Abstract for TOX-33

Toxicity Studies of 2-Chloronitrobenzene and 4-Chloronitrobenzene Administered by Inhalation to F344/N Rats and B6C3F1 Mice


  • 2-Chloronitrobenzene (CASRN 88-73-3)
  • 4-Chloronitrobenzene (CASRN 100-00-5)

Report Date: July 1993

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2-Chloronitrobenzene and 4-chloronitrobenzene are oily yellow solids that are used primarily as chemical intermediates in the production of dyes, lumber preservatives, drugs, and photographic chemicals. Although these chemicals are solids at room temperature, the vapor pressures of these chemicals are sufficiently high to result in significant inhalation exposure. Toxicity studies of 2-chloronitrobenzene and 4-chloronitrobenzene were performed by exposing male and female F344/N rats and B6C3F1 mice to the chemicals by whole-body inhalation 6 hours per day, 5 days per week, for 2 weeks or 13 weeks. Animals were evaluated for histopathology, clinical chemistry (rats), hematology (rats), and reproductive system effects. In separate studies, the dermal absorption of the chemicals was compared, and the absorption, distribution, metabolism, and excretion were partially characterized following oral administration to male F344/N rats. 2-Chloronitrobenzene and 4-chloronitrobenzene were also administered orally to CD-1 Swiss mice for evaluation of reproductive and developmental toxicity. Genetic effects were evaluated in Salmonella typhimurium, in Chinese hamster ovary cells, and in Drosophila melanogaster.

The highest exposure concentrations used in the 2 week and 13 week studies were limited by technical factors in vapor generation to 18 ppm (115.2mg/m3) for 2-chloronitrobenzene and 24 ppm (153.6 mg/m3) for 4-chloronitrobenzene. Other concentrations were 0, 1.1, 2.3, 4.5, and 9 ppm (0, 7, 14.7, 28.8, and 57.6 mg/m3) for 2-chloronitrobenzene and 0,1.5, 3, 6, and 12 ppm (0, 9.6, 19.2, 38.4, and 76.8 mg/m3) for 4-chloronitrobenzene.

In 2-week studies with 2-chloronitrobenzene, all rats survived to the end of the study. One of five male mice exposed to 18 ppm died, but weight gains of exposed rats and mice were not affected. Exposed rats and mice had concentration-related increases in liver weights, and spleen weights were increased in rats and mice exposed to 18 ppm. Histopathologic findings in rats were limited to hemosiderin deposition in the liver and spleen at the highest exposure concentration. Exposed mice, primarily those in the 18 ppm groups, had coagulative necrosis, hepatocytomegaly, and granulomatous inflammation in the liver. Splenic changes including increased hematopoietic cell proliferation and hemosiderin deposition occurred at concentrations as low as 4.5 ppm.

In 13-week studies with 2-chloronitrobenzene, all rats survived to the end of the study; 2 of 10 male mice exposed to 18 ppm died. Body weight gains of exposed rats and mice were similar to or somewhat higher than those of the respective controls. Methemoglobinemia occurred in rats and resulted in a normocytic, normochromic anemia that became responsive by the end of the study. Exposed rats and mice had increased liver weights, but these increases were not as great as those seen in the 2-week studies. Spleen weights were increased in exposed rats. Histopathologic changes in rats included increased basophilia of centrilobular hepatocytes, pigmentation and regeneration of the proximal convoluted tubules of the kidney, and hyperplasia of the nasal cavity respiratory epithelium. In mice, hepatocellular necrosis, cytomegaly, mineralization, and chronic inflammation occurred in the liver, primarily in mice in the 18 ppm group, and hematopoietic activity in the spleen was increased.

In 2-week studies with 4-chloronitrobenzene, all rats and mice survived to the end of the studies. Body weight gains of exposed rats were similar to those of the controls; body weight gains of exposed mice were greater than those of the controls. Liver and spleen weights were increased in exposed rats and mice. In rats, histopathologic changes in the liver were limited to an increase in hemosiderin pigment in Kupffer cells. The spleens of exposed rats were congested and had increased hematopoietic activity and hemosiderin deposition. Kidneys of exposed male rats had lesions consistent with hyaline droplet nephropathy. The proximal convoluted tubules of exposed female rats contained hemosiderin. Microscopic changes in exposed mice primarily involved increased hematopoietic activity in the spleen and hemosiderin pigmentation in the spleen, liver, and proximal convoluted tubules in the kidney.

In 13-week studies with 4-chloronitrobenzene, there were no deaths that were clearly related to exposure to 4-chloronitrobenzene. Body weight gains of exposed rats and mice were either equal to or greater than those of the controls. A more severe methemoglobinemia developed in rats exposed to 4-chloronitrobenzene than occurred in rats exposed to 2-chloronitrobenzene, and this methemoglobinemia resulted in a responsive macrocytic, hyperchromic anemia. Spleen weights were markedly greater in exposed rats and mice than in controls. In exposed rats, lesions in the spleen, liver, and kidney were similar to those described for the 2-week study. Additionally, increased hematopoietic cell proliferation in bone marrow, histiocytic hyperplasia in mediastinal lymph nodes, testicular atrophy, and chronic inflammation of the harderian gland occurred in exposed rats. In exposed mice, microscopic changes in the spleen and liver were similar to those noted in the 2-week study. Additional lesions included increased hematopoiesis and hemosiderin deposition in the bone marrow of exposed males and females and squamous cell hyperplasia of the forestomach epithelium in female mice.

In reproductive system assessments, there was evidence of decreased spermatogenesis in rats exposed to either 2- or 4-chloronitrobenzene. In mice, effects were limited to a decrease in sperm motility in males exposed to 2-chloronitrobenzene and an increase in estrous cycle length in females exposed to 4-chloronitrobenzene. In continuous breeding studies, a progressive decrease in fertility was noted in CD-1 Swiss mice receiving 4-chloronitrobenzene by oral gavage; fertility was not affected in mice administered 2-chloronitrobenzene by oral gavage.

Percutaneous absorption of [14C]-2-chloronitrobenzene and [14C]-4-chloronitrobenzene was demonstrated in rats. For doses ranging from 0.65 to 65 mg/kg of either chemical, 33% to 40% of 2-chloronitrobenzene and 51% to 62% of 4-chloronitrobenzene were absorbed under nonocclusive conditions. Oral absorption was somewhat higher than dermal absorption for both chemicals, and metabolism was complicated, with over 20 unidentified metabolites isolated from urine of rats given either 2- or 4-chloronitrobenzene.

2-Chloronitrobenzene and 4-chloronitrobenzene were mutagenic in Salmonella typhimurium with S9 activation. In addition, both compounds induced sister chromatid exchanges and chromosomal aberrations in Chinese hamster ovary cells; requirements for S9 activation varied among testing laboratories. Neither compound induced sex-linked recessive lethal mutations in germ cells of male Drosophila melanogaster treated as adults or as larvae.

In summary, inhalation exposure of rats and mice to 2- or 4-chloronitrobenzene resulted in methemoglobin formation and oxidative damage to red blood cells, leading to a regenerative anemia and a recognized spectrum of tissue damage and changes secondary to erythrocyte injury. In addition, numerous other lesions that were considered primary toxic effects occurred following exposure. These included renal hyaline droplet accumulation and testicular atrophy in male rats exposed to 4-chloronitrobenzene and hyperplasia of the respiratory epithelium in rats exposed to 2-chloronitrobenzene. A no-observed-adverse-effect-level (NOAEL) for rats was not achieved, as increases in methemoglobin and histopathologic changes occurred at exposure concentrations as low as 1.1 ppm for 2-chloronitrobenzene and 1.5 ppm for 4-chloronitrobenzene in the 13-week studies. The NOAEL for histopathologic injury in mice was 4.5 ppm for 2-chloronitrobenzene and 6 ppm for 4-chloronitrobenzene.