Potassium sodium 4,4'-bis[6-anilino-4-[bis(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 3a-A(N (Wollny H.E. 1998), the analogous dihydroxyethyl derivative disulphonated sodium salt. This substance has the same organic functionalities than CAS 70942-01-7, but a different salification. The presence of a complete sodium salification instead than a sodium/potassium mixture of the tested substance has no impact on general water toxicological properties and the percentage and chemical identity of organic impurities are also very similar.

The study was performed to investigate the potential of CAS 4193-55-9 to induce gene mutations using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and the Escherichia coli strain WP2 uvrA. The assay was performed in two independent experiments both with and without liver microsomal activation. The plates incubated with the test article showed normal background growth up to 5000 µg/plate with and without metabolic activation in both independent experiments. 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.

A further AMES test, conducted on the same analogous, 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.

Mammalian mutagenicity test according to OECD 476 (HPRT) was performed on 3a-DSA, the analogous dihydroxyethyl hexasulphonated sodium salt; the substance has the same functional groups and it is a representative of the group 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).

No specific information on diethylamino derivative on chromosomal aberration is available, therefore it has been decided to use the available data on 3a-MSA: it is a valid surrogate to assess genotoxicity in the Read Across approach from a point of view of structural similarity.

Indeed a partial reliablein vivo chromosomal aberration test, dominant lethal assay, was performed on analogous 3a-A(Na) (Lorke D. and Machemer L., 1973).

The chromosome aberration potential was investigated also for the analogous 3a-MSA, 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).

CAS 16470-24-9 is a similar substance within the category of Stilbene Fluorescent Whitening Agents, the analogous dihydroxyethyl derivative tetrasulphonated sodium salt. This substance has the same organic functionalities, with a higher 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 an issue to evaluate mammalian mutagenicity effects. Therefore the result for CAS 16470-24-9 can be applied also for CAS 70942-01-7 (justification for Read Across is reported within the Category Justification Report attached to the section 13 of the technical dossier).

 

A further in vivo chromosomal aberration test, dominant lethal assay, was performed on the analogous CAS 4193-559 (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.

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.

The test substance did not show any reasons of concern in the test performed.

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).