Tetrasodium 4,4'-bis[[4-[bis(2-hydroxypropyl)amino]-6-[(4-sulphonatophenyl)amino]-1,3,5-triazin-2-yl]amino]-stilbene-2,2'-disulphonate

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

The substance under registration OB 4-MSA is part of the Stilbene Fluorescent Whitening Agents category: within the whole category ten over fourteen registered substances (see data matrix in the Category Justification Report) 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 with 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.

Three in vitro studies (gene mutation in bacteria, gene mutation in mammalian cells and micronucleus), and an in vivo study (mammalian germ cell) are available for OB 4-MSA. The genetic toxicity of this substance was therefore well investigated and results obtained in these experiments are in line with the ones obtained within the category.

The bacterial reverse mutation test was performed according to the OECD Test Guideline No. 471 (report n. 19-263, 2019) Four indicator Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and one indicator Escherichia coli WP2 uvrA strain were used. The test item was diluted in water for injection and assayed in concentrations 50, 150, 500, 1500 and 5000 μg per plate, which were applied to plates in volume of 0.1 mL. The maximum test item concentration used has been determined with respect to results of previous cytotoxicity test. The first mutagenicity experiments were performed as plate incorporation test without and with the metabolic activation using a supernatant of rat liver (volume of S9 was 30μL per plate) and a mixture of cofactors by the plate incorporation test with a dose range of 50-5000 mg per plate. In the first series of mutagenicity experiments no cytotoxicity, precipitation or signs of mutagenicity were observed. In the second experiments the same concentrations were used but experiments were performed with 30 minutes of pre-incubation at 37±1 °C and the metabolic activation was slightly modified (volume of S9 was 50 μL per plate). Experimental conditions were changed in order to improve the contact of bacteria with the test item and metabolic activation, according to OECD requirements. The concurrent positive controls verified the sensitivity of the assay and the metabolising activity of the liver preparations. Average revertant colony counts for the vehicle controls were within the current historical control range for the laboratory. In the arrangement given above, the test item, OB 4-MSA, was non-mutagenic for all the used indicator strains in experiments with and without metabolic activation. Change of experimental conditions performed in the second experiments had no influence on study results. 

 

In the vitro mammalian cell gene mutation test (report n. 222/14/20, 2014), V79 hamster fibroblast were used and experiments were performed without as well as with of metabolic activation using the supernatant of rat liver and a mixture of cofactors, according to the OECD guideline 476. The experiment gives no evidence of the mutagenicity of test substance, thus the test substance resulted non-mutagenic for V79 cells without as well as with metabolic activation.

 

In vitro mammalian cell micronucleus test assayed genotoxicity of the test item according to the OECD Guideline No. 487 (report n. 20-4, 2020). The human peripheral blood lymphocytes from healthy donors were used for testing. The test item was dissolved in culture medium (RPMI 1640) and diluted in concentrations 100 – 6.25 mg/mL, which were applied to cultures in volume of 50mL (final test concentrations were 2000, 1000, 500, 250 and 125 µg/mL). At first, genotoxicity experiment was done to assess the genotoxicity potential of test concentrations with 3 hours (short) exposure with and without metabolic activation. The experiment with 3 hours exposure without metabolic activation was repeated, because of the positive control was out of historical control range. Because no genotoxicity was observed after short term exposure, the further genotoxicity test was performed to assess the genotoxicity in extended exposure 24 hours (without metabolic activation). Under the experimental design described above, the test item, OB 4-MSA, had no genotoxic effects in the human peripheral blood lymphocytes in experiment with metabolic activation as well as without metabolic activation in both times of exposure. The result of micronucleus test was negative, test item is then considered not able to induce chromosome breaks and/or chromosome gain or loss in this test system.

An in vivo study on the chromosomal aberration, dominant lethal assay, was performed on OB 4-MSA (report n. 7170, 1977). The compound was administered orally in single doses to mice (NMRI) which were then mated to untreated females. 5000 mg/kg bw were given by oral gavage; pre- and post implant losses were examined. All treated animals survived. The fertility of the male animals was not influenced by the test substance. No substance related post implantation losses were recorded and no influence on rates of implants, alive and dead implants per female was found; no significant changes between control and treated groups were recorded.

Furthermore, two in vivo test performed on the analogous substances (OB 3a-A(Na) and OB 3a-MSA) were reported in order to confirm the outcomes from in vitro test.

A dominant lethal assay was performed on the similar substance OB 3a-A(Na) (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 OB 3a-MSA: 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 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).