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Genetic toxicity in vitro

Description of key information

Short description of key information:  The genetic toxicity with regard to point mutation and/or chromosomal mutation of DTDMAC was evaluated using available experimental data from in vitro and in vivo studies on DTDMAC as well as read-across from data on the structural comparable dialkyldimethylammonium compounds didecyldimethylammonium chloride (CAS No. 7173-51-5) and DHTDMAC (CAS No. 92129-33-4). Potential point mutagenic effects were assessed in both, bacterial test systems and in mammalian cells. Gene mutation assays performed on Salmonella typhimurium TA 1535, TA 1537, TA1538, TA 98, TA 100 and on E. Coli WP2uvrA Ames test) with DTDMAC, as well as a mouse lymphoma assay in L5178Y cells according to OECD guideline 473 performed on didecyldimethylammonium chloride revealed no indications of point mutagenic effects. Additionally no indication of chromosomal mutation was found in an in vitro chromosomal aberration assay with DHTDAMC in V79 cells. In all studies negative results were reported, in the presence and absence of metabolic activation. Likewise, absence of mutagenic properties were also revealed in an micronucleus test in vivo. Based hereupon, a mutagenic potential of DTDMAC is not to be expected.  

Additional information

DTDMAC was investigated for genotoxic properties in vitro and in vivo. The evaluation is based on available mutagenicity data on DTDMAC itself as well as on read-across using data from the structural analogue compounds didecyldimethylammonium chloride and DHTDMAC.

DTDMAC was tested for point mutagenic effects in bacterial systems following the requirements of OECD test guideline 471in the direct plate incorporation method. The study is regarded to be valid with restrictions because a second independent assay was not performed. However, a repetition was not considered necessary because an unequivocally negative result was obtained. Test systems were Salmonella typhimurium strains TA 1535, TA 1537, TA 1538, TA 98 and TA 100 as well as Escherichia coli WP2uvrA with and without metabolic activation (S9 -mix of Aroclor 1254 pretreated rats). Following a preliminary toxicity test to define the dose-levels to be selected for the main experiment, DTDMAC was tested at the following concentrations: 0, 4, 20, 100, 500 and 1000 µg/plate in Salmonella typhimurium (three plates per dose level) and 0, 4, 20, 100, 500 and 2500 µg/plate in Escherichia coli (three plates per dose level). After 48 to 72 hours of incubation at 37°C the revertant colonies were scored. The evaluation of the toxicity was performed on the basis of the observation of the decrease in the number of revertant colonies and/or a thinning of the bacterial lawn. The number of revertants for the vehicle and positive controls was as specified in the acceptance criteria. The study was therefore considered valid. Toxicity was noted towards TA100 at 1000 µg/plate with and without S9, and towards TA1535, TA1537, TA1538 and TA98 at 1000 µg/plate without S9. Toxicity was also observed in WP2uvrA at 2500 µg/plate without S9. The test substance did not induce any significant increase in the number of revertants, both with or without S9 mix, in any of the five strains of Salmonella typhimurium and Escherichia coli WP2uvrA.

The absence of point mutagenic properties of DTDMAC is confirmed by read-across using data from an in vitro mammalian cell gene mutation test with didecyldimethylammonium chloride. The study was conducted to evaluate the potential of the test material (40% didecyldimethylammonium chloride (DDAC) in water) to induce mutations at the TK (thymidine kinase) locus in L5178Y mouse lymphoma cells and followed OECD guideline 476 in compliance with GLP. After a preliminary toxicity test, didecyldimethylammonium chloride was tested in two independent experiments, with and without a metabolic activation system (S9 -mix). The test item was dissolved in DMSO and the highest dose level for the main test was based on the level of toxicity according to the criteria specified in the international guidelines following a toxicity pretest. The test material was investigated at a concentration range of 0.07- 5.56 (without S9 mix) and 0.21- 50 (with S9 mix) µg DDAC /mL in the main test .A slight to strong toxicity was induced, depending on the dose-levels and the treatment duration. No increase in the mutation frequency was induced.

Negative findings with regard to mutagenic effects have been also observed with DHTDMAC. An in vitro chromosomal aberration test in V79 Chinese hamster cells in concentrations up to 50 microgram per mL with and without metabolic activation and fixation time points of 18 and 28 hours was negative. Higher concentrations proved to be cytotoxic.

In addition to these negative findings in the in vitro test systems, DTDMAC was also devoid of mutagenic effects in an in vivo micronucleus test in mice. In this assay eight mice per group were dosed twice within 24 hours with 0, 50, 500, or 1000 mg/kg body weight via gavage. The evaluation of 1000 polychromatic erythrocytes from each mouse for micronuclei revealed no significant differences between test groups and the negative controls. Based on the results of this test, DTDMAC is not mutagenic under in vivo conditions.

Taking into account all available data with regard to potential genotoxicity on ditallowalkyldimethylammonium chloride and structurally related dialkyldimethylammonium chlorides, there are no indications of a mutagenic potential of DTDMAC. The available data support the conclusion that, because of their closely related structures and similar physico-chemical properties, dialkyldimethylammonium chlorides possess similar human health-related effects and no additional testing is required.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Based on the results of the studies performed on DTDMAC as well as on the structural analogue compounds didecyldimethylammonium chloride and DHTDMAC, no indications of mutagenic properties were revealed. Thus, according to the criteria laid down in EU regulation (EC) 1272/2008 (CLP) and the EU directive 67/548/EEC (DSD), DTDMAC is not classified for genetic toxicity.