Benzenesulfonic acid, 2,2'-(1,2-ethenediyl)bis[5-[[4-(diethylamino)-6-[(4-sulfophenyl)amino]-1,3,5-triazin-2-yl]amino]-, sodium salt (1:4)

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

Additional information

Mutagenicity in bacterial reverse mutation assays (Ames test) was investigated in a complete study on the substance. The performed test was performed on Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and one indicator Escherichia coli WP2 uvrA strain were used. The test substance was assayed in doses of (10) 50-5000 µg per plate and the experiments were performed without as well as with metabolic activation with rat liver and a mixture of cofactors. The test substance was non-mutagenic for all the Salmonella typhimurium as well as Escherichia coli strains tested without metabolic activation (Täublová E, 2014).

Further AMES test on analogous substances were reported as supporting studies. OB 3a-A(Na) (the dihydroxyethylamino derivatives, disulphonated) did not induce gene mutations by base pair changes or frameshifcs in the genome of the Salmonella typhimurium and Escherichia coli (Wollny H.E., 1998 and Microtest Research Ltd., 1989).

OB 1 -DSA was tested to investigate the potential to induce gene mutations using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and TA1538 and it did not induce mutations in the strains of Salmonella typhimurium, when tested in the absence and presence of a rat liver metabolic activation system (Microtest Research Ltd., 1989). OB 1 -MSA and OB 1 -DSA share the common organic functional group diethylamino derivative, with different sulphonation degrees: tetrasulphonated (OB 1 -MSA) and hexasulphonated (OB 1 -DSA). The hexasulphonated is the best conservative representative in the group because of the structural similarity and the lower water solubility.

 

The mammalian cell gene mutation was assessed testing the in Vitro Mammalian Cell Gene Mutation Test, which 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 (Täublová E., 2015).

The same test was performed also on the OB 3a-DSA, which is the dihydroxyethylamino, hexasulphonated sodium salt. Also in this case no evidence of the mutagenicity was recorded (Täublová E., 2014).

Mammalian Cell Micronucleus Test was conducted on the test substance under registration. The test was performed according to the OECD Guideline 487. The human peripheral blood lymphocytes from healthy donors were used for testing. Experiments were performed without as well as with metabolic activation with a supernatant of rat liver and a mixture of cofactors. Under the test condition, the test substance does not induce chromosome breaks and/or gain or loss of chromosomes in cultured mammalian cells (Cacková L., 2014).

Furthermore, the chromosome aberration potential was investigated also for the analogous OB 3a-MSA (the dihydroxyethylamino, tetrasulphonated sodium salt), 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 (CCR- Cytotest Cell Research GmbH & Co. KG, 1991) and in the in vivo dominant lethal assay (Bayer AG, 1995); furthermore the substance did not induce micronuclei as determined by the micronucleus test with bone marrow cells of the mouse (CCR - Cytotest Cell Research GmbH & Co KG, 1991).

Mutagenicity is a non-threshold end-point, therefore mutagenicity potential is evaluated firstly based on the reactivity of the substance in itself, based on the chemical structure, functional groups and metabolism pathway, than on the bioavailability potential. Moreover this first screening in vitro is conservative regarding the end point, since the substance is put into the reaction plate even if potentially it will never be absorbed and will never express the mutagenic potential.

Within the whole category, ten over fourteen registered substances covering at least one member per group (see data matrix in the Category Justification Report attached to the section 13 of the technical dossier) were tested for bacteria reverse mutation and chromosomal aberration and none of the existing tests arisen any concern for mutagenicity or genotoxicity.

In order to assess the mutagenicity potential on mammalian cells, an in vitro Ames test in combination with the micronucleous assay has been planned. This choice has been determined based on the position of The Committee on Mutagenicity of Chemicals in Food, Consumer Products and the Environment (COM) that has a remit to provide UK Government Departments and Agencies with advice on the most suitable approaches to testing chemical substances for genotoxicity, the best strategy to assess genotoxicity in vitro is the combination of Ames test and micronucleous, because together with a better sensitivity, a better specificity is also demonstrated respect than testing mutagenicity in mammalian cells. More details are specified within the Category Justification Report attached to the section 13 of the dossier.

Furthermore, mammalian mutagenicity was performed on three representative members of the category (OB 2-A, OB 3a-DSA and OB 4-MSA), on the basis of three different levels of solubility and covering those groups which could be the most biologically reactive, based on chemical constitution and expected metabolism; in all cases no signs of genetic toxicity were recorded.

All substances of the category were modelled using 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 proteinsvianon-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. Based on all those considerations, the available studies on the analogous substances are representative also for the substance under registration that can then be considered as not genotoxic.

Read across within the same group is well justified in this case taking also into account the impurities of the involved substances: the identified organic impurities can have different substitution on the molecule, nevertheless the functional reactive groups are potentially the same, and molecules are of the same molecular size and polarity of the main component. As a consequence the systemic absorption and reactivity is practically the same than the main constituent and Read Across is justified.

REFERENCES

Snyder, R. D., Ewing, D. and Hendry, L. B. 2006. DNA intercalative potential of marketed drugs testing positive in in vitro cytogenetics assays.

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).