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

Endpoint summary

Additional information

Mutagenicity in bacterial reverse mutation assays (Ames test) was investigated in a complete study on the substance under registration (CAS 17958-73-5). The test substance was assayed for the mutagenicity by the Bacterial Reverse Mutation Test in Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and one indicator Escherichia coli WP2 uvrA strain. 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 studies are available on the similar substances.

The test on CAS 16324-27-9 is negative but it was not performed on all the required strains (Ciba-Geigy Ltd., 1979). The substance is the analogous monohydroxyethylamino, tetrasulphonated sodium salt. It has the same organic functionalities but is more sulphonated and more water soluble.

The other AMES test available was performed on the CAS 4193-55-9: the mutagenicity in bacterial reverse mutation assays (Ames test) was investigated in a complete study on the substance (RCC - Cytotest Cell Research GmbH, 1998). No genotoxic effects occurred in the test groups with and without metabolic activation. In this case the substance is the analogous disulphonated dihydroxyethylamino substituted, thus the structural difference interests the substitution on the triazino ring with dihydroxyethyl (CAS 4193-55-9) instead than monohydroxyethyl (CAS 17958-73-5). From a metabolic point of view the substances can be considered very similar because the monohydroxyl derivative is a metabolite of the dihydroxy derivative. In this respect the water solubilities of CAS 17958-73-5 and CAS 4193-55-9 are very similar (30.2 vs 48.2 g/l, respectively).

The mammalian cell gene mutation was assessed on the analogous CAS 68971-49-3. 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 (Täublová E., 2014).

CAS 68971-49-3 is the dihydroxyethylamino, hexasulphonated sodium salt.

The chromosome aberration potential was investigated also for the analogous CAS 16470-24-9, both in vivo and 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).

 

A further in vivo chromosomal aberration test, dominant lethal assay, was performed on the CAS 4193-55-9 (Lorke D. and Machemer L., 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.

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 dossier) 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 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 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. 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.

Justification for selection of genetic toxicity endpoint

Evaluation of the endpoint was performed with the integrated evaluation of the following studies: in vitro Ames test (Täublová E., 2014), in vitro gene mutation on mammalian cells (Täublová E., 2014), in vitro chromosomal aberration (CCR- Cytotest Cell Research GmbH & Co. KG, 1991) and in vivo Micronucleous study (CCR- Cytotest Cell Research GmbH & Co. KG, 1991). Since all studies are negative, the substance can be considered as not having mutagenic or genotoxicological properties.

Short description of key information:

Non genotoxic

Endpoint Conclusion: No adverse effect observed (negative)

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).