since these chemicals occur neoplasia. In: W. H. Butler and P. M. Newberne (eds.), Mouse Hepatic Neoplasia, pp. 21-57. New York: Elsevier Scientific Publishing Co., 1975. naturally and as food additives at no more than low ppm levels 22. Leung, A. Y. Encyclopedia of Common Natural Ingredients Used in Food, in the total food intake (31 ), they appear to make, at most, only Drugs, and Cosmetics, 409 pp. New York: John Wiley & Sons, Inc.. 1980. a very minor contribution to the burden of exogenous carci 23. Lichtenstein. E. P., and Casida, J. E. Myristicin, an insecticide and Synergist occurring naturally in the edible part of parsnips. J. Agrie. Food Chem.. ) 1: nogenic agents to which humans are exposed. Further meta 410-415, 1963. bolic and related studies are needed to determine whether or 24. Luff, B. D. W.,Perkin. W. H., Jr., and Robinson. R. XCV. m-Hemipinic and asaronic acids. J. Chem. Soc. (Lond.). 97: 1131-1140, 1910. not this view is correct. 25. Mauthner, F. Die Synthese des Elemicins und Isoelemicins. Ann., 414: 250- 255, 1917. 26. McCann, J., Choi, E., Yamasaki, E., and Ames. B. N. The detection of ACKNOWLEDGMENTS carcinogens as mutagens in the Salmonella microsome test: assay of 300 chemicals. Proc. Nati. Acad. Sei. U. S. A.. 72: 5135-5139, 1975. The authors appreciate the assistance of Randy Knibbs in the animal tests. 27. Miller, E. C.. Kadlubar. F. F., Miller. J. A., Pilot, H. C., and Drinkwater, N. R. The W-hydroxy metabolites of (V-methyl-4-aminoazobenzene and related Note Added in Proof dyes as proximate carcinogens in the rat and mouse. Cancer Res., 39: An experiment still in progress by R. W. Wiseman indicates that 1'-hydroxy- 3411-3418, 1979. elemicin at a high total dose (9.5 /imol) has about 5% of the hepatocarcinogenic 28. Miller. J. A.. Miller. E. C., and Phillips. D. H. The metabolic activation and activity of 1'-hydroxyestragole when administered prior to weaning to male Carcinogenicity of alkenylbenzenes that occur naturally in many spices. In: B6C3F, mice (see the protocol for Table 3). H. F. Stich (ed.). Carcinogens and Mutagens in the Environment. Vol. 1, pp. 83-96. Boca Raton, Fla.: CRC Press, Inc.. 1982. 29 Miller. J. A.. Swanson, A. B.. and Miller, E. C. The metabolic activation of REFERENCES safrole and related naturally occurring alkenylbenzenes in relation to carcinogenesis by these agents. In: E. C. Miller, J. A. Miller. I. Hirono. T. 1. Borchert, P.. Miller, J. A., Miller, E. C., and Shires, T. K. 1'-Hydroxysafrole, Sugimura, and S. Takayama, (eds.). Naturally Occurring Carcinogens-Mu- a proximate carcinogenic metabolite of safrole in the rat and mouse. Cancer tagens and Modulators of Carcinogenesis, pp. 111-123. Tokyo: Japan Res., 33. 590-600, 1973. Scientific Societies Press, 1979. 2. Borchert, P., Wislocki, P. G., Miller, J. 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36. Phillips, D. H . Miller. J. A.. Miller, E. C., and Adams, B. Structures of the 46. Steiner, L. F. Methyl eugenol as an artractant for oriental fruit fly. J. Econ. ONA adducts formed in mouse liver after administration of the proximate Entomol.. 45: 241-248, 1952. hepatocarcinogen 1'-hydroxyestragole. Cancer Res., 41: 176-186, 1981. 47. Steiner, L. F., Mitchell, W. C., Harris, E. J., Kozuma, T. T., and Fujimoto, M. 37. Phillips. D. H Miller, J. A., Miller, E. C . and Adams. B. The N -atom of S. Orientai fruit fly eradication by male annihilation. J. Econ. Entomol., 58: guanine and the W -Â¡ttumof adenine residues as sites for covalent binding 961-964, 1965. of metabolically activated 1'-hydroxysafrole to mouse-liver DNA in vivo. 48. Stillwell, W. G., Carman, M. J., Bell. L., and Horning, M. G. The metabolism Cancer Res., 41: 2664-2671, 1981. of safrole and 2',3'-epoxysafrole in the rat and guinea pig. Drug Metab. 38. Poirier, M. M., Miller, J. A., and Miller, E. C. The carcinogenic activities of Dispos., 2: 489-498. 1974. N-hydroxy-2-acetylaminofluorene and its metal chelates as a function of 49. Swanson, A. B.. Chambliss, D. D., Blomquist, J. C., Miller. E. C.. and Miller, retention at the injection site. Cancer Res., 25. 527-533, 1965. J. A. The mutagenicities of safrole, estragÃ³le, eugenol, frans-anethole, and 39. Roe, F. J. C. Neonatal induction of hepatic and other tumors. In: W. H. Butler some of their known or possible metabolites for Salmonella typhimurium and P. M. Newberne (eds.), Mouse Hepatic Neoplasia, pp. 133-141. New mutants. MutÃ¢t.Res., 60. 143-153, 1979. York: Elsevier Scientific Publishing Co., 1975. 50. Swanson, A. B.. Miller. E. C., and Miller, J. A. The side-chain epoxidation 40. Sharpe, I. 0., Miller. J. A., and Miller. E. C. Electrophilic activity of carcino and hydroxylation of the hepatocarcinogens safrole and estragÃ³le and some genic diaryl acetylenic carbamates. Proc. Am. Assoc. Cancer Res., 14: 19, related compounds by rat and mouse liver microsomes. Biochim. Biophys. 1973. Acta, 673. 505-516, 1981. 41. Shaver, T. N., and Bull, D. L. Environmental fate of methyl eugenol. Bull. 51. Tomatis, L., Partensky, C., and Montesano, R. The predictive value of mouse Environ. Contam. Toxicol., 24: 619-626, 1980. liver tumour induction in carcinogenicity testingâ€”a literature survey. Int. J. 42. Solheim, E.. and Scheline, R. R. Metabolism of alkenebenzene derivatives Cancer, 12: 1-20, 1973. in the rat. I. p-Methoxyallylbenzene (estragÃ³le) and p-methoxypropenylben- 52. Vesselinovitch, S. D., Rao. K. V. N., and Mihailovich. N. Transplacental and zene (anethole). Xenobiotica, 3: 493-510, 1973. lactational carcinogenesis by safrole. Cancer Res., 39: 4378-4380, 1979. 43. Solheim, E., and Scheline, R. R. Metabolism of alkenebenzene derivatives 53. Wislocki, P. G., Borchert, P., Miller. J. A., and Miler, E. C. The metabolic in the rat. II. Eugenol and isoeugenol methyl ethers. Xenobiotica, 6: 137- activation of the carcinogen 1'-hydroxysafrole in vivo and in vitro and the 150. 1976. electrophilic reactivities of possible ultimate carcinogens. Cancer Res., 36: 44. Solheim, E., and Scheline. R. R. Metabolism of alkenebenzene derivatives 1686-1695, 1976. in the rat. III. Elemicin and isoelemicin. Xenobiotica. 70. 371-380, 1980. 54. Wislocki, P. G., Miller. E. C., Miller, J. A.. McCoy, E. C.. and Rosenkranz. H. 45. Stahl, E. Variation in myristicin content of cultivated parsnip roots (Pastinaca S. Carcinogenic and mutagenic activities of safrole. 1'-hydroxysafrole, and sativa ssp. sativa var. hortensis). J. Agrie. Food Chem., 29:890-892.1981. some known or possible metabolites. Cancer Res., 37: 1883-1891, 1977.

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Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 1983 American Association for Cancer Research. Structure-Activity Studies of the Carcinogenicities in the Mouse and Rat of Some Naturally Occurring and Synthetic Alkenylbenzene Derivatives Related to Safrole and Estragole

Elizabeth C. Miller, Anne B. Swanson, David H. Phillips, et al.

Cancer Res 1983;43:1124-1134.

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