These studies were supported in part by funds from the Comprehensive Environmental Response, Compensation, and Liability Act trust fund (Superfund) by an interagency agreement with the Agency for Toxic Substances and Disease Registry, U.S. Public Health Service.
Toluene is used to back-blend gasoline, as a chemical intermediate, and as a solvent; 920 million gallons were produced in the United States in 1988. Toxicology studies were conducted by administering toluene (greater than 99% pure) in corn oil by gavage to groups of F344/N rats and B6C3F1 mice of each sex for 13 weeks or by whole-body inhalation exposure for 14 or 15 weeks. Toxicology and carcinogenesis studies were conducted by whole-body inhalation exposure of F344/N rats and B6C3F1 mice of each sex for 15 months or 2 years. Genetic toxicology studies were conducted in Salmonella typhimurium, mouse L5178Y lymphoma cells, and Chinese hamster ovary cells.
Thirteen-week gavage studies
All rats that received the top dose of 5,000 mg/kg died during the first week, and 8/10 male rats that received 2,500 mg/kg died early. The final mean body weight of male rats that received 2,500 mg/kg was 19% lower than that of vehicle controls. Relative liver, kidney, and heart (female only) weights for rats that received the higher doses were greater than those for vehicle controls. Necrosis of the brain and hemorrhage of the urinary bladder were seen at increased incidences in dosed rats.
All mice that received the top dose of 5,000 mg/kg died during the first week, and 40% of those that received 2,500 mg/kg died before the end of the 13-week gavage studies. The final mean body weight of males at 2,500 mg/kg was 16% lower than that of vehicle controls. At the higher doses, relative liver weights were increased for mice.
Fifteen-week and fourteen-week inhalation studies
Eight of 10 male rats exposed at the top exposure concentration of 3,000 ppm died during week 2. Final mean body weights of rats exposed at concentrations of 2,500 or 3,000 ppm were 14%-25% lower than that of controls. As in the gavage studies, the relative liver, kidney, and heart weights for rats exposed at the top two concentrations were increased compared with those for controls. No compound-related effects were seen on sperm; no adverse effects on the estrous cycle were observed.
Five of 10 male mice and all female mice exposed at 3,000 ppm and 70% of female mice at 2,500 ppm died during the first 2 weeks. Final mean body weights of all exposed groups were 7%-13% lower than those of controls. Relative liver weights for mice exposed at 625 ppm or higher, relative lung weights for mice exposed at 1,250 ppm or higher, and relative kidney weights for female mice exposed at 1,250 ppm or higher were greater than those for controls. Centrilobular hypertrophy of the liver was observed in all male mice exposed at 2,500 ppm and 70% of male mice exposed at 3,000 ppm. No effects on sperm or the estrous cycle were observed.
Fifteen-month and two-year inhalation studies
Long-term studies were conducted by exposing groups of 60 rats of each sex to 0, 600, or 1,200 ppm toluene by inhalation, 6.5 hours per day, 5 days per week. Groups of 60 mice of each sex were exposed at 0, 120, 600, or 1,200 ppm on the same schedule. Ten animals per group (except male mice) were removed for toxicologic evaluation after being exposed for 15 months. All other animals were exposed to toluene for 103 weeks.
In the 15-month inhalation studies, the incidences and severity of nonneoplastic lesions of the nasal cavity (degeneration of olfactory and respiratory epithelium and goblet cell hyperplasia) were increased in exposed rats. Minimal hyperplasia of the bronchial epithelium was seen in 4/10 female mice at 1,200 ppm. The severity of nephropathy was slightly increased in exposed female rats. No chemical-induced neoplasms were observed.
Two-year studies
Body weight and survival
Mean body weights of rats and mice were generally similar (yearly averages within 5%) among groups throughout the 2-year studies. No significant differences in survival were observed among rats or mice of either sex, although survival in all groups of male mice was lower than usual (male rats: control, 30/50; 600 ppm, 28/50; 1,200 ppm, 22/50; female rats: 33/50; 35/50; 30/50; male mice: control, 17/60; 120 ppm, 22/60; 600 ppm, 16/60; 1,200 ppm, 19/60; female mice: 30/50; 33/50; 24/50; 32/50). Scrotal, preputial, and penile lesions observed in male mice were associated with many of the early deaths and with animals killed in a moribund condition.
Nonneoplastic and neoplastic effects
Nephropathy was seen in almost all rats, and the severity was somewhat increased in exposed rats. A rare renal tubular cell carcinoma in a female rat and an equally uncommon sarcoma of the kidney in another female rat were seen in the 1,200-ppm exposure group. Erosion of the olfactory epithelium and degeneration of the respiratory epithelium were increased in exposed rats. Inflammation of the nasal mucosa and metaplasia of the olfactory epithelium were increased in exposed female rats. A rare squamous cell carcinoma of the nasal mucosa was seen in one female rat at 1,200 ppm. A squamous cell papilloma of the forestomach was observed in one female rat at 1,200 ppm, and a squamous cell carcinoma was observed in a second female rat at 1,200 ppm. No chemically related neoplasms were found in male rats, and the one nasal, two kidney, and two forestomach neoplasms observed in female rats were considered not to be associated with inhalation exposed to toluene.
For mice, no biologically important increases were observed for any nonneoplastic or neoplastic lesions.
Genetic toxicology
Toluene did not induce gene mutations in S. typhimurium strain TA98, TA100, TA1535, or TA1537 with or without exogenous metabolic activation. In the mouse lymphoma assay, toluene gave an equivocal response with and without exogenous metabolic activation. Toluene did not induce sister chromatid exchanges or chromosomal aberrations in Chinese hamster ovary cells in the presence or absence of exogenous metabolic activation.
Conclusions
Under the conditions of these 2-year inhalation studies, there was no evidence of carcinogenic activity for male or female F344/N rats exposed to toluene at concentrations of 600 or 1,200 ppm. There was no evidence of carcinogenic activity for male or female B6C3F1 mice exposed by inhalation to toluene at concentrations of 120, 600, or 1,200 ppm for 2 years.