title: NTP Toxicology and Carcinogenesis Studies of t -Butyl Alcohol (CAS No. 75-65-0) in F344/N Rats and B6C3F1 Mice (Drinking Water Studies).
abstract: t -Butyl alcohol is widely used in the manufacture of perfumes and a variety of cosmetics. It is also used as a raw material in the production of isobutylene, which may be used to produce methyl tertiary butyl ether, a common gasoline additive, or to produce butyl elastomers used in the production of automobile tires. Male and female F344/N rats and B6C3F1 mice were given t -butyl alcohol (greater than 99% pure) in drinking water for 13 weeks or 2 years. The genetic toxicity of t -butyl alcohol was assessed by testing the ability of the chemical to induce mutations in various strains of Salmonella typhimurium and in L5178Y mouse lymphoma cells, sister chromatid exchanges and chromosomal aberrations in cultured Chinese hamster ovary cells, and by measuring the frequency of micronucleated erythrocytes in mouse peripheral blood. 13-WEEK STUDY IN RATS: Groups of 10 male and 10 female F344/N rats were given 0, 2.5, 5, 10, 20, or 40 mg/mL t -butyl alcohol in drinking water for 13 weeks. All males and six females given 40 mg/mL died during the study. Final mean body weights of 10 and 20 mg/mL males and of 40 mg/mL females were 12%, 17%, or 21% less than those of the corresponding controls, respectively. Serum sorbitol dehydrogenase activities in 10 and 20 mg/mL males were greater than that in the controls after 13 weeks. Serum alanine aminotransferase activity in 40 mg/mL females was greater than that in the controls after 2 weeks and greater in all exposed females after 13 weeks. Urine volumes of 10, 20, and 40 mg/mL males and females decreased, and urine specific gravity values increased. Transitional epithelial hyperplasia and inflammation of the urinary bladder were observed in 20 and 40 mg/mL males and 40 mg/mL females. Absolute and relative liver weights of all exposed groups of females and relative liver weights of 5, 10, and 20 mg/mL males were significantly greater than those of the controls. Absolute and relative kidney weights of all exposed groups of males and females were significantly greater than those of the controls. Incidences of mineralization of the kidney were significantly increased in 10, 20, and 40 mg/mL males. The severity of nephropathy in 2.5, 5, 10, and 20 mg/mL males was significantly greater than that of the controls as was the accumulation of hyaline droplets in the kidney of 5, 10, and 20 mg/mL males. The incidences of nephropathy in 10, 20, and 40 mg/mL females were significantly greater than that of the controls. 13-WEEK STUDY IN MICE: Groups of 10 male and 10 female B6C3F1 mice were given 0, 2.5, 5, 10, 20, or 40 mg/mL t -butyl alcohol in drinking water for 13 weeks. The deaths of two males and one female in the 40 mg/mL group were attributed to exposure to t -butyl alcohol. The final mean body weights of 20 and 40 mg/mL males and 40 mg/mL females were significantly lower than those of the controls. There were no biologically significant differences in hematology parameters of exposed and control groups of mice. Transitional epithelial hyperplasia and inflammation were observed in the urinary bladder of 20 and 40 mg/mL males and 40 mg/mL females. 2-YEAR STUDY IN RATS: Groups of 60 F344/N rats were given 0, 1.25, 2.5, or 5 mg/mL t -butyl alcohol (males) or 0, 2.5, 5, or 10 mg/mL t -butyl alcohol (females) in drinking water for 2 years. These correspond to average daily doses of approximately 90, 200, or 420 mg t -butyl alcohol/kg body weight for males and approximately 180, 330, or 650 mg t -butyl alcohol/kg body weight for females. Ten rats per group were evaluated after 15 months of chemical administration. Survival, Body Weights, and Water Consumption: Survival rates of 5 mg/mL males and 10 mg/mL females were significantly lower than those of the controls. The final mean body weights of exposed groups of males were 15% to 24% lower than that of the controls, and the final mean body weight of 10 mg/mL females was 21% lower than that of the controls. Water consumption by males increased with dose; water consumption by females decreased with dose. Hematology and Urinalysis: At the 15-month inte. Hematology and Urinalysis: At the 15-month interim evaluation, there were no significant differences in hematology parameters in males and females, and there were no significant differences in urinalysis parameters in males. Females given 5 or 10 mg/mL had increased urine specific gravities and decreased urine volumes. Pathology Findings: At the 15-month interim evaluation, relative kidney weights of 2.5 and 5 mg/mL males and absolute and relative kidney weights of 2.5, 5, and 10 mg/mL females were significantly greater than those of the controls. At 2 years, the incidence of mineralization in the kidney increased with dose and that of 5 mg/mL males was significantly greater than that of the controls. In the standard evaluation at the end of the study, the incidences of focal renal tubule hyperplasia and of adenoma were increased in exposed males and a carcinoma was observed in one 5 mg/mL male. Renal tubule hyperplasia occurred in one 10 mg/mL female. An extended evaluation of the kidney identified additional male rats with hyperplasia (control, 11/50; 1.25 mg/mL, 13/50; 2.5 mg/mL, 11/50; 5 mg/mL, 19/50) and renal tubule adenoma (7/50, 8/50, 15/50, 10/50); renal tubule carcinomas were identified in two 1.25 mg/mL males and in one 2.5 mg/mL male. Renal tubule adenoma was identified in one 5 mg/mL male from the 15-month extended evaluation. In the standard and extended evaluations combined, there were dose-related increased incidences of hyperplasia and adenoma. The severity of nephropathy and the incidence and severity of transitional cell hyperplasia of the kidney were increased in exposed male and female rats. Linear foci of mineralization were present in the renal papilla of exposed males. 2-YEAR STUDY IN MICE: Groups of 60 male and 60 female B6C3F1 mice were given 0, 5, 10, or 20 mg/mL t -butyl alcohol in drinking water for 2 years. Exposure levels of 5, 10, or 20 mg/mL delivered average daily doses of approximately 540, 1,040, or 2,070 mg t -butyl alcohol/kg body weight to males and approximately 510, 1,020, or 2,110 mg/kg to females. Survival, Body Weights, and Water Consumption: Survival of 20 mg/mL males was significantly lower than that of the controls. The final mean body weights of exposed groups of males were similar to those of the controls. The mean body weights of females given 20 mg/mL were 10&percnt; to 15&percnt; lower than those of the controls from week 13 to the end of the study. Water consumption by exposed groups of males and females was similar to that by the controls. Pathology Findings: Incidences of thyroid gland follicular cell hyperplasia were significantly increased in all exposed groups of males and in 10 and 20 mg/mL females. The incidence of follicular cell adenoma or carcinoma (combined) was marginally increased in 10 mg/mL males (0 mg/mL, 1/60; 5 mg/mL, 0/59; 10 mg/mL, 4/59; 20 mg/mL, 2/57). The incidence of follicular cell adenoma was significantly increased in 20 mg/mL females (2/58, 3/60, 2/59, 9/59). The incidences of chronic inflammation and transitional epithelial hyperplasia of the urinary bladder were increased in 20 mg/mL males and to a lesser extent in 20 mg/mL females. GENETIC TOXICOLOGY: t -Butyl alcohol was tested for induction of genetic damage in vitro and in vivo, and all results were negative. In vitro, t -butyl alcohol was negative in Salmonella typhimurium and mouse lymphoma cell mutation tests, and it did not induce sister chromatid exchanges or chromosomal aberrations in cultured Chinese hamster ovary cells. These in vitro studies were conducted with and without metabolic activation (S9). In vivo, no increase in micronucleated erythrocytes was observed in peripheral blood samples from mice administered t -butyl alcohol in drinking water for 13 weeks. CONCLUSIONS: Under the conditions of these 2-year drinking water studies, there was some evidence of carcinogenic activity of t -butyl alcohol in male F344/N rats based on increased incidences of renal tubule adenoma or carcinoma (combined). There was no evidence of carcinogenic activity in female F344/N rats receiving 2.5, 5, or 10 mg/mL t -butyl alcohol. There was equivocal evidence of carcinogenic activity of t -butyl alcohol in male B6C3F1 mice based on the marginally increased incidences of follicular cell adenoma or carcinoma (combined) of the thyroid gland. There was some evidence of carcinogenic activity of t -butyl alcohol in female B6C3F1 mice based on increased incidences of follicular cell adenoma of the thyroid gland. Exposure to t -butyl alcohol was associated with mineralization and renal tubule hyperplasia in male rats, transitional epithelial hyperplasia and increased severity of nephropathy of the kidney in male and female rats, follicular cell hyperplasia of the thyroid gland in male and female mice, and chronic inflammation and hyperplasia of the urinary bladder in male mice and to a lesser extent in female mice. Synonyms: 2-Methyl-2-propanol, 2-methylpropan-2-ol, TBA, t -butanol, tertiary butyl alcohol, t -butyl hydroxide, trimethyl carbinol, trimethyl methanol