Tetrasodium 4,4'-bis[[4-[(2-hydroxyethyl)amino]-6-(m-sulphonatoanilino)-1,3,5-triazin-2-yl]amino]stilbene-2,2'-disulphonate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Not mutagenic

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Not mutagenic

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

The substance under registration OB 3c-MSA is part of the Stilbene Fluorescent Whitening Agents category. Within the whole category none of the existing tests assessing the genetic toxicity potential arisen any concern for mutagenicity or genotoxicity.

All substances of the category were modelled with the OECD Toolbox and the provisional results about mutagenicity alerts were calculated for all members and their metabolites. The same alert was reported based on the Hacceptor-path3-Hacceptor. This alert explores the possibility that a chemical interacts with DNA and/or proteins via non-covalent binding, such as DNA intercalation or groove-binding (Snyder et al. 2006). Among the descriptors potentially accounting for non-covalent interactions, the present molecular framework representing two bonded atoms connecting two H bond acceptors (calculated with software Leadscope Enteprise 2.4.15-6) resulted in an increased sensitivity/specificity for what concerns the Micronucleus training set. Experimental tests both in vivo and in vitro demonstrate that this alert is not expressed in none of the substances of the group.

The substance under registration was tested for mutagenic effects (bacteria reverse mutation assay) on histidine auxotrophic mutants of Salmonella typhimurium. The investigations were performed with the following concentrations of the trial substance with and without microsomal activation: 25, 75 225, 675 and 2025 µg/0.1 ml. Since the results obtained in the experiments with Strain TA 98 were equivocal, additional experiments were performed on Strains TA 98 and TA 1538. In the experiments performed with and without microsomal activation, comparison of the number of back-mutant colonies in the controls and the cultures treated with the various concentrations of test item revealed no marked deviations. An increase in the number of back-mutants in one experiment without activation on Strain TA 98 is attributed to unsterility. No evidence of the induction of point mutations by test item or by the metabolites of the substance formed as a result of microsomal activation was detectable in the strains of S. typhimurium used in the experiments (report n. 79/0229, 1979).

The mammalian cell gene mutation was assessed on the analogous OB 3a-DSA. The in Vitro Mammalian Cell Gene Mutation Test was performed with V79 hamster fibroblast. The test substance was tested at the concentrations of 0.15; 0.5; 1.5 and 5 mg/ml. Each concentration was tested in two replicates. Experiments were performed without as well as with of metabolic activation using the supernatant of rat liver and a mixture of cofactors. No evidence of the mutagenicity of test substance was recorded, thus the test substance resulted non-mutagenic for V79 cells without as well as with metabolic activation (report n. 223/14/20, 2015).

OB 3a-DSA was additionally tested in the in vitro mammalian cell micronucleus test, according to OECD Guideline No. 487 (report n. 19-493, 2020). The human peripheral blood lymphocytes from healthy donors were used for testing. The test item was dissolved in culture medium (RPMI 1640) and diluted in concentrations 100 – 6.25 mg/mL, which were applied to cultures in volume of 50 mL (final test concentrations were 2000, 1000, 500, 250 and 125 µg/mL). At first, genotoxicity experiment was done to assess the genotoxicity potential of test concentrations with 3 hours (short) exposure with and without metabolic activation. Because no genotoxicity was observed after short term exposure, the further genotoxicity test was performed to assess the genotoxicity in extended exposure 24 hours (without metabolic activation). Under the experimental design described above, the test item, OB 3a-DSA, had no genotoxic effects in the human peripheral blood lymphocytes in experiment with metabolic activation as well as without metabolic activation in both times of exposure. The result of micronucleus test was negative, test item is then considered not able to induce chromosome breaks and/or chromosome gain or loss in this test system.

 

The chromosome aberration potential was investigated for the analogous OB 3a-MSA, both in vivo ad in vitro and the results were used as supporting studies: no indication of a mutagenic effect were recorded in the in vitro test (report n. 183508, 1995) and in the in vivo dominant lethal assay (report n. T1059160, 1995); furthermore the substance did not induce micronuclei as determined by the micronucleus test with bone marrow cells of the mouse (report n. 213208, 1991).

A further in vivo chromosomal aberration test, dominant lethal assay, was performed on the OB 3a-A(Na)(report n. 4167, 1973). The compound was administered orally in single dose to NMRI mice by gavage at the concentration of 5000 mg/kg and did not shown any evidence of mutagenicity.

 

Justification for classification or non-classification

According to the CLP regulation (EC 1272/2008), 3.5 Germ cell mutagenicity section, a mutation means a permanent change in the amount or structure of the genetic material in a cell.

Evaluation of the endpoint was performed with the integrated evaluation of the following studies: in vitro Ames test, in vitro gene mutation on mammalian cells, in vitro micronuclesu test, in vitro chromosomal aberration and in vivo Micronucleous study. Since all studies are negative, the substance can be considered as not having mutagenic or genotoxicological properties.

In conclusion, the available experimental data are adequate for classification and labelling and the substance is not classified for genetic toxicity according to the CLP Regulation (EC 1272/2008).