Disodium 4,4'-bis[[4-anilino-6-[(2-carbamoylethyl)(2-hydroxyethyl)amino]-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 genotoxic

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Not genotoxic

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

The substance under registration OB 5-A is part of the Stilbene Fluorescent Whitening Agents category: within the whole category ten over fourteen registered substances (see data matrix in the Category Justification Report) were tested for bacteria reverse mutation and chromosomal aberration and none of the existing tests 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.

Five in vitro studies (two gene mutation in bacteria, gene mutation in mammalian cells, chromosome aberration and micronucleus are available for OB 5-A. The genetic toxicity of this substance was therefore well investigated and results obtained in these experiments are in line with the ones obtained within the category.

The bacterial reverse mutation test was performed according to the OECD Test Guideline No. 471 ((Iwahara S. and Kanatani H., 1980). All the strains tested (Salmonella Typhinurium TA 1535, TA 100, TA 1537, TA 1538, TA 98; Escherichia coll WP2 uvrA) with and without Metabolic Activation Tests, did not shown remarkable increase in the mutation colony.

A further AMES test confirmed these outcomes (report n. T8534, 1988): the results show that the test strains are sensitive to the positive control mutagens, indicating that under the assay conditions, the test strains were sensitive to the detection of potentially genotoxic agents. The test article was found to be soluble in DMSO and showed no apparent cytotoxicity at the concentrations tested. No increase in numbers of revertant colonies was observed on plates containing the test article without and with metabolic activation compared to negative control plates.

 

In the in vitro mammalian cell gene mutation test (report n. 387/14/20, 2015), V79 hamster fibroblast were used for testing. A preliminary cytotoxicity assay (3-hour treatment) was performed at first. First experiment, with 3 hours treatment by the test substance followed then. Four concentrations were used - the maximum concentration 5.0 mg/ml (in experiment with metabolic activation) and 3.0 mg/ml (without metabolic activation) and three lower concentrations, 1.5; 0.5 and 0.15 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. Results of first experiment were negative, so the modification of experiment was performed with extended treatment time of 24 hours. Concentrations of 1.5; 1.0; 0.5 and 0.25 mg/ml were used according to results of cytotoxicity test. Additional dose 5.0 mg/ml was tested in parallel with 24 hours experiment. Also the second expreriment gives no evidence of the mutagenicity of test substance.

The substance was also tested for its potential to cause chromosome aberrations in cultured Chinese hamster ovary cells with and without an exogenous S-9 activation mixture (report n. T8528, 1989). The Chromosome Aberration Assay was performed at five dose levels, 5000, 4000, 3000, 2000 and 1000 µg/ml, in the non-activated system and at four dose levels, 5000, 4000, 3000 and 2000 µg/ml, in the activated system. When compared to the solvent control data, the test doses did not cause a significant increase in the number of cells with chromosome aberrations in the non-activated or activated system. In the test was not reported a detectable dose response in the number of aberrations per cell or number of cells with aberrations in the activated or non-activated system.

In vitro mammalian cell micronucleus test assayed genotoxicity of the test item according to the OECD Guideline No. 487 (report n. 20-22, 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 50mL (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, the second 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 5-A, 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.

Furthermore, two in vivo test performed on the analogous substances OB 3a-A(Na) and OB 3a-MSA were reported in order to confirm the outcomes from in vitro test.

A dominant lethal assay was performed on the similar substance 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. Methylmethanesulfonate (MMS) and trimethyl phosphate (TMPO) were used as positive controls. Each of the 20 male mice in each group was mated with three untreated females directly after treatment. After insemination (determined by vaginal smear), or after a week, the females were isolated. This procedure was repeated weekly for eight weeks. On the fourteenth day of gestation the females were sacrificed and the numbers of fertile matings, implantations, resorptions, live foetuses, and corpora lutea were determined. The dominant lethal test investigation did not shown any evidence of mutagenicity caused by the test substance.

The chromosome aberration potential was investigated also for the analogous OB 3a-MSA: the substance did not induce micronuclei as determined by the micronucleus test with bone marrow cells of the mouse (report n. 313208, 1991).

Justification for classification or non-classification

According to the CLP Regulation (EC 1272/2008), for the purpose of the classification for germ cell mutagenicity, substances are allocated in one of two categories in consideration of the fact that they are:

- substances known to induce heritable mutations or to be regarded as if they induce heritable mutations in the germ cells of humans or substances known to induce heritable mutations in the germ cells of humans or

- substances, which cause concern for humans owing to the possibility that they may induce heritable mutations in the germ cells of humans.

 

On the basis of the results of the available studies, the substance can be considered as not having mutagenic or genotoxic 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).