title: Toxicology and Carcinogenesis Studies of 5,5-Diphenylhydantoin (CAS No. 57-41-0) (Phenytoin) in F344/N Rats and B6C3F1 Mice (Feed Studies).
abstract: 5,5-Diphenylhydantoin and its sodium salt are primarily used in the treatment of grand mal and psychomotor seizures, often in combination with other anticonvulsants, including phenobarbital. 5,5-Diphenylhydantoin is a suspected human carcinogen and was one of three compounds selected by the NTP to investigate the potential value of perinatal exposures in assessing chemical carcinogenicity. Chronic toxicity and carcinogenicity studies of 5,5-diphenylhydantoin were conducted in male and female F344/N rats and B6C3F1 mice. The studies were designed to determine the following: a) the effects of 5,5-diphenylhydantoin in the diet given to rats and mice during the adult (F1) period only (a typical carcinogenicity study), b) the toxic and carcinogenic effects of 5,5-diphenylhydantoin in rats and mice receiving perinatal (F0) exposure only (dietary exposure of dams prior to breeding and throughout gestation and lactation), and c) the effects of combined perinatal and adult exposure to 5,5-diphenylhydantoin. Genetic toxicology studies were conducted in Salmonella typhimurium, mouse Iymphoma cells, cultured Chinese hamster ovary cells, Drosophila melanogaster, and mouse bone marrow cells. STUDIES IN F344/N RATS: A 13-week toxicity study was conducted to select the exposure levels for adults in the 2-year study. The exposure levels for the 13-week study ranged from 300 to 4,800 ppm 5,5-diphenylhydantoin in the diet. The final mean body weights of males and females exposed to 2,400 or 4,800 ppm were significantly decreased. All groups showed a net weight gain over the study period, although the mean body weight gain of females in the 4,800 ppm group was only one-half that of the controls. Feed consumption also decreased with increasing exposure level. No chemical-related gross lesions were present in the tissues of exposed rats. Microscopically, centrilobular hypertrophy of hepatocytes was observed in the liver of rats in the 4,800 ppm groups. Based on these results, 2,400 ppm was selected as the highest exposure for the adult-only portion of the 2-year carcinogenicity study. A gestational study was performed to select the exposure levels for the perinatal portion of the 2-year study. The exposure levels ranged from 80 to 2,400 ppm 5,5-diphenylhydantoin in the diet of the dams. The 2,400 ppm exposure level was found to have reproductive and embryotoxic effects, as none of the sperm-positive females delivered litters. In the 800 ppm group, a greater number of pups died between postnatal day 1 and day 28 than in the control group. No gross external malformations were observed among fetuses or pups surviving to term in any exposure group, and no gross or histopathologic lesions were observed in the animals exposed to 800 ppm for 4 weeks following weaning. Based on these results, 630 ppm was selected as the highest exposure level for the perinatal portion of the 2-year carcinogenicity study. The eight F0:F1 exposure combinations selected for the 2-year study are listed in the table (contained in full report - page 6). In the 2-year study, male and female rats in the 630:2,400 ppm groups evaluated at 9 months had increased relative liver weights. Hematologic evaluations indicated mild but consistent chemical-related increases in erythrocyte and platelet counts in male and female rats. Mild decreases in triglyceride concentrations and alanine aminotransferase enzyme activity were seen generally in the high-exposure groups. In the 2-year study, the survival of exposed rats was similar to that of the controls. However, body weights of exposed rats were lower than those of the controls, and body weights were 11% to 35~ lower in rats receiving adult exposure of 2,400 ppm 5,5-diphenylhydantoin. Feed consumption was similar for exposed and control groups. Hepatocellular neoplasms, primarily adenomas, occurred with a positive trend in male rats fed 5,5-diphenylhydantoin only as adults (0:0 ppm, 0/50; 0:800 ppm, 2/50; 0:2,400 ppm, 4/50). There were no increased neoplasm incidences at other sites in exposed males or at any site in exposed females.emales. Perinatal-only or combined perinatal and adult exposure to 5,5-diphenylhydantoin did not enhance the overall incidences of liver neoplasms in male or female rats. However, the finding of 5/49 hepatocellular adenomas in the 630:2,400 male rat group was consistent with the marginally elevated liver neoplasm rate observed in the 0:2,400 group. Decreased incidences of a number of different neoplasms in exposed groups were most likely related to the lower body weights. STUDIES IN B6C3F1 MICE: A 13-week toxicity study was conducted to select the exposure levels for adults in the 2-year study. The exposure levels for the 13-week study ranged from 75 to 1,200 ppm 5,5-diphenylhydantoin in the diet. With the exception of one male, all mice exposed to 1,200 ppm died before the end of the study. No other chemical-related deaths occurred. All groups of mice except the 1,200 ppm groups gained weight over the 13-week period; however, an exposure related decrease in body weight gain was seen in males and females. Feed consumption by exposed and control groups was generally similar. Chemical related histomorphologic lesions were present in the liver of exposed mice, particularly 600 ppm males, and consisted of centrilobular hypertrophy of hepatocytes. Females appeared to be less sensitive than males to the effects of 5,5-diphenylhydantoin on growth and on histomorphologic liver lesions. Based on these results, 300 ppm (males) and 600 ppm (females) were selected as the highest exposure levels for the adult-only portion of the 2-year carcinogenicity study. A gestational study was performed to select the exposure levels for the perinatal portion of the 2-year study. The exposure levels for males and females ranged from 20 to 600 ppm 5,5-diphenylhydantoin in the diet. In general, reproductive performance and maternal care were poor in all groups, including the controls, thus restricting the sample size and sensitivity of this evaluation. There were no litters in the 600 ppm group, and maternal weight gain was depressed. There were no gross external malformations among pups surviving to term, and no gross or histopathologic lesions were observed in any mice exposed for 4 weeks following weaning. Based on these results, 210 ppm was selected as the highest exposure level for the perinatal portion of the 2-year carcinogenicity study. The F0:F1 exposure combinations selected for the 2-year study are listed in the following table (contained in full report - page 7). For mice evaluated at 9 months, males and females receiving the highest F0:F1 exposure levels had increased relative liver weights. In the 2-year study, the survival of exposed animals was similar to that of the controls; however, body weights were lower for exposed groups, and decreased body weights were most severe in adult females receiving 600 ppm 5,5-diphenylhydantoin. Feed consumption was similar for exposed and control groups. The incidences of hepatocellular neoplasms were increased in female mice receiving adult-only exposure (0:0 ppm, 5/48; 0:200 ppm, 14/49; 0:600 ppm, 30/50) or combined perinatal and adult exposure (210:200 ppm, 16/50; 210:600 ppm, 34/50). A marginally increased incidence of liver neoplasms (12/49) occurred in females in the perinatal-only (210:0) exposure group. There were no chemical-related increased incidences of liver neoplasms in males receiving adult-only or perinatal-only exposure. However, males receiving the high-exposure combined perinatal and adult exposure regimen (210:300 ppm) had an increased incidence of liver neoplasms (41/50) compared to the 0:0 (29/50), 0:300 (26/49), and 210:0 (33/50) groups. As a result, there was a significant enhancement (interaction) associated with combined perinatal and adult exposure. Such enhancement of neoplasia did not occur in female mice. Decreased incidences of malignant neoplasms in exposed groups were most likely related to the lower body weights. GENETIC TOXICOLOGY: In general, tests for genotoxic activity of 5,5-diphenylhydantoin were negative. All in vitro testing was performed in the presence and the absence of exogenous metabolic activation (S9). 5,5-Diphenylhydantoin did not induce mutations in Salmonella typhimurium, in L5178Y mouse Iymphoma cells, or in germ cells of male Drosophila melanogaster, nor did it induce chromosomal aberrations in cultured Chinese hamster ovary cells. A small but statistically significant increase was obtained in the cultured Chinese hamster ovary cell test for induction of sister chromatid exchanges in the presence of S9; without S9, no increase in sister chromatid exchanges was observed. In vivo, 5,5-diphenylhydantoin did not induce micronuclei polychromatic erythrocytes or chromosomal aberrations in bone marrow cells of male mice; equivocal results were obtained in an in vivo test for induction of sister chromatid exchanges in mouse bone marrow cells. CONCLUSIONS: Adult-Only Exposure: Under the conditions of these 2-year, adult-only, dietary exposure studies, there was equivocal evidence of carcinogenic activity of 5,5-diphenylhydantoin in male F344/N rats based on marginally increased incidences of hepatocellular neoplasms. There was no evidence of carcinogenic activity of 5,5-diphenylhydantoin in female F344/N rats given 240, 800, or 2,400 ppm. There was no evidence of carcinogenic activity of 5,5-diphenylhydantoin in male B6C3F1 mice given 30,100, or 300 ppm. There was clear evidence of carcinogenic activity of 5,5-diphenylhydantoin in female B6C3F1 mice based on increased incidences of hepatocellular neoplasms. Perinatal-Only Exposure: Perinatal exposure alone (through dietary administration of 210 ppm 5,5-diphenylhydantoin during the perinatal period) caused a marginal increase in the incidences of hepatocellular neoplasms in female B6C3F1 mice evaluated 2 years after cessation of exposure. In male and female F344/N rats, exposure to 630 ppm during the perinatal period did not influence the incidences of hepatocellular or other neoplasms. Similarly, exposure of male B6C3F1 mice to dietary levels of 210 ppm 5,5-diphenylhydantoin during the perinatal period did not affect neoplasm incidences. No teratologic effects were observed. Combined Perinatal and Adult Exposure: Combined perinatal and adult dietary exposure to 5,5-diphenylhydantoin confirmed the findings of the increased incidences of hepatocellular neoplasms for adult-only exposures in male F344/N rats and female B6C3F1 mice, although combined exposure did not enhance these neoplastic effects. However, in male B6C3F1 mice, combined perinatal and adult exposure resulted in increased incidences of hepatocellular neoplasms (hepatocellular carcinomas and multiple adenomas) that were not seen when dietary exposure was limited to the adult exposure period only. Synonyms: Diphenylhydantoin; 5,5-diphenyl-2,4-imidazolidinedione Trade names: Difhydan; Dihycon; Di-Hydan; Di-Lan; Dilabid; Dilantin; Ekko; Hydantol; Lehydan; Zentropil