Registration Dossier

Administrative data

Key value for chemical safety assessment

Additional information

The substance under registration (CAS 16324-27-9) was tested for mutagenic effects on histidine auxotrophic mutants of Salmonella typhimurium. The investigations were performed with the following concentrations of the trial substance with and without microsomal activation: 25, 75 225, 675 and 2025 µg/0.1 ml. Since the results obtained in the experiments with Strain TA 98 were equivocal, additional experiments were performed on Strains TA 98 and TA 1538. In the experiments performed with and without microsomal activation, comparison of the number of back-mutant colonies in the controls and the cultures treated with the various concentrations of test item revealed no marked deviations. An increase in the number of back-mutants in one experiment without activation on Strain TA 98 is attributed to unsterility. No evidence of the induction of point mutations by test item or by the metabolites of the substance formed as a result of microsomal activation was detectable in the strains of S. typhimurium used in the experiments (Ciba-Geigy Ltd., 1979).

In order to confirm the outcomes in the test on CAS 16324-27-9, the results from a complete AMES test performed on the analogous CAS 17958-73-5 were considered. The substance is the analogous monohydroxyethylamino, disulphonated sodium salt. It has the same organic functionalities, but is less soluble than the substance under registration (30.2 g/l vs 140 g/l, respectively); therefore it can be then considered as a good conservative representative for the considered end point.

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).

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., 2015).

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

The chromosome aberration potential was investigated for the analogous CAS 16470-24-9, 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).


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 technical 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 (Ciba-Geigy Ltd., 1979 and Täublová E., 2015), in vitro gene mutation on mammalian cells (Täublová E., 2015), 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).

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), 3.5 Germ cell mutagenicity section, a mutation means a permanent change in the amount or structure of the genetic material in a cell.

Evaluation of the endpoint was performed with the integrated evaluation of the following studies: in vitro Ames test, in vitro gene mutation on mammalian cells, in vitro chromosomal aberration and in vivo Micronucleous study. Since all studies are negative, the substance can be considered as not having mutagenic or genotoxicological 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).