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In vitro

A study was conducted to evaluate the genotoxic potential of amides, C12-18 (even-numbered) and C18-unsatd., N-hydroxyethyl in the Ames test.Salmonella typhimurium strains TA1535, TA1537, TA1538, TA98 and TA100 were treated with the test substance using the Ames plate incorporation method at up to five dose levels (4, 20, 100, 500 and 2,500 µg/plate test material per plate) both with and without metabolic activation. Amides, C12-18 (even-numbered) and C18-unsatd., N-hydroxyethyl was found to be non-mutagenic under the conditions of this test (Wallat, 1981).

A study was conducted to determine the mutagenic potential of structurally similar amides, C8-18 (even numbered) and C18-unsat., N-(hydroxyethyl) in the Salmonella typhimurium reverse mutation assay (strains TA 1535, TA 1537, TA98 and TA100) and in theEscherichia colireverse mutation assay strain WP2 uvrA. The test was conducted in the presence and absence of metabolic activation (S9 -mix). The study was conducted according to the most recent OECD and EC guidelines. In the dose range finding test, the test substance was tested up to concentrations of 5,000 µg/plate in the absence and presence of S9-mix in the strains TA100 and WP2 uvrA. The test substance precipitated on the plates at dose levels of 3,330 and 5,000 μg/plate. In tester strain TA100, toxicity was observed at dose levels of 333 μg/plate and upwards in the absence of S9-mix and at dose levels of 1,000 μg/plate and upwards in the presence of S9-mix. In tester strain WP2 uvrA, the bacterial background lawn was not reduced at any of the concentrations tested. No biologically relevant decrease in the number of revertants was observed up to the dose level of 3,330 μg/plate. Since the test substance precipitated heavily on the plates at the test substance concentration of 5,000 µg/plate, the number of revertant colonies of this dose level could not be determined. Based on the results of the dose range finding test, the test substance was tested in the first mutation assay at a concentration range of 3 to 666 µg/plate in the absence of S9-mix and at a concentration range of 10 to 1,000 µg/plate in the presence of 5 % (v/v) S9-mix in tester strains TA1535, TA1537 and TA98. The test substance did not precipitate on the plates at this dose level. Toxicity was observed in all tester strains, except in tester strain TA98 in the presence of S9-mix. In an independent repeat of the assay with additional parameters, the test substance was tested at a concentration range of 3 to 666 µg/plate in the absence of S9-mix and at a concentration range of 10 to 1,000 µg/plate in the presence of 10% (v/v) S9-mix in tester strains TA1535, TA1537, TA98 and TA100 and at 10 to 3,330 µg/plate in tester strain WP2 uvrA in the absence and presence of 10% (v/v) S9-mix. Precipitate on the plates was only observed at the dose level of 3,330 μg/plate in the absence of S9-mix. Toxicity was observed in all tester strains, except in tester strain TA1535 and TA1537 in the presence of S9-mix and in WP2 uvrA in the absence and presence of S9-mix. Since in the first experiment in tester strain TA98 and in the second experiment in tester strains TA1537 and WP2 uvrA no toxicity or precipitate on the plates was observed in the presence of S9-mix, a third mutation experiment was performed with these strains and tester strain TA98 in the presence of S9-mix. The test substance was tested up to 5,000 µg/plate. The test substance precipitated on the plates at dose levels of 3,330 and 5,000 μg/plate. Due to the precipitate of the test substance on the plates the bacterial background could not be determined at the dose levels of 3,330 and 5,000 μg/plate, except at tester strain WP2 uvrA. Cytotoxicity, as evidenced by a decrease in the number of revertants, was observed in tester strain TA98 in the presence of 5 and 10% (v/v) S9-mix. The test substance did not induce a significant dose-related increase in the number of revertant (His+) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in tester strain WP2 uvrA both in the absence and presence of S9-metabolic activation. The negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly. Based on the results of this study it is concluded that the test substance is not mutagenic in the Salmonella typhimuriumr everse mutation assay and in the Escherichia colir reverse mutation assay (Verspeek-Rip CM, 2014).

The mutagenic potential of structurally similar amides, C18-18 (even numbered) and C18-unsatd., N-(hydroxyethyl) was evaluated according to OECD guideline 476 and EU Method B.17. The test was performed in two independent experiments with L5178Y mouse lymphoma cells in the absence and presence of S9-mix. In the first experiment, test material was tested up to concentrations of 60 and 200µg/mL in the absence and presence of 8 % (v/v) S9-mix. The incubation time was 3 hours. Test material was tested up to cytotoxic levels of 74 and 77 % in the absence and presence of S9-mix, respectively. In the second experiment, test material was tested up to concentrations of 45 and 225µg/mL in the absence and presence of 12 % (v/v) S9-mix with incubation times of 24 hours and 3 hours, respectively. Test material was tested up to the cytotoxic level of 95 % (absence of S9-mix) and up to 72 % (presence of S9-mix). The spontaneous mutation frequencies in the solvent-treated control cultures were between the minimum and maximum value of the historical control data range and within the acceptability criteria of this assay. Mutation frequencies in cultures treated with positive control chemicals were increased by 8.2- and 16-fold for MMS (absence of S9-mix), and by 10 and 13 fold for CP (presence of S9-mix). The test conditions, both in the absence and presence of S9-mix, were appropriate and the metabolic activation system (S9-mix) functioned properly. In both the presence and absence of S9-mix, test material did not induce a significant increase in the mutation frequency in the first experiment. This result was confirmed in an independent repeat experiment with modifications in the duration of treatment time. Under the test conditions, amides, C18-18 (even numbered) and C18-unsatd., N-(hydroxyethyl) was not mutagenic in the TK mutation test system both with and without metabolic activation (Verspeek-Rip CM, 2009a).

In another study, the ability of structurally similar amides, C18-18 (even numbered) and C18-unsatd., N-(hydroxyethyl) to induce chromosome aberrations in cultured peripheral human lymphocytes according to OECD guideline 473 was evaluated. Peripheral human lymphocytes treated with the test material were evaluated for chromosome aberrations with vehicle and positive controls. In experiment 1, 3 hours exposure with a 24 hours fixation in the absence and presence of S9-mix (1.8 %) and in experiment 2, 24 hours exposure with a 24 hours fixation time and 48 hours exposure with a 48 hours fixation time in the absence of S9-mix and 3 hours exposure with a 48 hours fixation in presence of S9-mix were tested. Frequency of cells with aberrations in the vehicle control group was within the historical control data range. Both of the positive control materials induced significant increases in the frequency of aberrations indicating the satisfactory performance of the test and of the activity of the metabolising system. The test material did not induce any significant or biologically relevant increases in the frequency of cells with chromosome aberrations in the presence or absence of metabolic activation system, in either of the two independently repeated experiments. No effects on the number of polyploid cells and cells with chromosomes were observed both in the absence and presence of S9-mix. The test material did not disturb the mitotic processes, cell cycle progression and did not induce numerical chromosome aberrations therefore it was concluded that amides, C18-18 (even numbered) and C18-unsatd., N-(hydroxyethyl) can be considered to be non-clastogenic in cultured human lymphocytes in vitro (Verspeek-Rip CM, 2009b)

In vivo

In accordance with Annex VIII, column 2 adaptation of REACH, data on in vivo genetoxicity studies are not required since all available in vitro genotoxicity study results are negative.


Justification for selection of genetic toxicity endpoint
All data contribute to the overall endpoint conclusion.

Short description of key information:
Based on available data for the substance itself and structurally similar substances, amides, C12-18 (even-numbered) and C18-unsatd., N-hydroxyethyl was not considered have any genotoxic potential.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Available data suggests that amides, C12-18 (even numbered) and C18-unsatd., N-hydroxyethyl is not genotoxic. Therefore, no classification is required for this endpoint according to EC (67/548/EEC) and CLP (EC 1272/2008) criteria.