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Additional information

In vitro genotoxicity:

A study was conducted to determine the mutagenic potential of C12-14 alkylmorpholine according to the OECD Guideline 471 and 472 in compliance with GLP. Tester strains TA98, TA100, TA1535, TA1537 of Salmonella typhimurium and WP2uvrA of Escherichia coli were exposed to the test substance in the standard plate test (SPT) and the preincubation test (PIT). The test concentrations were as follows: 1st Experiment (all S. typhimurium and E.coli strains, standard plate test with and without S-9 mix, 3 plates/dose): 0; 20; 100; 500; 2500 and 5000 μg/plate; 2nd Experiment (all S. typhimurium strains, standard plate test with and without S-9 mix, 3 plates/dose): 0; 3.125; 6.25; 12.3; 25 and 50 μg/plate; 3rd Experiment (all S. typhimurium strains, standard plate test with and without S-9 mix, 3 plates/dose): 0; 0.625; 1.25; 2.5; 5 and 10 μg/plate; 4th Experiment (all S. typhimurium and E.coli strains, preincubation test with and without S-9 mix, 3 plates/dose): 0; 0.625; 1.25; 2.5; 5 and 10 μg/plate. When tested in all strains mentioned above under SPT and PIT conditions, with and without S9 mix, the test substance did not induce an increase in his+ or trp+ revertant colonies at concentrations up to 5000 μg/plate. A cytototoxic effect was observed in the SPT from about 6.25 - 12.5 μg/ plate onward for the Salmonella strains, and at doses >= 2500 μg/plate for E. coliWP2 uvrA. In the PIT, cytotoxicity was observed for all strain at about 5 to 10 μg/plate. Test substance precipitation was observed at 5000 μg/plate. The positive control substances induced the expected increases in revertant colonies, both with and without S9-mix. Thus, under the experimental conditions chosen, the test substance was not mutagenic in bacteria in the absence and presence of metabolic activation.

An in vitro assay was performed to assess the potential of C12-14 alkylmorpholine to induce mutations at the thymidine kinase locus of the mouse lymphoma cell line L5178Y in accordance with the OECD Guideline 476 and EU Method B.17 and in compliance with GLP. The assay was performed in two independent experiments, using two parallel cultures each. The first experiment was performed with and without liver microsomal activation and a treatment period of 4 h. The second experiment was performed with a treatment time of 4 h with and 24 h without metabolic activation. The dose range of the first experiment was set according to the cytotoxicity data generated in the pre-experiment and ranges from 2.5 to 60 µg/mL (without metabolic activation) and 2.5 to 80 µg/mL (with metabolic activation). The dose ranges for the second experiment were 1.3 to 30 µg/mL (without metabolic activation) and 2.5 to 40 µg/mL (with metabolic activation). No substantial and reproducible dose dependent increase in mutant colony numbers was observed in both main experiments. No relevant shift of the ratio of small versus large colonies was observed up to the maximal concentration of the test substance. Under the experimental conditions reported, the test substance did not induce mutations in the mouse lymphoma thymidine kinase locus assay using the cell line L5178Y in the absence and presence of metabolic activation.

The test substance C12-14 alkylmorpholine, dissolved in ethanol, was assessed for its potential to induce structural chromosomal aberrations in human lymphocytes in vitro according to the OECD Guideline 473 and EU Method B.10 in compliance with GLP. Three independent experiments were performed. In Experiment I the exposure period was 4 h with and without S9 mix. In Experiment IIA the exposure period was 4 h with S9 mix and 22 h without S9 mix. In Experiment IIB the exposure period was 22 h without S9 mix. The chromosomes were prepared 22 h after the start of treatment with the test substance. In each experimental group two parallel cultures were analyzed. At least 100 metaphases per culture were scored for structural chromosomal aberrations, except for the positive control in Experiment IIB, in the absence of S9 mix, where only 50 metaphases were evaluated. 1,000 cells were counted per culture for determination of the mitotic index. The highest treatment concentration used was 2,700 μg/mL (approx. 10 mM). No visible precipitation of the test substance in the culture medium was observed. Phase separation was observed in Experiment I at 164.5 μg/mL and above in the absence and presence of S9 mix. In Experiment IIA at 85.7 μg/mL and above in the absence of S9 mix and at 50.0 μg/mL and above in the presence of S9 mix. In Experiment IIB in the absence of S9 mix phase separation was observed at 150 μg/mL. No relevant influence on pH value was observed. The osmolality was slightly decreased in Experiment I at the highest applied concentration. In Experiment I and IIB in the absence of S9 mix and in Experiment IIA in the presence of S9 mix, cytotoxicity was observed at the highest evaluated concentration (38.4, 42.8 and 42.6 % of control, respectively). In Experiment I in the presence of S9 mix and in Experiment IIA in the absence of S9 mix, concentrations showing clear cytotoxicity were not evaluable for cytogenetic damage. In the absence and presence of S9 mix, no biologically relevant increase in the number of cells carrying structural chromosome aberrations was observed. The aberration rates of the cells after treatment with the test substance (0.0 – 3.0 % aberrant cells, excluding gaps) were slightly above the range of the solvent control values (0.0 – 1.5 % aberrant cells, excluding gaps) and within the range of the laboratory historical solvent control data. No evidence of an increase in polyploid metaphases was noticed after treatment with the test substance as compared to the control cultures. Under the study conditions, the test substance did not induce structural chromosomal aberrations in human lymphocytes in vitro, when tested up to cytotoxic or the highest evaluable concentrations (Bohnenberger S, 2013).

In vivo genotoxicity:

In accordance with Annex VIII Column 2 of REACH,in vivo testing needs to be considered, if positive results have been observed in any of the available in vitro studies. As in the present case all available in vitro studies were negative, the performance of additional in vivo studies is not indicated.


Justification for selection of genetic toxicity endpoint
For each in vitro endpoint, bacterial mutagenicity, mammalian mutagenicity and mammalian clastogenicity, a GLP compliant study is available.

Short description of key information:
C12-14 alkylmorpholine is negative in all the in vitro assays.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Based on the results of the in vitro genetic toxicity studies, the substance does not need to be classified for genotoxicity according to EU CLP Regulation (EC) No. 1272/2008.