Registration Dossier

Administrative data

Key value for chemical safety assessment

Additional information

Bacterial reverse mutation
In a key in vitro genetic toxicity study, the registered test substance containing 39.8% act.ingr. was assayed in a bacterial gene mutation assay (Ames test) using the strains TA100, TA1535, TA98 and TA1537 both in the absence and presence of metabolic activation (Grötsch and Sonnenschein, 1995). In two independent mutation experiments, cells were exposed to concentrations of 0.04, 0.20, 1.00, 5.00 and 25.00 (cytotoxic concentration) µL per plate in the absence and presence of S9. In both assays the test item induced neither cytotoxicity nor statistically significant increases in histidine-protrophy revertants in any tester-strains with and without metabolic activation in the tested concentration range. It was thus concluded that the test item did not induce gene mutations in the bacterial mutagenicity assay with and without metabolic activation in vitro when tested under the experimental conditions reported.
In conclusion, negative results were obtained for bacterial mutagenicity up to cytotoxic concentrations in a key study with registered substance.

 

Mammalian gene mutation
A key study with registered substance was conducted in cultured mammalian cells (V79, genetic marker HPRT) both in the presence (4 hours) and absence (4 and 24 hours) of metabolic activation (Flügge, 2013c). In the preliminary experiment without and with metabolic activation test item concentrations of 10, 25, 100, 250, 1000, 2500 µg solid matter/mL medium and the undiluted test item (ca. 4600 µg/mL medium) were employed. The test item was diluted with aqua ad iniectabilia. A correction factor of 2.49 was used as the supplied test item contains only 40.20% solid matter. Aqua ad iniectabilia served as the vehicle control. Pronounced cytotoxicity in form of decreased plating efficiency was noted starting at concentrations of 1000 µg/mL medium in the experiments without and with metabolic activation (24-h or 4-h exposure), respectively. Hence, 1000 µg solid matter/mL were employed as the top concentration for the mutagenicity tests in the absence and in the presence of metabolic activation Five concentrations 62.5, 125, 250, 500 or 1000 µg solid matter/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 concentration of 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 were 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. Under the present test conditions, the test item 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.
 
In conclusion, negative results were obtained for mammalian mutagenicity in a key study with registered substance tested up to cytotoxic concenrtations.

 

Chromosome aberration
A key Micronucleus test with registered substance was conducted in human peripheral lymphocytes both in presence (4 hours) and in absence (4 and 20 hours) of metabolic activation (Flügge, 2013d). In the preliminary experiment without and with metabolic activation test item concentrations of 10, 25, 100, 250, 1000, 2500 and 4020 µg test item /mL medium were employed. The test item was diluted with aqua ad iniectabilia. A correction factor of 2.49 was used as the supplied test item contains only 40.20% solid matter. Aqua ad iniectabilia served as the vehicle control.
Pronounced to complete cytotoxicity was noted at concentrations of 1000 µg test item/mL and higher in the experiment with metabolic activation. In the experiment without metabolic activation concentrations of 1000 µg test item/mL and higher caused complete cytotoxicity. Hence, 2500 µg/mL was employed as the top concentration for the mutagenicity tests with metabolic activation and 1000 µg test item/mL in the tests without metabolic activation. In the main study cytotoxicity was noted starting at concentrations of 500 or 1000 µg/mL in the experiments without and with metabolic activation, respectively. Mitomycin C and colchicine were employed as positive controls in the absence and cyclophosphamide in the presence of metabolic activation.
The micronucleus frequencies of cultures treated with the test item at concentrations of 62.5, 125, 250 or 500 µg test item/mL medium (4-h or 20-h exposure) in the absence of metabolic activation ranged from 0.5 to 8.0 micronuclei per 1000 binucleated cells. There was no dose related increase in micronuclei up to the cytotoxic concentrations. The dose level of 1000 µg test item/mL medium led to cytotoxicity, no cells of sufficient quality were available for evaluation. Vehicle and untreated control values fell within acceptation ranges.
The micronucleus frequencies of cultures treated with the test item at concentrations of 125, 250, 500 or 1000 µg test item/mL medium (4-h exposure) in the presence of metabolic activation ranged from 3.0 to 8.5 micronuclei per 1000 binucleated cells. There was no dose related increase in micronuclei up to the cytotoxic concentration. The dose level of 2500 µg test item/mL medium led to cytotoxicity, no cells of sufficient quality were available for evaluation. Vehicle and untreated control values fell within acceptation ranges. Under the present test conditions, Reaction products of ricinoleic acid with 2-aminoethanol and maleic acid and sodium hydrogensulfite tested up to cytotoxic concentrations, 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 chromosomal damage in the in vitro micronucleus test. In the same test, Mitomycin C and cyclophosphamide induced significant chromosomal damage and colchicine induced significant damage to the cell division apparatus, respectively. Therefore, the test is considered valid.
 
In conclusion, negative results were obtained for chromosome aberration in a key study with registered substance tested up to cytotoxic concenrtations.

 

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
Altough the Ames study was selected as main endpoint, other endpoints were equally important for genetic toxicity assessment.

Short description of key information:
Data from key studies were available for registered substance . Gene mutation was tested to be negative in an Ames bacterial reverse mutation assay in strains TA98, TA100, TA1535 and TA1537 with and without S9 up to cytotoxic concentrations of 25 µg/plate in the absence and presence of metabolic activation. Gene mutation was also negative in the HPRT-V79 mammalian cell mutagenicity test when tested up to cytotoxic concentrations of 1000 µg/mL in the absence and presence of metabolic ativation. In the micronucleus test in human peripheral lymphocytes the registered substance tested negative for chromosome aberration when tested up to cytotoxic concentrations of 500 µg/mL in the absence and 1000 µg/mL in the presence of metabolic activation.

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 item does not have to be classified and has no obligatory labelling requirement for genetic toxicity.