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Mutagenicity in bacterial reverse mutation assays (Ames test) was investigated in a complete study on the substance (RCC - Cytotest Cell Research GmbH, 1998); no toxic effects, evident as a reduction in the number of revertants, occurred in the test groups with and without metabolic activation. No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance. Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies.

A further AMES test confirmed these outcomes (Microtest Research Ltd., 1989): the substance resulted to be unable to induce mutation in five strains of Salmonella tvphimurium, when tested up to 5000 µg/plate in the absence and presence of a rat liver metabolic activation system.

 

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 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 two substances (CAS 68971-49-3 and CAS 4193-55-9) have the same organic functionality, both are dihydroxyethylamino derivative with different sulphonation degree. Based on experimental results the difference in solubility is not directly related to the sulphonation degree and in the in vitro test bioavailability, as soon as the substances are soluble in the medium, it is not impairing the evaluation of a non-treshold effect like mammalian mutagenicity. Therefore the results for CAS 68971-49-3 can be applied also for CAS 4193-55-9 (further details in the Category Justification Report attached to the section 13 of the dossier).

 

An in vivo chromosomal aberration test, dominant lethal assay, was performed on the substance under registration (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. 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 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).


Justification for selection of genetic toxicity endpoint
Evaluation of the endpoint has been performed with the integrated evaluation of the following studies: in vitro Ames test (RCC - Cytotest Cell Research GmbH, 1998), in vitro gene mutation on mammalian cells (Täublová, 2014), in vitro chromosomal aberration (CCR- Cytotest Cell Research GmbH & Co. KG, 1991). The in vivo Bone Marrow study (Kemira, 1973) is supported by an in vivo Micronucleous study (CCR- Cytotest Cell Research GmbH & Co. KG., 1991) 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.

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).