NTP Technical report on the toxicity studies of ortho-, meta-, and para- Nitrotoluenes (CAS Nos. 88-72-2, 99-08-1, 99-99-0) Administered in Dosed Feed to F344/N Rats And B6C3F1 Mice.

Nitrotoluenes are high production volume chemicals used in the synthesis of agricultural and rubber chemicals and in various dyes. Because of differences in the metabolism of the 3 isomers and their capability to bind to DNA, comparative toxicity studies of o-, m-, or p-nitrotoluene were conducted in F344 rats and B6C3F1 mice. Animals were evaluated for histopathology, clinical pathology, and toxicity to the reproductive system. The nitrotoluenes were also studied in several in vitro and in vivo assays for genetic toxicity. In 14-day studies, o-nitrotoluene, m-nitrotoluene, or p-nitrotoluene was administered in the feed to male and female rats and mice at concentrations ranging from 388 to 20000 ppm (5 animals/chemical/species/sex/dose). There were no effects on survival or clinical signs of toxicity in these studies, although animals at the higher doses showed decreases in body weight gains relative to controls. In the 13-week studies, o-, m-, or p-nitrotoluene was given to male and female rats and mice (10 animals/chemical/species/ sex/dose) in the feed at concentrations between 625 and 10000 ppm. The estimated daily doses based on measures of feed consumption were 40 to 900 mg nitrotoluene/kg body weight/day for rats and 100 to 2000 mg/kg/day for mice and were similar for each of the 3 isomers when compared for each dietary level/sex/species. There were no effects on survival in any of the studies, and clinical signs of toxicity were limited to decreases in feed consumption. Decreased body weight gains occurred in dosed rats and mice in all studies at the higher dose levels and were most pronounced in rats receiving o-nitrotoluene. In rats, histopathologic analyses after 13 weeks of dosing showed toxicity to kidney, spleen, and testis in animals receiving any of the 3 isomers, and toxicity to the liver and mesothelium in male rats given o-nitrotoluene. Kidney toxicity observed in male rats was characterized by the presence of hyaline droplets in tubular epithelial cells, attributed to an increase in the level of alpha-2μ-globulin. Pigment, possibly lipofuscin, and karyomegaly in the p-nitrotoluene study were present in the renal tubular epithelium of dosed male and female rats. In the spleen of treated male and female rats, there was a mild increase in hematopoiesis, hemosiderin deposition, and/or congestion; this effect was most severe with the para-isomer, followed by the ortho- and then the meta-isomer. Administration of o-, m-, or p-nitrotoluene impaired testicular function of the rat, shown by degeneration of the testis and reduction in sperm concentration, motility, and spermatid number. All 3 isomers increased the length of the estrous cycle in rats. Hepatic toxicity was characterized by cytoplasmic vacuolization and oval cell hyperplasia and by an increase in the level of serum bile acids, SDH, and ALT activities in male rats given o-nitrotoluene. There was no histopathologic evidence for liver toxicity in male or female rats with the m- or p-isomers, or in female rats with the o-isomer; but evidence of liver injury was observed in these groups, indicated by increases in relative liver weights and elevations in bile acids and liver enzymes in serum. Mesotheliomas of the tunica vaginalis were observed in 3/10 male rats receiving o-nitrotoluene at 5000 ppm, and mesothelial cell hyperplasia was observed in 2/10 male rats receiving o-nitrotoluene at 10000 ppm. The only histopathologic evidence for toxicity in mice in the 13- week studies occurred in the olfactory epithelium in mice receiving o-nitrotoluene, where the chemical caused degeneration and metaplasia. No liver lesions were noted in mice, but the 3 isomers caused increases in relative liver weights. There was no toxicity to the reproductive system in male or female mice treated with any of the nitrotoluene isomers. The 3 nitrotoluene isomers were not mutagenic in Salmonella typhimurium strains TA100, TA1535, TA1537, and TA98. Only p-nitrotoluene induced chromosomal aberrations in cultured Chinese hamster ovary (CHO) cells, and this required metabolic activation. Sister-chromatid exchanges were increased in CHO cells following exposure to each isomer; the requirement for metabolic activation varied. Only p-nitrotoluene was studied in the mouse lymphoma L5178Y test; it caused mutations with metabolic activation. Unscheduled DNA synthesis (UDS) was increased in in vitro incubations of hepatocytes isolated from both sexes of rats and mice after receiving a single in vivo oral dose of o-nitrotoluene. UDS was not increased in a similar study with male rats given m- or p-nitrotoluene. o-Nitrotoluene also induced s-phase DNA synthesis in hepatocytes of rats but not in those of mice. In summary, the 3 nitrotoluene isomers were toxic to the kidney, spleen and/or reproductive system in rats; o-nitrotoluene also caused lesions in the liver of male rats. No treatment-related lesions were noted in mice except with o-nitrotoluene where olfactory epithelium degeneration occurred. The increase in relative liver weights and the increase in UDS in liver indicate that all 3 isomers affected the liver of female rats and of male and female mice, even though histopathologic lesions were not observed. In general, the extent of the toxicity was most severe with the o-isomer in both rats and mice. o-Nitrotoluene was carcinogenic in male rats in 13-week studies, based on the occurrence of mesothelioma and mesothelial cell hyperplasia in dosed groups. Synonyms: o-NT, 2NT, 2-nitrotoluene, 2-methylnitrobenzene, 2-nitrotoluol; m-NT, 3NT, 3-nitrotoluene, 3-methylnitrobenzene, 3-nitrotoluol; p-NT, 4NT, 4-nitrotoluene, 4-methylnitrobenzene, 4-nitrotoluol.