title: NTP Toxicology and Carcinogenesis Studies of Benzethonium Chloride (CAS No. 121-54-0) in F344/N Rats and B6C3F1 Mice (Dermal Studies).
abstract: Benzethonium chloride is used primarily in cosmetics for its antimicrobial and cationic surfactant properties. Benzethonium chloride was nominated by the National Cancer Institute to the NTP for study from a class study of chemicals used as biocides. The chemical was selected based on a suspicion of carcinogenicity and its known widespread human exposure. Male and female F344/N rats and B6C3F1 mice were topically administered benzethonium chloride (greater than 98% pure) for 16 days, 13 weeks, or 2 years. Genetic toxicology studies were conducted in Salmonella typhimurium and cultured Chinese hamster ovary cells. 16-DAY STUDY IN RATS: Groups of five male and five female F344/N rats were topically administered 0, 6.3, 12.5, 25, 50, or 100 mg benzethonium chloride/kg body weight. Rats were administered a total of 12 doses in a fixed volume of 250 &mgr;L ethanol. All rats survived to the end of the study. The final mean body weights and body weight gains of rats administered 50 or 100 mg benzethonium chloride/kg body weight were significantly less than those of the controls. Clinical findings at necropsy included thickening or hardening of the skin at the site of application in all rats administered 50 or 100 mg/kg and in 25 mg/kg males. Lesions at the site of application appeared crusty or red-grey in color. Epithelial hyperplasia with or without inflammation occurred at the site of application in all groups of males and females administered benzethonium chloride. 16-DAY STUDY IN MICE: Groups of five male and five female B6C3F1 mice were topically administered 0, 6.3, 12.5, 25, 50, or 100 mg benzethonium chloride/kg body weight. Mice were administered a total of 12 doses in a fixed volume of 100 &mgr;L ethanol. One 100 mg/kg male mouse died on day 4 of the study. Final mean body weights of all groups of males and females were similar to those of the controls. Clinical findings included mild irritation at the site of application in 50 and 100 mg/kg males and females and in 25 mg/kg males. Epithelial hyperplasia with or without inflammation occurred at the site of application in all groups of males and females administered benzethonium chloride. 13-WEEK STUDY IN RATS: Groups of 10 male and 10 female F344/N rats were topically administered 0, 1.56, 3.13, 6.25, 12.5, or 25 mg benzethonium chloride/kg body weight, 5 days per week for 13 weeks. Doses were administered in ethanol at a volume not exceeding 300 &mgr;L. All rats survived to the end of the study. The final mean body weight and body weight gain of 25 mg/kg males were significantly lower than those of the controls. The final mean body weights of all other groups of males and of all groups of females were similar to those of the controls. Clinical findings included irritation at the site of application in groups administered 3.13 mg/kg or greater. There were no differences in absolute or relative organ weights considered to be related to chemical administration. Epithelial hyperplasia was observed at the site of application in all groups of males and females administered benzethonium chloride. Additionally, inflammation and ulceration were observed at the site of application in males and females administered 3.13 mg/kg or greater. Based on the lesions observed in the 13-week study, benzethonium chloride dose levels selected for the 2-year dermal study in male and female rats were 0.15, 0.5, and 1.5 mg/kg. 13-WEEK STUDY IN MICE: Groups of 10 male and 10 female B6C3F1 mice were topically administered 0, 1.56, 3.13, 6.25, 12.5, or 25 mg benzethonium chloride/kg body weight, 5 days per week for 13 weeks. Doses were administered in ethanol at a volume not exceeding 100 &mgr;L. All mice survived to the end of the study. The final mean body weights of all dosed groups of males and females were similar to those of the controls; the mean body weight gain of 25 mg/kg males was significantly less than that of the controls. Males administered 6.25, 12.5, or 25 mg/kg developed irritation, thickening of the skin, scales, and/or discoloration at the site of application, as did femaleale mice administered 12.5 or 25 mg/kg. Increased incidences of epithelial hyperplasia and inflammation were observed at the site of application in all groups of males and females administered benzethonium chloride. Based on the lesions observed in the 13-week study, benzethonium chloride dose levels selected for the 2-year dermal study in mice were 0.15, 0.5, and 1.5 mg/kg. 2-YEAR STUDY IN RATS: Groups of 60 male and 60 female F344/N rats were topically administered 0, 0.15, 0.5, or 1.5 mg benzethonium chloride/kg body weight 5 days per week for 103 weeks. Doses were administered in ethanol, and dose volumes were adjusted weekly according to the average body weights of the groups. As many as nine rats per group were evaluated after 15 months of chemical administration. Survival, Body Weights, and Clinical Findings: Survival of dosed rats was similar to that of the controls throughout the study. Mean body weights of all dosed groups of males and females were similar to those of the controls throughout the study. Reddening of the skin was observed at the site of application in all dosed groups of males and females. There were no other clinical findings considered to be related to chemical administration. Pathology Findings: There were no increased incidences of neoplasms in dosed male or female rats that were attributed directly to the administration of benzethonium chloride. Increased incidences of epithelial hyperplasia, sebaceous gland hyperplasia, and ulcers were observed at the site of application in dosed females. The incidence of epithelial hyperplasia was increased in 0.5 and 1.5 mg/kg males. 2-YEAR STUDY IN MICE: Groups of 60 male and 60 female B6C3F1 mice were topically administered 0, 0.15, 0.5, or 1.5 mg benzethonium chloride/kg body weight 5 days per week for 103 weeks. Doses were administered in ethanol, and dose volumes were adjusted weekly according to the average body weights of the groups. As many as 10 mice per group were evaluated after 15 months of chemical administration. Survival, Body Weights, and Clinical Findings: Survival of dosed mice was similar to that of the controls throughout the study. Mean body weights of all dosed groups of males and females were similar to those of the controls throughout the study. Reddening of the skin was observed at the site of application in all dosed groups of males and in 0.15 mg/kg females. There were no other clinical findings attributed to chemical administration. Pathology Findings: There were no increased incidences of neoplasms in dosed males or females that were related to administration of benzethonium chloride. Increased incidences of epithelial hyperplasia were observed at the site of application in dosed males and females. GENETIC TOXICOLOGY: Benzethonium chloride was not mutagenic in Salmonella typhimurium strains TA98, TA100, TA1535, or TA1537 and did not induce sister chromatid exchanges or chromosomal aberrations in cultured Chinese hamster ovary cells. All tests were conducted with and without S9 metabolic activation enzymes. CONCLUSIONS: Under the conditions of these 2-year dermal studies, there was no evidence of carcinogenic activity of benzethonium chloride in male or female F344/N rats receiving 0.15, 0.5, or 1.5 mg/kg. There was no evidence of carcinogenic activity in male or female B6C3F1 mice receiving 0.15, 0.5, or 1.5 mg/kg. Exposure of rats and mice to benzethonium chloride by dermal application in ethanol for 2 years resulted in epithelial hyperplasia in male and female rats and mice and sebaceous gland hyperplasia and ulcers in female rats at the site of application. Synonyms: Benzyldimethyl- p -(1,1,3,3-tetramethylbutyl) phenoxyethoxy-ethylammonium chloride; diisobutylphenoxyethoxyethyldimethyl benzyl ammonium chloride; p -tert-octylphenoxyethoxyethyldimethylbenzyl ammonium chloride Trade names: Anti-germ 77, Antiseptol, BZT, Diapp, Disilyn, Hyamine, Hyamine 1622, Phemeride, Phemithyn, Polymine D, Quatrachlor, Solamine