Hexasodium 2,2'-[vinylenebis[(3-sulphonato-4,1-phenylene)imino[6-(diethylamino)-1,3,5-triazine-4,2-diyl]imino]]bis(benzene-1,4-disulphonate)

Endpoint summary

Additional information

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.

An AMES test is available for the substance under registration. The study was performed to investigate the potential of the test item to induce gene mutations using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and TA1538; the test was performed according to the OECD guideline 471, except for fact that none of the E. coli WP2 uvrA, or E. coli WP2 uvrA (pKM101), or S. typhimurium TA102 was tested. The assay was performed in both with and without liver microsomal activation and the test substance did not induce mutations in the absence and presence of a rat liver metabolic activation system (Microtest Research Ltd., 1989).

As supporting study, a further AMES test complete and performed on the analogous CAS 42355-78-2 was reported. The study was performed to investigate the potential of test item to induce gene mutations using the 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 dissolved in dimethylsulfoxide and assayed in doses of (10) 50-5000 µg per plate, which were applied to plates in volume of 0.1 ml. 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).

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 technical dossier.

Furthermore, mammalian mutagenicity was performed on representative members of the category (CAS 68971-49-3 and CAS 67786-25-8), on the basis of different levels of solubility and covering those groups which could be the most biologically reactive, based on chemical constitution and expected metabolism.

The In Vitro Mammalian Cell Gene Mutation assay performed on the similar substance CAS 67786-25-8 (the bis(2-hydroxypropyl)amino derivative), was conducted in V79 hamster fibroblast and experiments were performed without as well as with of metabolic activation using the supernatant of rat liver and a mixture of cofactors. The test substance was non-mutagenic (Täublová E., 2014).

The same test was also performed on the CAS 68971-49-3 (the dihydroxyethylamino hexasulphonated sodium salt) and, as well as in the first case, the test substance was non-mutagenic for V79 cells with as well as without metabolic activation (Täublová E., 2015).

The chromosomal aberration potential was assessed taking into account the test results, both in vitro and in vivo, on the analogous substances CAS 42355-78-2 (the diethyl derivative, tetrasulphonated sodium salt (group 1)), CAS 16470-24-9 (the dihydroxyethyl derivative, tetrasulphonated salt) and CAS 4193-55-9 (the dihydroxyethyl derivative, disulphonated salt).

The In Vitro Mammalian Cell Micronucleus Test was performed in human peripheral blood lymphocytes from healthy donors were used for testing. The test substance was suspended in culture medium and assayed in concentrations of 250-5000 µg/ml, which were applied to cultures in volume of 50 µl. 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. It means that under the experimental design described, the test substance had no genotoxic effects in the human peripheral blood lymphocytes in experiments both without and with metabolic activation (Cacková L., 2014).

The chromosome aberration potential was further investigated for the analogous CAS 16470-24-9, both in vivo ad in vitro and the results were used: 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.

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.

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 selection of genetic toxicity endpoint

Evaluation of the endpoint have been performed with the integrated evaluation of the following studies: in vitro Ames test (Microtest Research Ltd., 1989), in vitro chromosomal aberration (Täublová E., 2014) performed on a similar substance of the Stilbene Fluorescent Whitening Agents and in vitro mammalian cell gene mutation assay (Täublová E., 2014)

The in vivo results are consistent: the two studies on chromosomal aberration, dominant lethal assay (Bayer AG, 1995 and Kemira, 1973) and a Micronucleous study (Voelkner W., 1991) reported were performed on a similar substance of the Stileben Fluorescent Whitening Agents. Since all studies are negative, the substance can be considered as not having mutagenic or genotoxic properties.

Detailed justification for Read Across is indicated within any endpoint and in the Category Justification Report attached to Section 13.

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).