title: NTP Toxicology and Carcinogenesis Studies of 4,4'-Thiobis(6- t -butyl- m -cresol) (CAS No. 96-69-5) in F344/N Rats and B6C3F1 Mice (Feed Studies).
abstract: 4,4'-Thiobis(6- t -butyl- m -cresol) (TBBC) is used in the rubber and plastics industries as an antioxidant. TBBC is also used as a stabilizer in polyethylene and polyolefin packaging materials for foodstuffs. Toxicology and carcinogenesis studies were conducted by administering TBBC (99% pure) in feed to groups of male and female F344/N rats and B6C3F1 mice for 15 days, 13 weeks, and 2 years. Genetic toxicology studies were conducted in Salmonella typhimurium and cultured Chinese hamster ovary cells. 15-DAY STUDY IN RATS: Groups of 10 male and 10 female F344/N rats were fed diets containing 0, 1,000, 2,500, 5,000, 10,000 or 25,000 ppm TBBC for 15 days. Rats given to 1,000, 2,500, 5,000, or 10,000 ppm received approximate doses of 95, 235, 335, or 365 mg TBBC per kilogram body weight per day (males) or 85, 220, 325, or 270 mg/kg per day (females). Approximate doses for rats receiving 25,000 ppm could not be calculated due to early deaths. All 25,000 ppm rats and three male and four female 10,000 ppm rats died. Surviving rats in the 10,000 ppm groups had a significant weight loss and the final mean body weights of 5,000 and 10,000 ppm male and female rats were significantly lower than those of the controls. Male and female rats exposed to 5,000, 10,000, or 25,000 ppm TBBC consumed markedly less feed than the controls. Diarrhea occurred in 5,000, 10,000, and 25,000 ppm males and females. The principal lesions attributed to the administration of TBBC were renal papillary and tubule necroses which occurred in 10,000 ppm rats. Focal necrosis or erosions of the glandular stomach also occurred in some 10,000 ppm rats. Changes observed in the thymus and spleen were attributed to debilitation or stress; bone marrow depletion was attributed to nutrient deficiency accompanying weight loss. 15-DAY STUDY IN MICE: Groups of 10 male and 10 female B6C3F1, mice were fed diets containing 0, 1,000, 2,500, 5,000, 10,000, or 25,000 ppm TBBC for 15 days. Mice given 1,000, 2,500, or 5,000 ppm received approximate doses of 285, 585, or 475 mg TBBC per kilogram body weight per day (males) or 360, 950, or 1,030 mg/kg per day (females). Approximate doses for mice given 10,000 or 25,000 ppm could not be calculated due to early deaths. All 10,000 and 25,000 ppm mice died, as did eight males and eight females given 5,000 ppm. A significant weight loss occurred in surviving 5,000 ppm males and females and the final mean body weights of 2,500 ppm females and 5,000 ppm males and females were significantly lower than those of the controls. Feed consumption by mice given 5,000, 10,000, or 25,000 ppm was markedly reduced. Diarrhea occurred in all 25,000 ppm mice and in most male and female mice given 5,000 or 10,000 ppm. Renal tubule necrosis occurred in eight males and three females in the 5,000 ppm groups. Lymphocytic depletion of Iymphoid tissues in many 5,000 ppm males and females was attributed to debilitation and stress or to nutrient deficiency accompanying weight loss. 13-WEEK STUDY IN RATS: Groups of 10 male and 10 female F344/N rats were fed diets containing 0, 250, 500, 1,000, 2,500, or 5,000 ppm TBBC for 13 weeks. These exposure levels delivered approximate doses of 15, 30, 60, 165, or 315 mg TBBC per kilogram body weight per day (males) or 15, 35, 70, 170, or 325 mg/kg per day (females). All rats survived to the end of the study. The final mean body weight of 5,000 ppm males was 40% lower than that of the controls; the final mean body weight of 5,000 ppm females was 27% lower than that of the controls. Feed consumption by male and female rats exposed to 5,000 ppm TBBC was markedly lower than that by the controls throughout the study. The absolute and relative liver weights of 5,000 ppm females were significantly greater than those of the controls. Serum alkaline phosphatase (ALP) levels were significantly higher in 2,500 and 5,000 ppm males and slightly higher in 5,000 ppm females. Serum alanine aminotransferase levels were significantly higher in 2,500 and 5,000 ppm males and females. Hematocrit and hemoglobin concentrations and mean erythroions and mean erythrocyte volume (MCV) values were significantly lower in 1,000, 2,500, and 5,000 ppm males than in controls; MCV values were also significantly lower in 5,000 ppm females. A dose-related significant increase in forelimb and hindlimb grip strength was observed in exposed male and female rats. Histopathologic findings in the liver of 2,500 and 5,000 ppm males and females included hypertrophy of Kupffer cells, bile duct hyperplasia, and individual cell necrosis of hepatocytes; centrilobular hepatocyte hypertrophy also occurred in males and females exposed to 5,000 ppm TBBC. Macrophages were increased in size and number in the mesenteric Iymph nodes of males and females exposed to 5,000 ppm, and to a lesser extent in 2,500 ppm male and female rats. Pigmentation and degeneration of the renal cortical tubule epithelial cells was also present in males and females in the 2,500 and 5,000 ppm groups; cortical tubule necrosis occurred in 5,000 ppm males and females. 13-WEEK STUDY IN MICE: Groups of up to 10 male and 10 female B6C3F1 mice were fed diets containing 0, 100, 250, 500, 1,000, or 2,500 ppm TBBC for 13 weeks. These exposure levels delivered approximate doses of 15, 30, 65, 145, or 345 mg TBBC per kilogram body weight per day (males) or 10, 35, 60, 165, or 340 mg/kg per day (females). All mice survived to the end of the study. The final mean body weights of 2,500 ppm males and of 500,1,000, or 2,500 ppm females were significantly lower than those of the controls. Feed consumption by 2,500 ppm males averaged 24&percnt; lower than that by controls through week 3 and was similar to that by controls for the remainder of the study. Feed consumption by females receiving 2,500 ppm averaged 27&percnt; less than that by the controls during most of the study. The absolute and relative liver weights of males and females exposed to 2,500 ppm TBBC were slightly but significantly greater than those of the controls. Males exposed to 500, 1,000, or 2,500 ppm and females exposed to 2,500 ppm had significantly increased absolute and relative spleen weights. No clinical findings in mice were considered chemical related. Hematocrit concentrations and erythrocyte counts of males receiving 1,000 or 2,500 ppm were significantly less than those of the controls; hemoglobin concentration in males receiving 2,500 ppm was significantly less and mean erythrocyte volume was significantly less in males receiving 2,500 ppm. Females in the 1,000 and 2,500 ppm groups had significantly decreased hematocrit concentrations and erythrocyte counts; 2,500 ppm females also had significantly decreased hemoglobin concentrations and mean erythrocyte volumes. Kupffer cell hypertrophy, bile duct hyperplasia, and an increase in size and number of macrophages in mesenteric Iymph nodes were present in 2,500 ppm male and female mice. 2-YEAR STUDY IN RATS: Doses selected for the 2-year study of TBBC were based on the lower body weights and liver and kidney toxicity observed at 5,000 ppm in the 13-week study. Groups of 115 male and 75 female F344/N rats were fed diets containing 0, 500, 1,000, or 2,500 ppm TBBC for 2 years. Based on average daily feed consumption, these exposure levels resulted in a daily ingestion of TBBC of approximately 20, 40, or 100 mg/kg body weight for males and 20, 45, or 120 mg/kg body weight for females. Hematology, clinical chemistry, and urinalysis evaluations were performed on 15 male and 15 female rats from each group at 3, 9, and 15 months. Also at 15 months, an additional 10 male and 10 female rats from each group were evaluated for histopathology, hematology, and clinical chemistry. Forty male rats per group were evaluated for neurotoxic effects. Survival, Body Weights, Feed Consumption, and Clinical Findings: Two-year survival rates and mean body weights of exposed male and female rats were generally similar to those of the controls. The mean body weights of 2,500 ppm male rats were slightly lower than those of the controls throughout the study. At week 65, the mean body weight of 2,500 ppm females was 14&percnt; lower than that of the controls, but the final mean body weight of this group was 6&percnt; lower than that of the control group. Feed consumption, behavior, and general health and appearance of exposed male and female rats were similar to those of the controls. Hematology and Clinical Chemistry: Results of the hematology evaluation were not uniformly consistent at 3, 9, and 15 months in one set of rats, nor were they consistent between the two sets of rats evaluated at 15 months. Slight but significant decreases in hematocrit levels, hemoglobin concentrations, and erythrocyte counts were observed in the 1,000 and 2,500 ppm groups in one set of males at 15 months. Similar significant decreases in hematocrit level and hemoglobin concentration occurred in 2,500 ppm females at 9 months. Mean erythrocyte hemoglobin and mean erythrocyte hemoglobin concentration of 2,500 ppm females were also significantly lower than those of controls at 9 months and in both sets of female rats evaluated at 15 months. Platelet counts of 2,500 ppm male and female rats were slightly but significantly higher than those of controls at 3 and 9 months. Platelet counts were also slightly but significantly increased in 2,500 ppm males of one set evaluated at 15 months, and in 2,500 ppm females of the second set evaluated at 15 months. Serum activities of alkaline phosphatase, alanine aminotransferase, and sorbitol dehydrogenase in 2,500 ppm males were significantly greater than those in the controls at 3, 9, and 15 months. Alkaline phosphatase activities in both sets of 1,000 ppm males evaluated at 15 months were also significantly greater than those of controls. Serum activities of alanine aminotransferase and sorbitol dehydrogenase in 2,500 ppm females were also significantly greater than those in controls at 3, 9, and 15 months. Neurotoxicity Findings: There were no significant inhibitory effects of TBBC on motor nerve excitability or conduction, neuromuscular transmission, or muscle contractility. There were no microscopic lesions in the sciatic nerve, quadriceps muscle, or teased nerve preparations of sciatic nerve that could be attributed to TBBC administration. Pathology Findings: At the 15-month interim evaluation, the absolute and relative liver weights of 2,500 ppm female rats were significantly greater than those of controls; at 15 months and at the end of the study, the incidences of Kupffer cell hypertrophy, hepatocyte cytoplasmic vacuolization, and mixed cell foci were also significantly increased. At the end of the study, the incidence of hepatocellular fatty change was significantly increased in 2,500 ppm females. The incidence of Kupffer cell hypertrophy was significantly increased in 2,500 ppm males at 15 months and at 2 years; the incidence of cytoplasmic vacuolization was significantly increased in all exposed males at 15 months but only moderately increased in 1,000 and 2,500 ppm males at 2 years; the incidence of basophilic foci was significantly increased in 2,500 ppm males at 15 months and the incidence of mixed cell foci was significantly increased in 1,000 and 2,500 ppm male rats at 2 years. The incidences of hepatocellular adenoma or carcinoma (combined) in exposed male rats were not significantly greater than that in the controls (0 ppm, 1/50; 500 ppm, 3/50; 1,000 ppm, 3/50; 2,500 ppm, 5/49), were within the historical control range, and were not considered chemical related. The severity of nephropathy was significantly increased in 2,500 ppm female rats. There was a significant negative trend in the incidence of mammary gland fibroadenoma, adenoma, or carcinoma (combined) in female rats (32/50, 24/50, 11/50, 16/50), and the incidences of fibroadenoma in 1,000 and 2,500 ppm females were significantly less than that of the controls. 2-YEAR STUDY IN MICE: Because of the reduction in body weights, the increase in liver and spleen weights, and the accompanying histopathologic changes in the liver of 2,500 ppm male and female mice in the 13-week study, the doses selected for the 2-year study were 250, 500, and 1,000 ppm. Groups of 80 male and 80 female mice were fed diets containing 0, 250, 500, or 1,000 ppm TBBC for 2 years. Based on average daily feed consumption, these exposure levels resulted in the daily ingestion of approximately 30, 60, or 145 mg TBBC/kg body weight for males and 45, 110, or 255 mg TBBC/kg body weight for females. Nine or 10 animals from each exposure group were evaluated at 3, 9, and 15 months. Survival, Body Weights, Feed Consumption, and Clinical Findings: Two-year survival rates of exposed male and female mice were similar to those of the controls. The final mean body weights of male and female mice exposed to 1,000 ppm were 8&percnt; and 18&percnt; lower than those of the controls, respectively. The final mean body weights of females exposed to 250 or 500 ppm were 8&percnt; to 9&percnt; lower than that of the controls. Feed consumption by exposed males was similar to that by controls, and there were no clinical findings attributed to TBBC administration. Hematology and Clinical Chemistry: Hematocrit level, hemoglobin concentration, and erythrocyte count in 1,000 ppm male mice were significantly lower than those in controls at the 15-month interim evaluation. Serum alkaline phosphatase activities in 1,000 ppm males were slightly but significantly greater than those in controls at 3 and 9 months, as was the serum alkaline phosphatase activity in 1,000 ppm females at 9 months. Serum levels of total bilirubin in all exposed groups of males were significantly greater than those in controls at 9 and 15 months. Pathology Findings: In the liver of male mice, negative trends in the incidences of fatty change, clear cell foci, and adenoma or carcinoma combined occurred at the end of the 2-year study. There were no compound-related increased incidences of neoplasms or nonneoplastic lesions in mice receiving TBBC for 2 years. A negative trend in the incidence of fatty change in the liver of male mice also occurred at 15 months. GENETIC TOXICOLOGY: 4,4'-Thiobis(6- t -butyl- m -cresol) was not mutagenic in Salmonella typhimurium strains TA98, TA100, TA1535, or TA1537 with or without exogenous metabolic activation (S9). Sister chromatid exchanges were induced in cultured Chinese hamster ovary cells treated with TBBC, with and without S9, but no increases in chromosomal aberrations were noted in cultured Chinese hamster ovary cells after treatment with TBBC. CONCLUSIONS: Under the conditions of these 2-year feed studies, there was no evidence of carcinogenic activity of 4,4'-thiobis(6- t -butyl- m -cresol) in male or female F344/N rats administered 500, 1,000, or 2,500 ppm or in male or female B6C3F1, mice administered 250, 500, or 1,000 ppm. Nonneoplastic lesions associated with exposure to TBBC included: Kupffer cell hypertrophy, cytoplasmic vacuolization, and mixed cell foci in the liver of male and female rats, fatty change in the liver of female, rats, and an increase in the severity of nephropathy in the kidney of female rats. In addition, decreased incidences of fibroadenoma, adenoma, or carcinoma (combined) were observed in the mammary gland of female rats. Decreases also occurred in the incidences of fatty change, clear cell foci, and adenoma or carcinoma (combined) in the liver of male mice. Synonyms: 4,4'-Thiobis(6- t -butyl-3-cresol); bis(3- t -butyl-4-hydroxy-6-methylphenyl)sulfide