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In a GLP compliant study performed according to OECD 471, metanilic acid was investigated using the Salmonella/microsome test for point mutagenic effects in doses of up to 5000 µg per plate en four Salmonella typhimurium LT2 mutants (Bayer AG 1992). These comprised the histidine-auxotrophic strains TA 1535, TA 100, TA 1537 and TA 98. Doses of up to and including 200 μg per plate did not cause any bacteriotoxic effects: Total bacteria counts remained unchanged and no inhibition of growth was observed. At higher doses, the substance had a weak strain-specific bacteriotoxic effect, so that this range could nevertheless be used for assessment purposes.

Evidence of mutagenic activity of the test substance was not seen. No biologically relevant increase in the mutant count, in comparison with the negative controls, was observed. Therefore, metanilic was considered to be non-mutagenic without and with S9 mix in the Salmonella/microsome test. The positive controls sodium azide, nitrofurantoin, 4-nitro-1,2-phenylene diamine and 2-aminoanthracene had a marked mutagenic effect, as was seen by a biologically relevant increase in mutant colonies compared to the corresponding negative controls.

Furthermore, 3 -aminobenzenesulfonic acid was not was not mutagenic in Salmonella typhimurium TA100, TA98, TA1535, TA1537 and Escherichia coli WP2 uvrA (NIHS 2008). No toxicity was observed up to a concentration of 5000 µg/plate with or without metabolic activation.

In a GLP complaint study according to the Japanese Guidelines for Screening Mutagenicity Testing of Chemicals 3 -Aminobenzenesulfonic acid was tested in the in vitro chromosomal aberration test in Chinese hamser CHL/IU cells (NIHS 2008, Morita 2012, Kusakabe 2002). In order to examine the metabolic activation of the test substance, the proliferating cells were treated with the test substance for 6 h in serum-free MEM with S9 mix (S9(+)), or without S9 mix (S9(-)), then cultured a further 18 h in the fresh MEM with serum. Moreover, the cells were also treated with the test substance for 24 and 48 h continuously in the absence of S9 mix. Duplicate cultures were used for each dose. For the test substance tested for 6 h with metabolic activation, the color of the culture medium shifted from orange or red (pH 7-8) to yellow (pH<6) just after applying the treatment solution, suggesting that CA was generated by the non-physiological culture conditions. Culture medium with low pH (pH<6.4) was known to induce structural CA in CHL/IU cells, but it could not be assumed without confirmation that structural CA in the current tests were caused by lowering pH. At the concentration of 0.41 mg/mL and 0.83 mg/mL short-term treatments with S9 mix resulted in 6.5% and 17% of the cells, respectively demonstrating structural aberrations (including gaps). However, it was suggested that acidic pH may have been related to the induction of chromosomal aberration.

Lowest concentration producing cytogenetic effects in vitro:

without metabolic activation (continuous treatment ): > 1.70 mg/mL

without metabolic activation (short-term treatment): > 4.4 mg/mL

with metabolic activation (short-term treatment): 0.41 mg/mL (clastogenicity)

3-Aminobezenesulfonic acid induced CAs with S9 mix (5.0 and 16.5% at 2.4 and 4.8 mM (0.83 mg/mL), respectively). The pH of the medium was 5.8 or 6.3 at the beginning of the 6-h treatment and 6.2 or 6.5 just after it at 4.8 or 2.4 mM. respectively. Relative cell growth, as measured by monolayer confluence, was about 100% or 90% at 4.8 or 9.5 mM, respectively. However, there were no metaphases at 9.5 mM. Without S9 mix, no CA induction was observed after 6- or 24-h treatment. The reason for this will be due to the short duration in Iow pH culture condition: the pH of the medium without S9 mix was 5.8 or 6.5 at the beginning of the 6-h treatment and 6.6 or 6.9 just after it at 4.8 or 2.4 mM, respectively. Initial pHs of the medium were similar, but the pHs after the treatment without S9 mix were higher than that with S9 mix. The window of the induction of CAs by Iow pH is narrower without S9 mix than that with S9 mix, generally. 3-Aminobenzenesulfonic acid does not possess any DEREK structural alerts. The CAs observed are considered as irrelevant as they were only seen at low pH and it is supported by all other available data. Thus the Ievel of concern is negligible.

 

To support this assumption and since no Micronucleus assay is available with 3-aminobenzenesulfonic acid a read-across with the structural analogue 4-Aminotoluene-3-sulfonic is applied (read across justification, see also attached document at chapter 13).

4-Aminotoluene-3-sulfonic was tested negative in a GLP Mouse Micronucleus test that was performed in accordance with OECD TG 474 (OECD SIDS 2003). The C57BL/6JfCD-1/Alpk mice received single doses orally of 3125 mg/kg and 5000 mg/kg 4-Aminotoluene-3-sulfonic. Though slight cytotoxicity was observed on polychromatic erythrocytes at 5000 mg/kg in males, it did not show a statistically significant increase on the ratio of micronucleated polychromatic erythrocytes at extended count.  

The evaluation of 1000 polychromatic erythrocytes 24 hours after dosing revealed that the mean percentage of polychromatic erythrocytes containing micronuclei was 38.1% and 34.9% in the group (female and male) at 3125 mg/kg, respectively, and 16.2% and 23.1% after 24 hours in animals (female and male) receiving the high dose. After 48 hours a mean percentage of 28.0% and 29.7% was observed in animals (female and male) receiving the high dose. At 72 hours de percentage for females and males receiving the high dose was 34.1% and 29.7% respectively.

Positive control gave the expected increase in the frequency of micronucleated polychromatic erythrocytes (MPE). Overall, there was no indication of a clastogenic activity.


Short description of key information:
3-Aminobenzenesulfonic acid was considered to be non-mutagenic in the Salmonella/microsome test, without and with S9 mix. 3-Aminobezenesulfonic acid induced CAs with S9 mix in the in vitro Chromosomal Aberration test. However the aberration is due to acidity and not to physiological DNA damage. No Micronucleus assay is available with 3-aminobenzenesulfonic acid. Read-across with the structural analogue 4-Aminotoluene-3-sulfonic is applied. Under the conditions of a chromosome aberration test with 4-Aminotoluene-3-sulfonic, the test substance is considered to be non-mutagenic in vivo.

Endpoint Conclusion:

Justification for classification or non-classification

Based on the available data, the substance does not need to be classified for genotoxicity according to Directive 67/548/EEC and according to the EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.