National Toxicology Program

TOX-33
Toxicity Studies of 2-Chloronitrobenzene (CAS No. 88-73-3) and 4-Chloronitrobenzene (CAS No. 100-00-5)Administered by Inhalation to F344/N Rats and B6C3F₁ Mice

2-Chloronitrobenzene and 4-chloronitrobenzene are oily yellow solids thatare used primarily as chemical intermediates in the production of dyes,lumber preservatives, drugs, and photographic chemicals. Although thesechemicals are solids at room temperature, the vapor pressures of thesechemicals are sufficiently high to result in significant inhalationexposure. Toxicity studies of 2-chloronitrobenzene and4-chloronitrobenzene were performed by exposing male and female F344/N ratsand 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 forhistopathology, clinical chemistry (rats), hematology (rats), andreproductive system effects. In separate studies, the dermal absorption ofthe chemicals was compared, and the absorption, distribution, metabolism,and excretion were partially characterized following oral administration tomale F344/N rats. 2-Chloronitrobenzene and 4-chloronitrobenzene were alsoadministered orally to CD-1® Swiss mice for evaluation of reproductive anddevelopmental toxicity. Genetic effects were evaluated in Salmonellatyphimurium, in Chinese hamster ovary cells, and in Drosophilamelanogaster.

The highest exposure concentrations used in the 2 week and 13 week studieswere limited by technical factors in vapor generation to 18 ppm (115.2mg/m³) for 2-chloronitrobenzene and 24 ppm (153.6 mg/m³) for4-chloronitrobenzene. Other concentrations were 0, 1.1, 2.3, 4.5, and9 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) for4-chloronitrobenzene.

In 2-week studies with 2-chloronitrobenzene, all rats survived to the endof the study. One of five male mice exposed to 18 ppm died, but weightgains of exposed rats and mice were not affected. Exposed rats and micehad concentration-related increases in liver weights, and spleen weightswere increased in rats and mice exposed to 18 ppm. Histopathologicfindings in rats were limited to hemosiderin deposition in the liver andspleen at the highest exposure concentration. Exposed mice, primarilythose in the 18 ppm groups, had coagulative necrosis, hepatocytomegaly,and granulomatous inflammation in the liver. Splenic changes includingincreased hematopoietic cell proliferation and hemosiderin depositionoccurred at concentrations as low as 4.5 ppm.

In 13-week studies with 2-chloronitrobenzene, all rats survived to the endof the study; 2 of 10 male mice exposed to 18 ppm died. Body weight gainsof exposed rats and mice were similar to or somewhat higher than those ofthe respective controls. Methemoglobinemia occurred in rats and resultedin a normocytic, normochromic anemia that became responsive by the end ofthe study. Exposed rats and mice had increased liver weights, but theseincreases were not as great as those seen in the 2-week studies. Spleenweights were increased in exposed rats. Histopathologic changes in ratsincluded increased basophilia of centrilobular hepatocytes, pigmentationand regeneration of the proximal convoluted tubules of the kidney, andhyperplasia of the nasal cavity respiratory epithelium. In mice,hepatocellular necrosis, cytomegaly, mineralization, and chronicinflammation 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 tothe end of the studies. Body weight gains of exposed rats were similar tothose of the controls; body weight gains of exposed mice were greater thanthose of the controls. Liver and spleen weights were increased in exposedrats and mice. In rats, histopathologic changes in the liver werelimited to an increase in hemosiderin pigment in Kupffer cells. Thespleens of exposed rats were congested and had increased hematopoieticactivity and hemosiderin deposition. Kidneys of exposed male rats hadlesions consistent with hyaline droplet nephropathy. The proximalconvoluted tubules of exposed female rats contained hemosiderin.Microscopic changes in exposed mice primarily involved increasedhematopoietic activity in the spleen and hemosiderin pigmentation in thespleen, liver, and proximal convoluted tubules in the kidney.

In 13-week studies with 4-chloronitrobenzene, there were no deaths thatwere clearly related to exposure to 4-chloronitrobenzene. Body weightgains of exposed rats and mice were either equal to or greater than thoseof the controls. A more severe methemoglobinemia developed in ratsexposed to 4-chloronitrobenzene than occurred in rats exposed to2-chloronitrobenzene, and this methemoglobinemia resulted in a responsivemacrocytic, hyperchromic anemia. Spleen weights were markedly greater inexposed rats and mice than in controls. In exposed rats, lesions in thespleen, liver, and kidney were similar to those described for the 2-weekstudy. Additionally, increased hematopoietic cell proliferation in bonemarrow, histiocytic hyperplasia in mediastinal lymph nodes, testicularatrophy, and chronic inflammation of the harderian gland occurred inexposed rats. In exposed mice, microscopic changes in the spleen and liverwere similar to those noted in the 2-week study. Additional lesionsincluded increased hematopoiesis and hemosiderin deposition in the bonemarrow of exposed males and females and squamous cell hyperplasia of theforestomach epithelium in female mice.

In reproductive system assessments, there was evidence of decreasedspermatogenesis in rats exposed to either 2- or 4-chloronitrobenzene. Inmice, effects were limited to a decrease in sperm motility in males exposedto 2-chloronitrobenzene and an increase in estrous cycle length in femalesexposed to 4-chloronitrobenzene. In continuous breeding studies, aprogressive decrease in fertility was noted in CD-1® Swiss mice receiving 4-chloronitrobenzene by oral gavage; fertility was not affected in miceadministered 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 somewhathigher 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 Salmonellatyphimurium with S9 activation. In addition, both compounds induced sisterchromatid exchanges and chromosomal aberrations in Chinese hamster ovarycells; requirements for S9 activation varied among testing laboratories.Neither compound induced sex-linked recessive lethal mutations in germcells 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 damageto red blood cells, leading to a regenerative anemia and a recognizedspectrum of tissue damage and changes secondary to erythrocyte injury. Inaddition, numerous other lesions that were considered primary toxic effectsoccurred following exposure. These included renal hyaline dropletaccumulation and testicular atrophy in male rats exposed to 4-chloronitrobenzene and hyperplasia of the respiratory epithelium in ratsexposed to 2-chloronitrobenzene. A no-observed-adverse-effect-level(NOAEL) for rats was not achieved, as increases in methemoglobin andhistopathologic changes occurred at exposure concentrations as low as 1.1ppm for 2-chloronitrobenzene and 1.5 ppm for 4-chloronitrobenzene in the13-week studies. The NOAEL for histopathologic injury in mice was 4.5 ppmfor 2-chloronitrobenzene and 6 ppm for 4-chloronitrobenzene.

2-Chloronitrobenzene Synonyms: o-Cloronitrobenzene; 2-chloro-1-nitrobenzene; ONCB
4-chloronitrobenzene Synonyms: p-Chloronitrobenzene; 4-chloro-1-nitrobenzene; PNCB

Report Date: July 1993

Growth & Survival Curves for NTP 13-Week Toxicity Studies

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