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Additional information

There is only data available on the mutagenicity of Reaction products of oleoyl sarcosine with sodium hydroxide in bacteria. In order to fulfil the standard information requirements set out in Annex VII and VIII, 8.4., in accordance with Annex XI, 1.5., of Regulation (EC) No 1907/2006, read-across from structurally related substances was performed for the other endpoints of genetic toxicity.

In accordance with Article 13 (1) of Regulation (EC) No 1907/2006, "information on intrinsic properties of substances may be generated by means other than tests, provided that the conditions set out in Annex XI are met.” In particular for human toxicity, information shall be generated whenever possible by means other than vertebrate animal tests, which includes the use of information from structurally related substances (grouping or read-across).

Having regard to the general rules for grouping of substances and read-across approach laid down in Annex XI, Item 1.5., of Regulation (EC) No 1907/2006, whereby substances may be predicted as similar provided that their physicochemical, toxicological and ecotoxicological properties are likely to be similar or follow a regular pattern as a result of structural similarity, the substance listed below are selected as reference substances for hazard assessment.

Discussion

Genetic toxicity (mutagenicity) in bacteria in vitro

A bacterial gene mutation assays (Ames test) was performed with Reaction products of oleoyl sarcosine with sodium hydroxide following OECD guideline 471 and in compliance with GLP (Paulus, 2012).

The strains Salmonella typhimurium TA97a, TA98, TA 100, TA 102 and TA 1535 were tested in two independent experiments according to the plate incorporation and preincubation procedure in the absence and presence of a metabolic activation system (Aroclor 1254-induced rat liver S9-mix). The experiments were conducted each in 4 replications at concentrations from 50 to 5004 µg/plate (vehicle: water). No increase in the number of revertant colonies was noted in any of the bacterial strains, with and without metabolic activation system. No cytotoxicity was observed up to the highest dose tested. The included positive and negative controls showed the expected results.

Under the conditions of the study, the test substance did not induce mutations in the bacterial mutation assay in the absence and presence of a metabolic activation system in any of the strains tested.

Genetic toxicity (cytogenicity) in mammalian cells in vitro

No studies are available investigating the cytogenicity in mammalian cells with Reaction products of oleoyl sarcosine with sodium hydroxide. In order to fulfil the standard information requirements set out in Annex VIII, 8.4.2. and in accordance with Annex XI, 1.5., of Regulation (EC) No 1907/2006, read-across from the structurally related category members Sodium N-lauroylsarcosinate (CAS 137-16-6) and (Z)-N-methyl-N-(1-oxo-9-octadecenyl) glycine (CAS 110-25-8) was conducted.

An in vitro mammalian chromosome aberration test was conducted with Sodium N-lauroylsarcosinate (CAS 137-16-6) in accordance with OECD guideline 473 under GLP conditions (Morris, 2010). The induction of structural chromosome aberrations was evaluated in vitro in lymphocytes of fresh heparised human whole blood cultures, incubated for 4 and 24 h with and without a metabolic activation system (S9-mix from rats treated with phenobarbitone and beta-naphthoflavone).

Concentrations of 22-360 µg/mL (4 h incubation) and 22.5-270 µg/mL (24 h incubation) of the test substance in Minimal Essential Media (MEM) were applied. The negative as well as the positive controls showed the expected results and were within the historical control data. In the preliminary toxicity test, haemolysis of the cultures was observed at 732.5 µg/mL and above in the 4 and 24 h exposure groups.

In the main experiment, haemolysis was seen at the end of exposure at and above 270 µg/mL in the exposure groups. The test material did not induce a statistically significant increase in the numbers of polyploid cells at any dose level in any of the exposure groups. No statistically or biologically significant increase in the incidence of chromosome aberrations was observed.

Therefore, under the conditions of the study, the test substance did not show clastogenic activity in this chromosomal aberration test with and without metabolic activation performed in peripheral human lymphocytes in vitro.

An in vitro mammalian chromosome aberration test was conducted with (Z)-N-methyl-N-(1-oxo-9-octadecenyl) glycine (CAS 110-25-8) in accordance with OECD guideline 473 under GLP conditions (Paulus, 2011). The induction of structural chromosome aberrations was evaluated in vitro in lymphocytes of fresh heparised human whole blood cultures, incubated for 4 h with and without and for 22 h without a metabolic activation system (S9-mix from rats treated with Aroclor 1245).

Concentrations of 10-40 µg/mL (4 h incubation) and 0.5-50 µg/mL (22 h incubation) of the test substance in the vehicle DMSO were applied. The negative as well as the positive controls showed the expected results. In the pre-experiment cytotoxicity was observed from 55.3 µg/mL with and without metabolic activation. In the main experiments a reduction in the mitotic index was observed from 30 µg/mL without and from 40 µg/mL with metabolic activation. No statistically or biologically significant increase in the incidence of chromosome aberrations was observed.

Therefore, under the conditions of the study, the test substance did not show clastogenic activity in this chromosomal aberration test with and without metabolic activation performed in peripheral human lymphocytes in vitro.

Genetic toxicity (mutagenicity) in mammalian cells in vitro

No studies are available investigating the mutagenicity in mammalian cells of with Reaction products of oleoyl sarcosine with sodium hydroxide. In order to fulfil the standard information requirements set out in Annex VIII, 8.4.3. and in accordance with Annex XI, 1.5., of Regulation (EC) No 1907/2006, read-across from the structurally related category members Sodium N-lauroylsarcosinate (CAS 137-16-6), and (Z)-N-methyl-N-(1-oxo-9-octadecenyl) glycine (CAS 110-25-8) was conducted.

The in vitro mammalian cell gene mutation study of Sodium N-lauroylsarcosinate (CAS 137-16-6) was carried out according to OECD guideline 476 under GLP conditions (Brown, 2010). Gene mutations in the thymidine kinase locus were investigated in L5178Y mouse lymphoma cells in the presence and absence of a metabolic activation system (Phenobarbital/beta-naphtoflavone-induced rat liver, S9).

In the first experiment, cells were exposed for 4 h to test substance at concentrations of 6.25-70 µg/mL and 12.5-100 µg/mL without and with metabolic activation, respectively. Concentrations of the second experiment without metabolic activation for an exposure time of 24 h ranged from 3.13 to 80 µg/mL. The vehicle and positive controls in the study showed the expected results and were within the range of historical control data of the laboratory. In the short- and long-term exposure experiments, cytotoxicity was observed from 60 and 70 µg/mL (4 h) without and with metabolic activation and from 12.5 µg/mL (24 h), respectively. There was no significant increase in the number of forward mutations at the thymidine kinase locus of L5178Y mouse lymphoma cells treated with the test material, neither in the presence nor in the absence of a metabolic activation system.

Under the conditions of the study, Sodium N-lauroylsarcosinate (CAS 137-16-6) did not show gene mutation activity in this test performed in L5178Y mouse lymphoma cells in vitro.

The in vitro mammalian cell gene mutation study of (Z)-N-methyl-N-(1-oxo-9-octadecenyl) glycine (CAS 110-25-8) was carried out according to OECD guideline 476 under GLP conditions (Wollny, 2010).

Gene mutations in the HPRT locus were investigated in Chinese hamster lung fibroblasts (V79) cells in the presence and absence of a metabolic activation system (Phenobarbital/beta-naphtoflavone-induced rat liver, S9). V79 cells were incubated with the test material at 0.25, 0.5, 1, 2 and 4 µg/mL for 4 h in the absence and at 4, 8, 16, 32 and 48 µg/mL in the presence of a metabolic activation system. Concentrations of the second experiment without metabolic activation for an exposure time of 24 h ranged from 4 to 96 µg/mL, and concentrations with metabolic activation for an exposure period of 4 h ranged from 4 to 56 µg/mL. The vehicle and positive controls in the study showed the expected results and were within the range of historical control data of the laboratory. Cytotoxicity was apparent at concentrations from 4 µg/mL and 48 µg/mL in experiment I without and with metabolic activation, respectively. In the second experiment, cytotoxicity was observed from 96 µg/mL and 56 µg/mL without and with metabolic activation, respectively. In the second experiment, precipitation was observed from 237.5 µg/mL after 24 h without metabolic activation. There was no significant increase in the number of forward mutations at the HPRT locus of V79 cells treated with the test material, neither in the presence nor in the absence of a metabolic activation system.

Under the conditions of the study, (Z)-N-methyl-N-(1-oxo-9-octadecenyl) glycine (CAS 110-25-8) did not show gene mutation activity in this test performed in V79 cells in vitro.


Justification for selection of genetic toxicity endpoint
No study was selected, since all available studies were negative. Hazard assessment is conducted by means of an Ames test available for the substance itself and read-across from structurally related category members. The available data are adequate and reliable based on the identified similarities in structure and intrinsic properties among the category members.

Short description of key information:
Negative results in Salmonella typhimurium TA 97a, TA 98, TA 100, TA 102 and TA 1535, with and without metabolic activation (OECD 471, GLP)
Negative results in mammalian chromosomal aberration test with cultured peripheral human lymphocytes (OECD 473, GLP, category approach)
Negative results in mammalian cell gene mutation tests using mouse lymphoma cells and Chinese Hamster V79 cells, with and without metabolic activation (OECD 476, GLP, category approach)

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

The available data on genetic toxicity from the test substance Reaction products of oleoyl sarcosine with sodium hydroxide and from its structurally related category members do not meet the criteria for classification according to Regulation (EC) No 1272/2008 or Directive 67/548/EEC, and are therefore conclusive but not sufficient for classification.

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