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There are several data available on the genotoxicity of 2-nitrotoluene in vitro and in vivo studies. The information in bacteria indicates that 2-nitrotoluene is not mutagenic in any of several strains of Salmonella typhimurium with or without metabolic activation enzymes (S9). However, the addition of norharman produced a positive result in the presence of S9 mix. On Bacillus subtilis, 2-nitrotoluene had a genotoxic effect. In cytogenetic tests on Chinese Hamster Ovary cells, 2-nitrotoluene increased the Sister Chromatid Exchange rate, being more pronounced in the presence of S9mix. In the Chromosomal Aberrations test the result was negative with or without S9 mix. There was an increase in polyploidy cells when 2-nitrotoluene was tested in cultures of Chinese hamster lung (CHL) cells in the absence of S9 mix. 2-nitrotoluene induced chromosomal aberrations in human lymphocytes. No induction of unscheduled DNA synthesis was observed in isolated rat spermatids, spermatocytes or hepatocytes. 2-nitrotoluene was found to be clastogenic when tested in larvae of mosquito Culex fatigans in vivo. However, for a Dominant Lethal test in the same species was negative. 2-Nitrotoluene induced adducts in haemoglobin and hepatic DNA in male Wels-Fohm rats dosed chronically 5 days a week for 12 weeks. However, Wistar rats dosed with a single dose of 2-nitrotoluene by oral gavage not induced DNA adducts, nevertheless formed haemoglobin adducts. 2-nitrotoluene did not induce a significant increase in the frequency of micronuclei in bone marrow polychromatic erythrocytes of male rats or male mice when administered by intraperitoneal injection. Results of a peripheral blood micronucleus test were equivocal for male mice and negative for female mice administered o-nitrotoluene in feed for 13 weeks. Positive results were found in the UDS test for both male and female rats administered 2- nitrotoluene. Males were more sensitive to genotoxicity of 2-nitrotoluene. A sex difference in biliary excretion may explain the sex difference in the genotoxicity of 2-nitrotoluene In addition, 2NT did not induce DNA repair in germ-free animals, whereas DNA repair was induced in Charles River Altered Schaedler Flora-associated animals. Male and female F344 rats were shown to have similar populations of intestinal bacteria; however at the doses used, females were resistant to the genotoxic action of 2NT. These results indicate the obligatory role of intestinal bacteria in the metabolic activation of 2NT, showing that the genotoxic potential of 2-NT is dependent upon the sex of the animal under study. Gene mutations inras, p53 and ß-catenin genes were observed in hemangiosarcomas from B6C3F1mice exposed to 2-nitrotoluene, for 2 years, in feed. These in vivo data suggest 2- nitrotoluene is metabolized to mutagenic intermediates and that could be the reason why most of the in vitro genotoxicity tests were negative. 2-nitrotoluene has a number of potentially active metabolites that could account for the mutation profile observed in these tumours, as described in the proposal pathway for bioactivation. In conclusion, 2-nitrotoluene is mutagenic in somatic cells. In addition, it reaches the germ cells since toxicity was observed in testis and epididymis.

Short description of key information:
Bacterial studies
2-nitrotoluene has been tested for genotoxicity in several studies in bacteria.
Chiu et al. (1978) tested 2-nitrotoluene for mutagenicity in Salmonella typhimirium TA98 and TA100 without metabolic activation, at 10, 1 and 0.1 µmol/plate. The results were negative.
Tokiwa et al. (1981) investigated mutagenicity of 2-nitrotoluene in strains TA98 and TA100 with and without metabolic activation, using liver S9 fraction prepared from rats treated with Aroclor 1254. The protein concentration of S9 fraction was 39 mg/ml. The S9 fraction was used at 0.15 ml/plate. The doses of 2-nitrotoluene were 1000 and 100 µg/plate. The results were negative.
Miyata et al. (1981), using Salmonella typhimirium, strains TA92 TA94, TA98, TA100, TA1535 and TA1537, tested 2-nitrotoluene in the absence and in the presence of a hepatic 9000xg supernatant (S9) containing NADPH-generating system. The doses were 30, 100, 300, 1000 and 3000 µg/plate. The results were negative, 1000 and 3000 µg/plate doses were toxic in the absence of S9 mix, 3000 µg/plate was toxic in the presence of S9 mix.
The mutagenicity of 2-nitrotoluene was investigated by Spanggord et al. (1982) with five strains of Salmonella typhimirium, TA98, TA100, TA1535, TA1537 and TA1538. The results were negative with and without metabolic activation. The range tested was 10-5000 µg.
Haworth et al. (1983), tested 2-nitrotoluene in the Ames test, using preincubation, strains TA98, TA100, TA1535 and TA1537 of Salmonella typhimirium. Liver S9 fractions were prepared from male Sprague-Dawley rats and male Syrian hamsters that were injected, i.p., with Aroclor 1254. The concentrations of 2-nitrotoluene used were 3.3, 10.0, 33.0, 100.0 and 333.0 µg/plate. The results were negative, 333.0 µg/plate resulted toxic for the strains TA 100, TA 1537 and TA 98 without S9 mix.

Suzuki et al. (1983), studied the mutagenicity of mono-nitrobenzene derivatives, among them, 2-nitrotoluene, in order to evaluate the effect of norharman, a substance found in tobacco tar.

The mutagenicity assay used wasSalmonella typhimirium,strains TA 98 and TA100. The concentrations used were 100, 200 and 300 µg/plate, with and without S9 mix and 200 µg/plate of norharman. The result was positive for the ortoisomers of every nitro-compound, among them 2-nitrotoluene, in the presence of norharman when Salmonella typhimirium TA 98 with S9 mix was used. This study is important from the viewpoint of environmental carcinogenesis, since norharman is found in tobacco tar and nitro aromatics are found in atmosphere particulate matter and in the exhaust gas from diesel engines.

Nohmiet al (1984) tested the mutagenicity of 2-nitrotoluene with th eSalmonella/microsome test according to the method of with preincubation during 20 min. at 37ºC.

S. typhimurium TA98 and TA100 were used, the concentration of 2-nitrotoluene was 10 mM.

The result was negative, both with and without metabolic activation.

Mutagenicity of 2-nitrotoluene was tested by pre-incubation method using Salmonella Typhimirium TA100 and TA98 strains with and without S9 mix. The result was negative (Kawai et al.,1987).

Negative results were found when 2-nitrotoluene was tested inS. typhimirium TA100 and TA98 strains with and without S9 at dose levels of 0.5-5.0mM per plate (Gupta et al., 1987).

A mutagenicity assay was carried out with 2-nitrotoluene according to the guidelines of Ministry of Labour (JETOC, 1996). Salmonella typhimiriumstrains TA98, TA100, TA102, TA104, TA1535, TA1537 and TA1538 were obtained from Dr. Ames and Escherichia coliWP2uvrA and WP2uvrA/pKM101 were from Dr. Ishizawa. DMSO was added to give a final concentration of 2.8% (v/v). Metabolic activation (S9 mix) was obtained using sodium phenobarbital and 5,6-benzoflavone as an inducer of the rat metabolic activation system. A preincubation procedure was performed. The concentrations used were 0.0763, 0.305, 1.22, 4.88, 19.5, 78.1, 313, 1250 and 5000µg/plate. The mutagenicity results for 2- nitrotoluene in bacterial test was negative, both Salmonella typhimiriumandEscherichia coli.

In the recombination assay onBacillus subtilis, strains H17 and M45, without addition of S9 mix, 2-nitrotoluene showed a weakly effect at 5ml/plate (Shimizu and Yano, 1986).

2-nitrotoluene was tested for its ability to inactivate Bacillus subtilis transforming DNA.

Incubation with 5 and 10 mM of 2-nitrotoluene dissolved in DMSO, during 60 min at 37ºC, did not inactivate transforming DNA (Nohmi et al., 1984).


NTP, 1992, in vivo testing, rat, mammalian cell study

Groups of 6 male F344/ rats/dose and 6 female F344 rats/dose were administered oral doses of o-Nitrotoluene in corn oil via gavage; 0; 100; 200; 500 mg/kg for males and 0; 200; 500 and 750 mg/kg bw for females, respectively, according to a method similar to OECD guideline 486 (total of 90 cells scored/ dose, positive control not listed in guideline) Perfusion of liver and preparation of hepatocytes was performed 12h and 24h after gavage.

o-Nitrotoluene induced DNA damage in mammalian liver cells in vivo (male and female F344 rats) that could be repaired by unscheduled DNA synthesis in vitro; Male rats (>= 100 mg/kg bw), Female rats (>= 200 mg/kg bw).

Endpoint Conclusion: Adverse effect observed (positive)

Justification for classification or non-classification

Harmonised classification:

It is reasonable to assume that a somatic cell mutagen also has the potential to cause mutations in germ cells. The likelihood of mutagenicity occurring in the germ cells in vivo is determined by the toxicokinetics of the substance and its ability to reach the target tissues in sufficient amounts to elicit the effect. Therefore, although there is not direct evidence relating to mutagenicity in the germ cells, in view of the above exposed, the substance is classified in Category 1B (H340) according to the Regulation (EC) No. 1272/2008 (CLP).