4-nitrotoluene-2-sulphonic acid

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

negative with and without metabolic activation (Ames, Chromosome aberration, micronucleus)

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

In-vitro Studies: Bacterial systems

In an Ames-Test (GINC, Japan) a dose-dependent tendency in the increase of mutated colonies was observed in strain TA100, TA 98 and TA1537 without S9 mix and in strain TA100 with S9 mix. In the trials which were repeated twice, the number of mutated colonies in the TA1535 group was 1.5 to 2 times higher than that of the solvent control, but it could not be determined to be definitely positive. A confirmative test was carried out with regard to the TA1535 strains with and without S9 mix to confirm its reproducibility, but a clear mutagenicity-inducing effect was not observed. On the other hand, the positive control substance induced two times or more of mutated colonies in each tester strain, compared to the solvent control group. In addition, remarkable changes such as precipitation were not observed during the tests.

A second study was conducted and further to the strains suggested in the OECD Guideline 471, nitroreductase deficient strains were tested. The role of nitroreduction in any test article related mutagenic response was assessed by using the nitroreductase deficient strains TA98NR and TA100NR alongside parent nitroreductase competent strains TA98 and TA100. The S. typhimurium strains TA98NR and TA100NR are variants of strains TA98 and
TA100 respectively that are deficient in “classical” nitroreductase activity. TA98NR is also deficient in “supplementary” nitroreductase activity. Comparison of the responses seen between the strains with and without the classical nitroreductase pathway was used to determine whether the bacterial nitroreduction enzymes play a significant role in any mutagenicity of the test compound.

The substance was assayed for mutation in seven histidine-requiringstrains (TA98, TA100, TA1535, TA1537, TA102, TA98NR and TA100NR) of Salmonella typhimurium, both in the absence and in the presence of metabolic, in two separate experiments. All substance treatments were performed using formulations prepared in DMSO.

Mutation Experiment 1 treatments of all the tester strains were performed using a pre-incubation treatment methodology in the absence and in the presence of S-9, using final concentrations at 5, 16, 50, 160, 500, 1600 and 5000 μg/plate. Following these treatments, evidence of cytotoxicity was observed at 5000 μg/plate in the absence and presence of S-9 in all strains except TA1537 (where no evidence of cytotoxicity was observed), and also at 1600 μg/plate in strain TA102 in the presence of S-9.

Mutation Experiment 2 treatments of all the tester strains were performed in the absence and in the presence of S-9 using a standard plate incorporation treatment methodology. The maximum test concentration of 5000 μg/plate was retained for all strains. Narrowed concentration intervals were employed covering the range 75-5000 μg/plate.  Following these treatments, evidence of cytotoxicity was again observed but was limited to 5000 μg/plate treatments of strain TA102 in the absence and presence of S-9.

No precipitation of test article was observed on any test plates in either experiment. Vehicle and positive control treatments were included for all strains in each experiment. The mean numbers of revertant colonies were comparable with acceptable ranges for vehicle control treatments, and the responses with the positive control treatments were sufficient to confirm the correct strain and assay functioning.

Following substance treatments of all the test strains in the absence and presence of S-9, no notable or concentration-related increases in revertant numbers were observed. It was concluded that the substance did not induce mutation in seven histidine-requiring strains (TA98, TA100, TA1535, TA1537, TA102, TA98NR and TA100NR) of Salmonella typhimurium when tested under the conditions of this study.

In-vitro Studies: Mammalian cell gene mutation test

An HPRT Locus Assay (BASF SE, 2010) with CHO cells was performed with 4-nitrotoluene-2-sulphonic acid. In the study the test substance did not lead to a relevant increase in the number of mutant colonies either without S9 mix or after the addition of a metabolizing system in two experiments performed independently of each other. The mutant frequencies at any concentration were within or slightly above the range of the concurrent negative control values and within the range of the historical negative control data.

In-vitro studies: Chromosomal aberrration and micronucleus tests

In the in vitro Chromosomal Aberration Test in Chinese hamster cells with 4-nitrotoluene-2 -sulphonic acid, no tendency of inducing the chromosomal structural aberration and ploidy cells was observed in either dose (GINC, Japan). On the other hand, a remakable induction of chromosomal structural aberrration was recognized in cells treated with MMC (positive control). In case of the test substance group, no tendency of inducing the chromosomal structural aberrration and ploidy cells was observed in either dose in the absence and presence of S9 mix. On the other hand, a remarkable induction of chromosomal structural aberration was recognized only in the presence of S9 mix for the cells treted with CP (positive crontrol). In each test system of continous treatment and shortterm treatment, a decrease in pH value for the culture fluid was recognized in all test doses, it has returned to the neutral range at the exchange of culture fluid or the completion of culture.

Short description of key information:
4-nitrotoluene-2-sulphonic acid showed a tendency toward mutagenicity in an Ames test in Salmonella typhimurium strains TA100, TA98 and TA1537 without an exogenous metabolic activation system, and in strain TA100 with an exogenous metabolic activation system (GINC, Japan).

A second study with the same strain tested and also NR deficient TA 100 and TA98 resulted as negative with and without metabolic activation.

4-nitrotoluene-2-sulphonic acid did not induce structural chromosomal aberrrations or polyploidy in CHL cells, with or without an exogenous metaboic activation system (GINC, Japan).
4-Nitrotoluene-2sulphonic acid was not mutagenic in the HPRT locus assy in CHO Cells (BASF SE, 2010).

Justification for classification or non-classification

Based on the available studies no classification as per the CLP Regulation EC No.1272/2008 is proposed.