p-Nitrobenzoic acid is produced in large volumes for organic synthesis and as an intermediate in the manufacture of pesticides, dyes, and industrial solvents. Groups of male and female F344/N rats and B6C3F1 mice were exposed to p-nitrobenzoic acid (>99% pure) in feed for 14 days, 13 weeks, or 2 years for toxicity and carcinogenicity studies. Genetic toxicology studies were conducted in in vitro assays with Salmonella typhimurium and cultured Chinese hamster ovary cells, and in studies of erythrocyte micronucleus formation in mice in the 13-week study.
Fourteen-day study in rats
Groups of five male and five female rats were given 0, 2,500, 5,000, 10,000, 20,000, or 40,000 ppm p-nitrobenzoic acid in feed for 14 days. All rats survived until the end of the study. Male and female rats given 20,000 and 40,000 ppm lost weight. The final mean body weights of 10,000, 20,000, and 40,000 ppm males were 82%, 60%, or 52% that of the controls, and the final mean body weights of 10,000, 20,000, and 40,000 ppm females were 87%, 68%, and 65% that of the controls. There were no clinical findings that were characteristic of organ-specific toxicity.
Absolute and relative spleen weights were significantly increased in rats exposed to 10,000, 20,000, and 40,000 ppm. There were decreases in erythrocyte count and hemoglobin and hematocrit values and increases in reticulocyte count, nucleated erythrocytes, and methemoglobin concentration that were most pronounced in the 20,000 and 40,000 ppm groups. Congestion of the spleen occurred in 10,000 ppm males and in 20,000 and 40,000 ppm females. Hypertrophy of the follicular epithelium of the thyroid gland was present in male and female rats exposed to 10,000, 20,000, or 40,000 ppm p-nitrobenzoic acid, while follicular hyperplasia was observed in the 40,000 ppm males and females. Atrophy of the testis was observed in 20,000 and 40,000 ppm males. Other lesions observed in 20,000 and 40,000 ppm rats included atrophy of the thymus in males and atrophy of the ovary, bone marrow, and thymus in females.
Fourteen-day study in mice
Groups of five male and five female mice were given 0, 2,500, 5,000, 10,000, 20,000, or 40,000 ppm p-nitrobenzoic acid in feed for 14 days. Three males and two females given 40,000 ppm died during the study. All other animals survived until the end of the study. Male mice given 20,000 and 40,000 ppm and females given 20,000 ppm lost weight. Mean body weight gains of 20,000 and 40,000 ppm males and 10,000, 20,000, and 40,000 ppm females were significantly lower than those of the controls. There were no clinical findings related to organ-specific toxicity although lethargy and ataxia were observed in 40,000 ppm mice.
Relative liver weights were significantly increased in 20,000 and 40,000 ppm males and females and in 10,000 ppm females. Absolute and relative thymus weights of 20,000 and 40,000 ppm males and of 10,000, 20,000, and 40,000 ppm females were reduced. No significant differences in hematology parameters occurred in exposed mice. Testicular degeneration was observed in three 20,000 ppm and two 40,000 ppm males. Bone marrow hemorrhage and atrophy occurred in 40,000 ppm females.
Thirteen-week study in rats
Groups of 10 male and 10 female rats were given 0, 630, 1,250, 2,500, 5,000, or 10,000 ppm pnitrobenzoic acid in feed for 13 weeks resulting in approximate daily doses of 40, 70, 160, 310, or 660 mg/kg to males and 40, 80, 170, 340, or 680 mg/kg to females. All rats survived until the end of the study. Mean body weight gains and final mean body weights were significantly less than those of the controls in 2,500, 5,000, and 10,000 ppm males and in 5,000 and 10,000 ppm females. There were no clinical findings related to organ-specific toxicity.
Differences in spleen weights and hematology parameters characteristic of regenerative anemia were observed in males and females, primarily in groups given 10,000 ppm. The absolute and relative spleen weights were significantly increased in 10,000 ppm males and females and the relative spleen weights were significantly increased in 5,000 ppm males and females. Methemoglobin, Heinz bodies, and reticulocyte counts were increased and erythrocyte counts, hemoglobin, and hematocrit values were decreased in 10,000 ppm males and females.
Congestion, pigmentation, and accumulation of macrophages in the spleen and pigmentation in the kidney occurred in 2,500, 5,000, and 10,000 ppm males. Congestion and pigmentation of the spleen occurred in 10,000 ppm females. A yellowish brown pigment (hemosiderin) in the spleen and kidney was associated with hemolytic anemia. Mild cytoplasmic hyaline droplet accumulation was present in renal tubule epithelial cells in 10,000 ppm males while karyomegaly was present in male and female rats exposed to 2,500, 5,000, and 10,000 ppm p-nitrobenzoic acid. A chemical-related testicular lesion, consisting of atrophy of the seminiferous tubules, occurred in 10,000 ppm males.
Thirteen-week study in mice
Groups of 10 male and 10 female mice were given 0, 1,250, 5,000, 10,000, or 20,000 ppm pnitrobenzoic acid in feed for 13 weeks resulting in approximate daily doses of 170, 330, 670, 1,900, or 4,000 mg/kg body weight to males and 240, 460, 970, 2,500, or 4,900 mg/kg to females. All mice survived until the end of the study, except one 1,250 ppm female that was killed accidentally. Final mean body weights and mean body weight gains of all exposed males and of 5,000, 10,000, and 20,000 ppm females were significantly lower than those of the controls. No clinical findings or differences in organ weights or histopathology related to organ-specific toxicity were observed in exposed mice.
Two-year study in rats
Groups of 60 male and 60 female rats were given 0, 1,250, 2,500, or 5,000 ppm p-nitrobenzoic acid in feed for 2 years. Ten males and 10 females from each exposure group were evaluated at 15 months.
Survival, body weights, feed consumption, and clinical findings
Two-year survival rates of 1,250 and 2,500 ppm males were similar to that of the controls. Two-year survival of 5,000 ppm males was marginally greater than that of the controls and was attributed in part to a decrease in the severity of nephropathy and a decrease in the incidence of mononuclear cell leukemia. Survival of exposed females was similar to that of the controls. Mean body weights of 5,000 ppm males were 2% to 8% lower than those of the controls through week 80. Final mean body weights of exposed males were similar to that of the controls. Mean body weights of 5,000 ppm females were 2% to 9% lower than those of the controls during the first year of the study and were 10% to 16% lower during the second year of the study. Final mean body weights of exposed females were 97% (1,250 ppm), 92% (2;500 ppm), and 84% (5,000 ppm) that of the controls. Feed consumption by exposed males and females was similar to that by the controls. Dietary levels of 1,250, 2,500, or 5,000 ppm p-nitrobenzoic acid delivered approximately 50, 100, or 210 mg/kg body weight per day to males and 60, 125, or 250 mg/kg per day to females. There were no clinical findings attributable to organ-specific toxicity.
Pathology findings
There were increases in the incidences of clitoral gland adenoma and of clitoral gland adenoma or carcinoma (combined) (4/50, 14/49, 15/49, 15/50) in exposed females. The incidences of clitoral gland adenoma or carcinoma (combined) in the exposed groups (29% to 31%) exceeded the historical control mean incidence (11%) and range (2% to 21%) in female F344/N rats in recent 2-year NTP feed studies. The increased incidences of clitoral gland neoplasms were considered to be some evidence of carcinogenic activity in female rats exposed to p-nitrobenzoic acid. The incidences of hyperplasia of the clitoral gland in exposed females were marginally lower than that of the controls (10/50, 6/49, 6/ 49, 7/50).
There was a chemical-related decrease in the severity of nephropathy in male rats. Male rat kidneys were examined using both single and step-section analyses, and the incidences of renal tubule neoplasms were not statistically greater than those of the controls. Mild hyaline droplet accumulation was observed in renal tubule epithelial cells in 10,000 ppm males in the 13-week study, but this effect was not severe enough to lead to a chemical-related neoplastic response in the 2-year study as has been observed with other chemicals.
At the 15-month interim evaluation, hematologic parameters characteristic of a mild regenerative anemia and significant differences in spleen weights were noted in 5,000 ppm females. These differences included decreases in erythrocyte count, hemoglobin, and hematocrit, increases in spleen weights, and hemosiderin accumulation in splenic macrophages.
At 2 years, significant decreases in the incidences of mononuclear cell leukemia were observed in 5,000 ppm males and 2,500 and 5,000 ppm females (males: 29/50, 35/50, 26/50, 2/50; females: 17/50, 11/50, 3/50, 0/50). While the mechanism for this decrease is unknown, decreases in the incidence of mononuclear cell leukemia have also been observed in 2year studies with other amine/nitro compounds.
Two-year study in mice
Groups of 60 male and 60 female mice were given 0, 1,250, 2,500, or 5,000 ppm p-nitrobenzoic acid in feed for 2 years. Ten males and 10 females from each exposure group were evaluated at 15 months.
Survival, body weights, feed consumption, and clinical findings
Two-year survival rates of exposed mice were similar to those of the controls. Mean body weights of 5,000 ppm males were 6% to 12% lower than those of the controls after week 17, and mean body weights of 5,000 ppm females were 12% to 24% lower than those of the controls after week 16. The final mean body weight of 5,000 ppm females was 19% less than that of the controls; final mean body weights of males were similar to that of the controls. Feed consumption by exposed mice was similar to that by the controls. Dietary levels of 1,250, 2,500, or 5,000 ppm p-nitrobenzoic acid delivered approximately 150, 300, or 675 mg/kg per day to males and 170, 365, or 905 mg/kg per day to females. There were no clinical findings of organ-specific toxicity. No chemical-related effects on hematology parameters were noted at the 15-month interim evaluation.
Pathology findings
There were no increases or decreases in neoplasms in male or female mice that were considered to be related to chemical administration.
Genetic toxicology
p-Nitrobenzoic acid was mutagenic in Salmonella typhimurium strain TA100 with and without S9. No mutagenic activity was noted in strains TA98, TA1535, or TA1537, with or without S9. p-Nitrobenzoic acid induced sister chromatid exchanges and chromosomal aberrations in cultured Chinese hamster ovary cells in the absence of S9; with S9, results of both tests were negative. In vivo, no increase in micronuclei was observed in peripheral blood erythrocytes of male or female mice administered p-nitrobenzoic acid in dosed feed for 13 weeks.
Conclusions
Under the conditions of these 2-year feed studies, there was no evidence of carcinogenic activity of p-nitrobenzoic acid in male F344/N rats exposed to 1,250, 2,500, or 5,000 ppm. There was some evidence of carcinogenic activity of p-nitrobenzoic acid in female F344/N rats based on increases in the incidences of clitoral gland adenoma and of clitoral gland adenoma or carcinoma (combined). There was no evidence of carcinogenic activity of p-nitrobenzoic acid in male or female B6C3F1 mice exposed to 1,250, 2,500, or 5,000 ppm.
There were chemical-related decreases in the incidences of mononuclear cell leukemia in exposed male and female rats. p-Nitrobenzoic acid caused mild hematologic toxicity in female rats.