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Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In a key Ames test with registered substance C18 unsaturated fatty acids, reaction products with 1-aminopropan-2-ol, maleic anhydride and sodium bisulfite no increase in mutations were observed in Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA with and without metabolic activation up to 5000 µg/plate. In a supporting Ames test with read-across substance Butanedioic acid, 2(or 3)-sulfo-, 4-[2-[(1-oxo(C12-C18(even numbered) and C18unsaturated)alkyl))amino]ethyl]esters, disodium salts (CAS 68784-08-7) no increase in mutations were observed in Salmonella typhimurium strains TA1535, TA1537, TA98, TA100 and TA102 with and without metabolic activation up to 5000 µg/plate. In a key mammalian gene mutation test in HPRT cells, the read-across test item CAS 68784-08-7did not induce mutations in the absence and presence of metabolic activation when tested up to cytotoxic concentrations of 125 µg and 1000 µg/mL, respectively. Finally, in a key in vitro Micronucleus study in human peripheral lymphocytes with CAS 68784-08-7, no chromosome damage was observed in the absence and presence of metabolic activation when tested up to cytotoxic concentrations of 125 µg/mL (4 and 20h exposure time) and up to 500 µg/mL (4h exposure), respectively.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vitro:  

Read-across substance Butanedioic acid, 2(or 3)-sulfo-, 4-[2-[(1-oxo(C12-C18(even numbered) and C18unsaturated)alkyl))amino]ethyl]esters, disodium salts (CAS 68784-08-7) was examined for bacterial and mammalian gene mutation as well as for chromosomal aberration by means of a liquid test item containing 41.5% active ingredient.

 

Bacterial mutagenicity

In a key test for bacterial mutation, 5 Salmonella typhimurium strains TA 98, TA 100, TA 102, TA 1535 and TA 1537 in two independent experiments were tested without and with metabolic activation with test item dissolved in aqua ad iniectabilia based on a correction factor of 2.41 for active ingredient (Flügge, 2013a). In a preliminary test, ten concentrations ranging from 0.316 to 5000 µg act.ingr./plate were tested without metabolic activation in strain TA 100. No signs of cytotoxicity were noted up to the top concentration of 5000 µg/plate. In the main study, six concentrations ranging from 3.16 to 5000 µg act.ingr./plate were employed in the plate incorporation test and in the preincubation test, each carried out without and with metabolic activation. No signs of cytotoxicity were noted without and with metabolic activation up to the top concentration of 5000 µg act.ingr./plate in all test strains. Under the present test conditions the test item tested up to a concentration of 5000 µg act.ingr./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 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 the preliminary experiment without and with metabolic activation test item concentrations of 10, 25, 100, 250, 1000, 2500 and 4150 µg/mL medium were employed. Pronounced cytotoxicity in form of decreased plating efficiency was noted starting at concentrations of 100 or 1000 µg in the experiments without and with metabolic activation (24-h or 4-h exposure), respectively. Hence, 125 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 7.81,15.63, 31.3, 62.5 or 125 and 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 125 or 1000µg/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.

 

Chromosome aberration

A key in vitro micronucleus test (Flügge, 2013c) was conducted using human peripheral lymphocytes both in the presence and absence of metabolic activation, employing 2 exposure times (4 hours and 20 hours). The experiment with S9 mix was carried out threefold with one exposure time of 4 hours employing two different concentration ranges. The harvesting time was 24 hours after the end of exposure. The test item was completely dissolved in aqua ad iniectabilia, which also served as the vehicle control. A correction factor of 2.41 was used in order to correct for a content of the solid material of 41.5% only.

Based on a preliminary experiment, cytotoxicity was noted starting at a concentration of 100 µg test item/mL in the experiment without and with metabolic activation. Hence, 125 µg/mL were employed as the top concentration for the mutagenicity tests without and with metabolic activation in two independent experiments, each (4-hour and 20-hour exposure). In a third experiment without and with metabolic activation (4-hour exposure) 500 µg/mL were employed as the top concentration for the mutagenicity tests. A third experiment with two higher concentrations was added as it was thought that a concentration of 125 µg/mL had not resulted in sufficient clear-cut cytotoxicity.

In the main study cytotoxicity was noted starting at a concentration of 125 µg 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. Positive controls induced significant increases in micronuclei in both experiments with/without metabolic activation.

Both in the tests without metabolic activation (4- and 20-hour exposure) and with metabolic activation (4 -h exposure), there was no increase in micronuclei up to the cytotoxic concentrations when compared to control. Under the present test conditions, the test item 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 any chromosomal damage 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 classification or non-classification

Based on these results and according to the CLP Guidance (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.