p-Chloro-α,α,α-trifluorotoluene is a solvent used in paints and coatings and as an industrial intermediate in the production of other chemicals (e.g., herbicides, dyes, pharmaceuticals). p-Chloro-α,α,α-trifluorotoluene was nominated for study by the National Cancer Institute and Kowa American Corporation for study because of its high import volume and lack of occupational exposure limits. Male and female Hsd:Sprague Dawley SD rats and B6C3F1/N mice were exposed to p-chloro-α,α,α-trifluorotoluene (purity greater than 99.5%) by inhalation for 3 months or 2 years. Genetic toxicology studies were conducted in Salmonella typhimurium, Escherichia coli, and rat and mouse peripheral blood erythrocytes.
Three-month study in rats
Groups of 10 male and 10 female rats were exposed by whole body inhalation to p-chloro-α,α,α-trifluorotoluene vapor at concentrations of 0, 125, 250, 500, 1,000, or 2,000 ppm for 6 hours plus T90 (15 minutes) per day, 5 days per week for 14 weeks. There was no exposure-related effect on survival. The final mean body weights and mean body weight gains of females exposed to 500 ppm or greater were significantly greater than those of the chamber controls. Alanine aminotransferase and sorbitol dehydrogenase activities were significantly increased in 2,000 ppm males and females. Alkaline phosphatase activities were significantly increased in all exposed male groups and in females exposed to 250 ppm or greater, and bile salt concentrations were significantly increased in females exposed to 500 ppm or greater. There were significant exposure concentration-dependent increases in cholesterol and triglyceride concentrations in males exposed to 500 ppm or greater. In females, cholesterol concentrations were significantly increased in groups exposed to 250 ppm or greater and triglyceride concentrations were significantly increased in the 1,000 and 2,000 ppm groups. There were significant exposure concentration-related increases in the absolute and relative liver weights of males exposed to 250 ppm or greater and of females exposed to 500 ppm or greater. The absolute kidney weights were significantly increased in all exposed groups of males, and relative kidney weights were significantly increased in males exposed to 250 ppm or greater.
In the liver, there were significantly increased incidences of centrilobular hepatocyte hypertrophy in males exposed to 250 ppm or greater and in females exposed to 1,000 or 2,000 ppm. In the Harderian gland, there were significantly increased incidences of degeneration in males and females exposed to 250 ppm or greater. There were significantly increased incidences of cytoplasmic vacuolization in the adrenal cortex of 2,000 ppm males and 1,000 and 2,000 ppm females. In the kidney of males, the severities of hyaline droplet accumulation and chronic nephropathy generally increased with increasing exposure concentration. There were significantly increased incidences of mammary gland hyperplasia in 1,000 and 2,000 ppm females.
Males exposed to 2,000 ppm had significantly decreased left cauda and left epididymis weights and numbers of sperm per cauda epididymis. Sperm motility was also significantly decreased in 1,000 and 2,000 ppm males. These findings were associated with histopathologic changes in the testes and epididymides. Female rats exposed to 2,000 ppm had decreased frequency of estrus and increased frequency of diestrus, a significantly higher probability of extended diestrus, a fewer number of cycles, and a fewer number of rats exhibiting cycles. Based on these findings, p-chloro-α,α,α-trifluorotoluene exhibited the potential to be a reproductive toxicant in both male and female rats.
Three-month study in mice
Groups of 10 male and 10 female mice were exposed by whole body inhalation to p-chloro-α,α,α-trifluorotoluene vapor at concentrations of 0, 125, 250, 500, 1,000, or 2,000 ppm for 6 hours plus T90 (15 minutes) per day, 5 days per week for 14 weeks. All mice survived to the end of the study. The final mean body weights of males exposed to 500 ppm or greater and females exposed to 250 ppm or greater were significantly greater than those of the chamber control groups. The absolute liver weights were significantly increased in an exposure concentration-dependent manner in males and females exposed to 250 ppm or greater. Relative liver weights were significantly increased in males exposed to 250 ppm or greater and females exposed to 500 ppm or greater. In males, absolute kidney weights were significantly increased in groups exposed to 500 ppm or greater, and relative kidney weights were significantly increased in the 1,000 and 2,000 ppm groups. The absolute thymus weight was significantly decreased in 2,000 ppm females, and the relative thymus weights were significantly decreased in 2,000 ppm males and females.
In the liver, there were significantly increased incidences of centrilobular hepatocyte hypertrophy in males exposed to 250 ppm or greater and females exposed to 500 ppm or greater. There were also significantly increased incidences of centrilobular hepatocyte necrosis and multinucleated hepatocytes in males exposed to 500 ppm or greater and females exposed to 1,000 or 2,000 ppm. There were significantly increased incidences of epithelium hyperplasia in the forestomach of males and females exposed to 500 ppm or greater. In 2,000 ppm males and females, there were significantly increased incidences of granulomatous inflammation in the forestomach. In the adrenal cortex, there were significantly increased incidences of zona fasciculata hypertrophy in 2,000 ppm males and females and X-zone degeneration in 2,000 ppm females. In the red pulp of the spleen, there were significantly increased incidences of megakaryocyte and erythrocytic hematopoietic cell proliferation in females exposed to 250 ppm or greater. In males, there were significantly increased incidences of megakaryocyte hematopoietic cell proliferation at 1,000 and 2,000 ppm, and a significantly increased incidence of erythrocytic hematopoietic cell proliferation at 2,000 ppm.
All exposed groups of males evaluated displayed significant exposure concentration-related decreases in sperm motility. In 1,000 and 2,000 ppm females, there were significant increases in estrous cycle length. In all exposed female groups evaluated, there were significantly higher probabilities of extended estrus. Based on these findings, p-chloro-α,α,α-trifluorotoluene exhibited the potential to be a reproductive toxicant in both male and female mice.
Two-year study in rats
Groups of 50 male and 50 female rats were exposed by whole body inhalation to p-chloro-α,α,α-trifluorotoluene at concentrations of 0, 100, 300, or 1,000 ppm for 6 hours plus T90 (12 minutes) per day, 5 days per week for 104 to 105 weeks. There was a significant negative trend in survival of males and survival of 1,000 ppm males was significantly less than that of the chamber controls; survival of other exposed groups of males and all exposed groups of females was similar to that of the chamber controls. Mean body weights of 100 and 300 ppm males and females were similar to those of the chamber controls throughout the study. The mean body weights of 1,000 ppm males were approximately 10% less than the chamber controls at the beginning of the study and subsequently were within 5% to 10% of the chamber controls for the remainder of the study. Female body weights in the 1,000 ppm group were within 10% of the chamber controls until week 72 and were approximately 10% less than the chamber controls for the remainder of the study.
In the thyroid gland, there were significantly increased incidences of C-cell adenoma in 1,000 ppm males and 100 and 1,000 ppm females.
There were occurrences of alveolar/bronchiolar adenoma and alveolar/bronchiolar carcinoma in the 100 and 1,000 ppm groups of male rats; these neoplasms were not observed in the chamber controls. There were significantly increased incidences of chronic inflammation of the lung in all exposed groups of males and females. There were significantly increased incidences of fibrosis of the lung in all exposed male groups and in 300 and 1,000 ppm females and of hemorrhage in all groups of exposed males and 1,000 ppm females.
In the adrenal medulla, there was a significantly increased incidence of benign pheochromocytoma in 1,000 ppm females, and the incidences of medullary hyperplasia were significantly increased in 300 and 1,000 ppm females.
In the uterus, there were positive trends in the incidences of adenocarcinoma and atypical hyperplasia of the endometrium and a significantly increased incidence of stromal polyp in 300 ppm females.
In the liver, there were significantly increased incidences of centrilobular hepatocyte hypertrophy in all exposed groups of males and in 300 and 1,000 ppm females and of fatty change in 300 and 1,000 ppm males and females. There were significantly increased incidences of eosinophilic focus in 1,000 ppm males and mixed cell focus and clear cell focus in 1,000 ppm females.
The severity of kidney nephropathy increased with increasing exposure concentration in male rats.
There was a significantly increased incidence of exudate in the nose of 1,000 ppm males.
Two-year study in mice
Groups of 50 male and 50 female mice were exposed by whole body inhalation to p-chloro-α,α,α-trifluorotoluene at concentrations of 0, 100, 200, or 400 ppm for 6 hours plus T90 (12 minutes) per day, 5 days per week for 104 to 105 weeks. Survival of 400 ppm males was significantly less than that of the chamber controls; survival of 400 ppm females was similar to that of 400 ppm males, but was not significantly different from the chamber controls. Survival of other exposed groups of males and females was similar to that of the chamber controls. Mean body weights of exposed groups of males were similar to those of the chamber control group throughout the study. The mean body weights of 100 ppm females were at least 10% greater than those of the chamber control group generally after week 69. The mean body weights of 200 and 400 ppm females were at least 10% greater than those of the chamber controls after weeks 13 and 2, respectively. Clinical observations included increased occurrences of distended abdomen at removal. Thinness also occurred in 400 ppm males and females.
In the liver of 200 and 400 ppm groups, there were significantly increased incidences of multiple hepatocellular adenoma in males and hepatocellular adenoma (including multiples) in females. There were significantly increased incidences of hepatocellular carcinoma in all exposed groups of males and in 400 ppm females. There were significantly increased incidences of hepatoblastoma in 400 ppm males and females. There were significantly increased incidences of eosinophilic focus in 400 ppm males and in 200 and 400 ppm females. There were significantly increased incidences of centrilobular hepatocyte hypertrophy in all exposed groups of males and in 200 and 400 ppm females. There were significantly increased incidences of multinucleated hepatocytes in 200 and 400 ppm males and in 400 ppm females. The incidences of hepatocyte necrosis were significantly increased in 400 ppm males and females. There were also significantly increased incidences of intrahepatocellular erythrocytes in 200 and 400 ppm males.
In the Harderian gland, there was a significantly increased incidence of adenoma in 400 ppm females, and significantly increased incidences of adenoma or adenocarcinoma (combined) in 200 and 400 ppm females.
There were significantly increased incidences of alveolar/bronchiolar epithelium hyperplasia and peribronchiolar fibrosis of the lungs in all exposed groups of males and females.
There was a significantly increased incidence of epithelium hyperplasia in the forestomach of 400 ppm females. In males, there were significantly increased incidences of inflammation in the 100 and 400 ppm groups.
In the larynx, there was a significantly increased incidence of squamous epithelium hyperplasia in 400 ppm males.
Genetic toxicology
p-Chloro-α,α,α-trifluorotoluene was not mutagenic in S. typhimurium strains TA98, TA100, TA1535, or TA1537 or in E. coli strain WP2 uvrA/pKM101 when tested without exogenous metabolic activation or with 10% induced hamster or rat liver S9 mix (only activation system used for the E. coli strain). In vivo, no significant increases in micronucleated immature or mature erythrocytes were observed in peripheral blood samples from male and female rats exposed to p-chloro-α,α,α-trifluorotoluene by inhalation for 3 months. In male and female mice, small but statistically significant increases in micronucleated mature erythrocytes were seen at the highest exposure concentration (2,000 ppm), but the observed values for the female mice were within historical control ranges and were not considered to be biologically significant. For male mice, the observed response was judged to be positive. An exposure concentration-related increase in the percentage of immature erythrocytes in peripheral blood was seen only in female mice, suggesting that p-chloro-α,α,α-trifluorotoluene may have stimulated erythropoiesis in female mice. No significant changes in the percentage of immature erythrocytes were seen in male or female rats or male mice.
Conclusions
Under the conditions of these 2-year inhalation studies, there was some evidence of carcinogenic activity of p-chloro-α,α,α-trifluorotoluene in male Hsd:Sprague Dawley SD rats based on increased incidences of C-cell adenoma in the thyroid gland (see summary of the Peer Review Panel comments and the public discussion on this Technical Report in Appendix M). The combined occurrences of alveolar/bronchiolar adenoma or carcinoma in the lung of male rats may have been related to treatment. There was some evidence of carcinogenic activity of p-chloro-α,α,α-trifluorotoluene in female Hsd:Sprague Dawley SD rats based on increased incidences of C-cell adenoma in the thyroid gland, increased incidences of benign pheochromocytoma in the adrenal medulla, increased incidences of adenocarcinoma in the uterus, and increased incidences of stromal polyp in the uterus. There was clear evidence of carcinogenic activity of p-chloro-α,α,α-trifluorotoluene in male B6C3F1/N mice based on increased incidences of hepatocellular carcinoma and hepatoblastoma in the liver. There was clear evidence of carcinogenic activity of p-chloro-α,α,α-trifluorotoluene in female B6C3F1/N mice based on increased incidences of hepatocellular adenoma, hepatocellular carcinoma, and hepatoblastoma in the liver. The combined incidences of adenoma or adenocarcinoma in the Harderian gland of female mice were also considered to be related to treatment.
Exposure to p-chloro-α,α,α-trifluorotoluene caused increased incidences of nonneoplastic lesions in the lung and liver of male and female rats and mice, in the nose of male rats, in the adrenal medulla and uterus of female rats, in the forestomach of male and female mice, and in the larynx in male mice. Exposure to p-chloro-α,α,α-trifluorotoluene caused increased severity of nonneoplastic lesions in the kidney of male rats.
National Toxicology Program (NTP). 2018. NTP Technical report on the toxicology and carcinogenesis studies of p-Chloro-α,α,α-trifluorotoluene (CASRN 98-56-6) in Sprague Dawley (Hsd:Sprague Dawley SD) rats and B6C3F1/N mice (inhalation studies). Research Triangle Park, NC: National Toxicology Program. Technical Report 594. https://doi.org/10.22427/NTP-TR-594