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Salmonella and E.Coli test in vitro (AmesTests):

FeNaEDDHA was tested for the ability to induce mutagenic effect in histidine-requiring strains of Salmonella mutagenic (TA 98, TA 100, TA 102, TA 1535, TA 1537) and in a tryptophan-requiring strain of Eschericia coli (WP2 uvr A). The test compound was dissolved in bidistilled water and tested at five concentrations ranging from 312.5 to 5000 ug/plate with and without metabolic activation.

Suitable positive controls were used for each strain. All experiments were repeated in order to confirm the results.

The results revealed no increased incidence of mutants by the test item with and without metabolic activation. Therefore, it was concluded that the test compound did not show mutagenic activity in S. typhimurium and E. coli. The positive controls induced mutagenic activity. In conclusion, FeNaEDDHA provoked no mutagenic activity in this test system.

In-vitro Mammalian Cell Gene Mutation Test (Mouse Lymphoma Assay):

FeNaEDDHA was tested for the ability to provoke mutations at the tk locus in L5178Y mouse lymphoma cells in vitro. The test compound was dissolved in DMSO. The range finding experiments showed that 1000upg/mL was the highest concentration which could be used. Higher concentrations (greater than 100 mg/ml) produced precipitates in the vehicle.

The results of the toxicity experiment which revealed cytotoxicity at the two highest concentrations with metabolic activation (Liver S9-fraction from Arochlor 1254 treated rats). In absence of metabolic activation no toxicity was noted.

For the mutagenicity experiment concentrations ranging from 0 to 125 ug/mL with metabolic activation and from 0 to 1000 ug/mL without metabolic activation were used. In the confirmatory experiment with metabolic activation concentrations ranging from 0 to 250 ug/mL were applied. The same concentrations (0 to 1000 ug/mL) were used in the confirmatory experiment without metabolic activation.

Corresponding positive controls (N-Nitrosodimethylamine, with metabolic activation and Ethylmethansulfonate without metabolic activation) were included. The mouse lymphoma cells were treated for 4 hours. After two days expression time, mutations at the tk locus were selected by resistance to 5-trifluorothymidine. Two types of colonies were selected, large colonies (base-pair substitutions and deletions) and small colonies (chromosome aberrations). The results showed no increase incidence of mutations at the tk locus of mouse lymphoma L5178Y cells in presence or absence of metabolic activation. Positive controls showed mutagenic activity. Conclusion: FeNaEDDHA was not mutagenic in this test system in vitro.

In-vitro Mammalian Chromosome Abberation Test

The test compound FeNaEDDHA was tested for the ability to provoke clastogenic effects in Chinese hamster ovary cells (CCL61) in vitro. The compound was dissolved in DMSO and tested without metabolic activation at concentrations of 0, 7.81,15.63 and 31.25 ug/mL for 18 and 42 hours. With metabolic activation (liver S9 fraction from Aroclor 1254 induced rat liver) concentrations of 0. 31.25, 62.5 and 125 ug/mL were applied for 3 hours followed by 15 hours recovery or 3 hours followed by 39 hours recovery. Higher concentrations could not be reached due to solubility limitations.

Three independent experiments of each with and without metabolic activation were performed. Two replicate culture per concentration and 200 cells per concentration were evaluated.

The results showed in both experiments with and without metabolic activation no increased number of metaphases with chromosomal aberrations. In contrast, the positive controls (Mitomycin 0.2 ug/mL and Cyclophosphamide 20 ug/mL) induced clastogenic effects. In conclusion, the test substance provoked no clastogenic activity in this test in vitro.

Conclusion: FeNaEDDHA provoked no mutagenic activity, when tested for gene mutation in bacteria and mammalian cells in vitro and chromosome aberration in mammalian cells in vitro. Therefore, it is concluded that the test item is devoid of a mutagenic activity.

Short description of key information:
FeNaEDDHA was examined in three different in vitro genetic toxicity studies, all three with and without metabolic activation. The test item did not induce gene mutations by frameshift or base-pair substitution in the examined strains in the Ames test. FeNaEDDHA tested up to cytotoxic concentrations did not induce structural chromosome aberrations in Chinese Hamster ovary cells and was therefore not considered clastogenic in the tested system. Finally the test substance showed no mutagenic effect in a Mouse Lymphoma assay. Overall, FeNaEDDHA was considered non genotoxic.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. The outcome of all genetic toxicity tests was negative for the test item. As a result the substance is not considered to be classified as mutagenic under Regulation (EC) No 1272/2008, as amended for the ninth time in Regulation (EC) No 2016/1179.