Methyleugenol is used as a flavoring agent in jellies, baked goods, nonalcoholic beverages, chewing gum, candy, pudding, relish, and ice cream. It is also used as a fragrance in perfumes, creams, lotions, detergents, and soaps. Methyleugenol has also been used as an insect attractant in eradication programs and as an anesthetic in rodents. Methyleugenol was nominated for testing because of its widespread use and because of its structural resemblance to safrole, a known carcinogen, and isosafrole and estragole. Male and female F344/N rats and B6C3F1 mice received methyleugenol (approximately 99% pure) in 0.5% methylcellulose by gavage for 14 weeks or 2 years. Genetic toxicology studies were conducted in Salmonella typhimurium, cultured Chinese hamster ovary cells, and mouse peripheral blood erythrocytes.
Fourteen-week study in rats
Groups of 9 or 10 male and 10 female F344/N rats were administered 0, 10, 30, 100, 300, or 1,000 mg methyleugenol/kg body weight in 0.5% methylcellulose by gavage 5 days per week for 14 weeks. A water control group of 10 male and 10 female rats received deionized water by gavage. All rats survived until the end of the study. The final mean body weights of 300 and 1,000 mg/kg males and of all dosed groups of females were significantly less than those of the vehicle controls. Erythrocyte microcytosis was demonstrated by decreased mean cell volumes in 300 mg/kg males and 1,000 mg/kg males and females. There was evidence of a thrombocytosis at all time points, demonstrated by increased platelet counts in the 100 mg/kg or greater groups. The serum activities of alanine aminotransferase and sorbitol dehydrogenase were increased in the 100 mg/kg or greater rats at various time points, suggesting hepatocellular injury. Additionally, bile acid concentrations were generally increased in the 300 and 1,000 mg/kg groups at all time points, consistent with cholestasis or altered hepatic function. A hypoproteinemia and hypoalbuminemia, evidenced by decreased total protein and albumin concentrations, occurred in rats in the 300 and 1,000 mg/kg groups at all time points. Liver weights of 100, 300, and 1,000 mg/kg males and 300 and 1,000 mg/kg females and testis weights of 1,000 mg/kg males were significantly increased. Increased incidences of liver lesions occurred in 300 and 1,000 mg/kg males and females and hepatocellular adenoma occurred in one 1,000 mg/kg male. The incidences of atrophy and chronic inflammation of the mucosa of the glandular stomach were significantly increased in rats administered 300 or 1,000 mg/kg. Increased incidences of adrenal gland cortical hypertrophy and/or cytoplasmic alteration in the submandibular gland occurred in the 100 mg/kg or greater groups.
Fourteen-week study in mice
Groups of 10 male and 10 female B6C3F1 mice received methyleugenol in 0.5% methylcellulose by gavage at doses of 0, 10, 30, 100, 300, or 1,000 mg/kg, 5 days per week for 14 weeks. A water control group of 10 male and 10 female mice received deionized water by gavage. All but one male and all females receiving 1,000 mg/kg died before the end of the study. The mean body weight gains of mice in the 300 mg/kg groups were significantly less than those of the vehicle controls. The only clinical finding was toxicity manifested as generalized morbidity in mice administered 1,000 mg/kg. Liver weights of 30, 100, and 300 mg/kg males and of 300 mg/kg females were significantly increased. Male mice administered 10 or 30 mg/kg had significantly lower cauda epididymis, epididymis, and testis weights; males receiving 100 mg/kg had significantly lower spermatozoal concentrations. Increased incidences of liver lesions occurred in 1,000 mg/kg males and 300 and 1,000 mg/kg females. The incidences of lesions of the glandular stomach were increased in one or more groups administered 30 mg/kg or greater.
Two-year study in rats
Groups of 50 male and 50 female rats received methyleugenol in 0.5% methylcellulose by gavage at doses of 37, 75, or 150 mg/kg, 5 days per week for 105 weeks; groups of 60 male and 60 female rats received the 0.5% methylcellulose vehicle only. Stop-exposure groups of 60 male and 60 female rats received 300 mg/kg in 0.5% methylcellulose by gavage for 52 weeks followed by just the 0.5% methylcellulose vehicle for the remaining 53 weeks of the study. Special study groups of 10 male and 10 female rats administered 36, 75, 150, or 300 mg/kg were designated for toxicokinetic studies.
Survival and body weights
All 150 and 300 mg/kg males died before the end of the study, and survival of 150 mg/kg females was slightly less than that of the vehicle controls. Mean body weights of all dosed groups of rats were less than those of the vehicle controls throughout most of the 2-year study.
Pathology findings
Chemical-related liver neoplasms occurred in all dosed groups of rats and included hepatocellular adenoma, hepatocellular carcinoma, hepatocholangioma, and hepatocholangiocarcinoma; at 2 years, there were positive trends in the incidences of hepatocellular adenoma, carcinoma, and adenoma or carcinoma (combined) in core study rats and in the numbers of rats with multiple liver neoplasms. Nonneoplastic lesions included eosinophilic and mixed cell foci, hepatocellular hypertrophy, oval cell hyperplasia, cystic degeneration, and bile duct hyperplasia (females); the incidences of these lesions in dosed groups of male and female rats were increased at 6 months, 12 months, and/or 2 years.
Chemical-related neoplasms and nonneoplastic lesions of the glandular stomach included benign and malignant neuroendocrine tumors in the 150 and 300 mg/kg groups and females in the 75 mg/kg group. In all dosed groups of rats at all time points, the incidences of mucosal atrophy were significantly greater than in the vehicle controls. Neuroendocrine cell hyperplasia was observed in females at 6 months and males and females at 12 months and at 2 years. In core study female rats, there was a positive trend in the incidences of squamous cell papilloma or carcinoma (combined) of the forestomach, and the incidence in the 150 mg/kg group exceeded the historical control range.
The incidences of renal tubule proliferative lesions in male rats were suggestive of a neoplastic effect in the kidney. Therefore, additional step sections of the kidneys of male rats were prepared. The incidences of renal tubule hyperplasia and adenoma in the extended evaluation and the combined incidences of standard and step sections in the 75, 150, and 300 mg/kg groups were greater than those in the vehicle controls. The incidences of nephropathy were increased in all dosed groups of females, and the increase was significant in the 300 mg/kg group.
In dosed groups of male rats, there was a positive trend in the incidences of malignant mesothelioma, and the incidences were significantly greater in 150 and 300 mg/kg males than in the vehicle controls. The incidences of mammary gland fibroadenoma in 75 and 150 mg/kg males were significantly increased. The incidences of fibroma of the subcutaneous tissue in 37 and 75 mg/kg males and the combined incidences of fibroma or fibrosarcoma in 37, 75, and 150 mg/kg males were significantly increased.
Two-year study in mice
Groups of 50 male and 50 female mice received methyleugenol in 0.5% methylcellulose by gavage at doses of 0, 37, 75, or 150 mg/kg for 105 weeks. Special study groups of 10 male and 10 female mice administered 37, 75, or 150 mg/kg were designated for toxicokinetic studies.
Survival and body weights
Survival of all dosed groups of male mice was similar to that of the vehicle controls. Survival of dosed groups of females was significantly less. Mean body weights of dosed mice were generally less than those of the vehicle controls throughout the studies.
Pathology findings
Chemical-related increases in the incidences of liver neoplasms and nonneoplastic lesions in mice included hepatocellular adenoma and carcinoma, hepatoblastoma, hepatocholangiocarcinoma, eosinophilic foci, oval cell hyperplasia, bile duct hyperplasia, hemosiderin pigmentation, chronic active inflammation, and hematopoietic cell proliferation. In all dosed groups ofmales and females, the incidences of hepatocellular neoplasms and the multiplicity of neoplasms were generally greater than in the vehicle controls. The incidences of hepatoblastoma were significantly increased in all dosed groups of females and slightly increased in 150 mg/kg males. Hepatocholangiocarcinoma was observed in 150 mg/kg females. The incidences of eosinophilic foci, oval cell hyperplasia, portal hypertrophy, hepatocyte necrosis, hematopoietic cell proliferation, bile duct hyperplasia, and hemosiderin pigmentation were significantly increased in two or more dosed groups of male and/or female mice.
The incidences of glandular ectasia, mucosal atrophy, chronic active inflammation, epithelial hyperplasia, and neuroendocrine cell hyperplasia of the glandular stomach were increased in one or more dosed groups of male and female mice. In addition, malignant neuroendocrine tumors were observed in the glandular stomach of two 150 mg/kg male mice; one male in this group had a carcinoma.
Toxicokinetic studies
Methyleugenol is rapidly absorbed following oral administration to rats and mice. The kinetic data are consistent with rapid clearance from the blood, metabolism in the liver, and excretion of the parent and various metabolites in the urine.
Genetic toxicology
Methyleugenol was not mutagenic in S. typhimurium strain TA98, TA100, TA1535, or TA1537, with or without exogenous metabolic activation (S9). In cytogenetic tests with cultured Chinese hamster ovary cells, methyleugenol induced sister chromatid exchanges in the presence of S9, but no induction of chromosomal aberrations was noted in cultured Chinese hamster ovary cells following exposure to methyleugenol, with or without S9. In vivo, no increase in the frequency of micronucleated normochromatic erythrocytes was seen in male or female mice administered methyleugenol by gavage for 14 weeks.
Physiologically based pharmacokinetic model
A physiologically based pharmacokinetic (PBPK) model resulting from intravenous and oral exposure was created to characterize tissue concentrations of methyleugenol in rats and mice. Data used to create the model were obtained from the literature or from current studies. The primary conclusions that can be reached from the PBPK model are: 1) absorption of oral doses of methyleugenol in rats and mice is rapid and complete, 2) distribution of methyleugenol to tissues is not hampered by capillary permeability, and 3) metabolism of methyleugenol is saturable and must have some extrahepatic component in the mouse. Model-based plasma methyleugenol concentrations were not found to be good dosimeters for evaluating neoplasm dose-response data.
Conclusions
Under the conditions of these 2-year gavage studies, there was clear evidence of carcinogenic activity of methyleugenol in male and female F344/N rats based on the increased incidences of liver neoplasms and neuroendocrine tumors of the glandular stomach in male and female rats and the increased incidences of kidney neoplasms, malignant mesothelioma, mammary gland fibroadenoma, and subcutaneous fibroma and fibroma or fibrosarcoma (combined) in male rats. A marginal increase in the incidence of squamous cell neoplasms of the forestomach may have been related to methyleugenol administration in female rats. There was clear evidence of carcinogenic activity of methyleugenol in male and female B6C3F1 mice based on the increased incidences of liver neoplasms. Neuroendocrine tumors of the glandular stomach in male mice were also considered related to methyleugenol administration.
In male and female rats and mice, methyleugenol administration caused significant increases in nonneoplastic lesions of the liver and glandular stomach.