Registration Dossier

Administrative data

Key value for chemical safety assessment

Additional information

Bacterial mutagenicity

A key study was conducted wiht registered substance. A liquid test item containing 34.4% active ingredient was examined in the 5 Salmonella typhimurium strains TA 98, TA 100, TA 102, TA 1535 and TA 1537 in two independent experiments, each carried out without and with metabolic activation (Flügge, 2013a). The first experiment was carried out as a plate incorporation test and the second as a preincubation test. The test item was completely dissolved in aqua ad iniectabilia ; a correction factor of 2.91 was used. In a preliminary cytotoxicity test without metabolic activation in test strain TA 100 employing a plate incorporation test, ten concentrations of 0.316 up to 5000 µg test item/plate were tested. No signs of cytotoxicity were noted up to the top concentration of 5000 µg/plate, hence six concentrations of 31.6, 100, 316, 1000, 3160 and 5000µg test item/plate were employed in the plate incorporation test and in the preincubation test without and with metabolic activation. No signs of cytotoxicity were noted in the plate incorporation and preincubation test. No increase in revertant colony numbers as compared with control counts was observed for the test item, tested up to a concentration of 5000 µg/plate, in any of the 5 test strains in two independent experiments without and with metabolic activation, respectively (plate incorporation and preincubation test).

In conclusion, the registered substance tested up to a concentration of 5000 µg/plate, caused no mutagenic effect in the Salmonella typhimurium strains TA 98, TA 100, TA 102, TA 1535 and TA 1537 neither in the plate incorporation test nor in the preincubation test each carried out without and with metabolic activation.

Mammalian mutagenicity

A key study was conducted with registered substance. A liquid test item containing 34.4% active ingredient was tested in cultured mammalian cells (V79, genetic marker HPRT) both in the presence (4 hours) and absence (4 and 24 hours) of metabolic activation (Flügge, 2013b). In this preliminary experiment without and with metabolic activation test item concentrations of 10, 25, 100, 250, 1000, 2500 and 50000 µg/mL medium were employed. Cytotoxicity was noted in form of decreased plating efficiency starting at concentrations of 250 or 1000 µg test item/mL in the experiment without and with metabolic activation, respectively. Hence, 250 µg test item/mL was employed as the top concentration for the mutagenicity tests in the absence and 1000 µg/mL in the presence of metabolic activation. Five concentrations 15.63, 31.3, 62.5, 125 or 250 or 62.5, 125, 250, 500 or 1000 µg test item/mL were selected for the experiments without and with metabolic activation, respectively. In the main study cytotoxicity in form of decreased plating efficiency (PE1) and (PE2)was noted in the first and second experiments at the top concentrations 250 or 1000 µg/mL in the absence and presence of metabolic activation, respectively. Both in the experiments with and without metabolic activation, the mutation frequencies of treated cell cultures within the normal range of the vehicle controls. The positive controls caused a pronounced increase in the mutation frequencies, indicating the validity of this test system.

In conclusion, the registered substance tested up to cytotoxic concentrations in the experiments without and with metabolic activation, was negative in the HPRT-V79 mammalian cell mutagenicity test under conditions where positive controls exerted potent mutagenic effects. 

Chromosome aberration

A key study was conducted with registered substance. A liquid test item containing 34.4% active ingredient was assayed in an in vitro micronucleus test using human peripheral lymphocytes both in the presence and absence of metabolic activation (Flügge,2013c).The test was carried out employing 2 exposure times without S9 mix: 4 and 20 hours, and 1 exposure time with S9 mix: 4 hours. The experiment with S9 mix was carried out twice. The harvesting time was 24 hours after the end of exposure. The study was conducted in duplicate.The test item was completely dissolved in aqua ad iniectabilia. A correction factor of 2.91 was used as the supplied test item contains only 34.40% active matter.The concentrations employed were chosen based on the results of a cytotoxicity study, showing cytotoxicity and haemolysis at a concentration of 1000 µg active ingredient/mL.Hence, 1000 µg/mL were employed as the top concentration for the mutagenicity tests without and with metabolic activation.In the main study cytotoxicity and haemolysis were noted at the top concentration of 1000 µg of active ingredient/mL in the experiments without and with metabolic activation.Mitomycin C and colchicine were employed as positive controls in the absence and cyclophosphamide in the presence of metabolic activation. In the main test without metabolic activation (4- and 20-hour exposure), the micronucleus frequencies of cultures treated with active ingredient at concentrations of 125, 250, 500 or 1000 µg/mL medium (4-h and 20-h exposure) ranged from 8.0 to 16.5 micronuclei per 1000 binucleated cells and treated groups did not differ from controls. In the main test with metabolic activation (4-hour exposure), the micronucleus frequencies of cultures treated with active ingredient at concentrations of 125, 250, 500 or 1000 µg/mL medium ranged from 11.0 to 21.0 micronuclei per 1000 binucleated cells and treated groups were als not different from controls.

In conclusion, the registered substance tested up to a cytotoxic concentration of 1000 µg/mL medium, in the absence and in the presence of metabolic activation employing two exposure times (without S9) and one exposure time (with S9) revealed no indications of mutagenic properties in the in vitro micronucleus test. In the same test, Mitomycin C and cyclophosphamide induced significant damage.


Conclusion

Standard information requirements according to REACH Guidance Part 3 R7a were fulfilled for genotoxicity testing, including bacterial and mammalian mutagenicity and chromosomal aberration. Based on the available results, there were no indications of mutagenicity or genotoxicity, and no further testing is needed. The substance can be considered to have no mutagenic or genotoxic potential.


Justification for selection of genetic toxicity endpoint
Allthough the Ames test was selected, the mammalian gene mutation test and micronucleus test are equivalent endpoints.

Short description of key information:
In a key Ames test no increase in mutations were observed up to tested concentrations of 5000 µg/plate in 5 different Salmonella typhimureum strains with and without metabolic activation. In a keymammalian gene mutation test in HPRT cells, the test item did not induce mutations in the absence and presence of metabolic activation when tested up to cytotoxic concentrations of 250 and 1000 µg/mL, respectively. In a key in vitro Micronucleus study in human peripheral lymphocytes, no chromosome damage was observed with and without metabolic activation when tested up to cytotoxic concentration of 1000 µg/mL.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Based on these results and according to the EC Directive (No.93/21/EEC) and CLP (No. 1272/2008 of 16 December 2008), the test substance does not have to be classified and has no obligatory labelling requirement for genetic toxicity.