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Key value for chemical safety assessment

Additional information

Ames test

In the Salmonella typhimurium and E. coli reverse mutation test performed according to OECD 471 FAT 40854/A TE was tested up to 5000 µg/plate.

In the absence of S9-mix, FAT 40854/A TE induced a 3.3-fold dose related increase in tester strain WP2uvrA after the pre-incubation treatment. The increase observed was above the laboratory historical control data range and was more than three times the concurrent control.

In the presence of S9-mix, FAT 40854/A TE induced dose related increases in two tester strains (TA100 and WP2uvrA) after the pre-incubation treatment. The increases observed in tester strain WP2uvrA were above the laboratory historical control data range, in two independently repeated experiments and were up to 6.4-fold the concurrent controls. The increases observed in tester strain TA100 were above the laboratory historical control data range in two independently repeated experiments and were up to 6.8-fold the concurrent controls. The increases observed in tester strain TA100 were only in the third experiment above the laboratory historical control data range, however the increase was more than two times the concurrent control in both experiments.

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, except for TA100 in the presence of S9-mix (second and third experiment; solvent control) and WP2uvrA in the presence of S9-mix (third experiment; positive control). Although these values were outside the limit of the range, clear mutagenic responses after treatment with FAT 40854/A TE were observed in these tester strains, therefore the validity of the test was considered to be not affected.

Since 3.3- to 6.8-fold, dose related increases were observed in two tester strains, both in the absence and presence of S9-mix (WP2uvrA) and in the presence of S9-mix (TA100) and the results obtained in the presence of S9-mix were reproducible in the repeat assay, these increases are considered biologically relevant, and FAT 40854/A TE is mutagenic in the absence and presence of S9-mix.

Chromosome aberration test

A chromosome aberration study with FAT 40854/A TE was tested up to cytotoxic concentrations according to OECD 473 in cultured peripheral human lymphocytes in two independent experiments. The number of cells with chromosome aberrations found in the solvent control was within the laboratory historical control data range. Positive controls produced an appropriate response.

FAT 40854/A TE did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in the absence and presence of metabolic activation. Based on these results, it is concluded that FAT 40854/A TE is not clastogenic.

Mouse lymphoma test

In a mouse lymphoma L5178Y assay FAT 40854/A TE was tested up to cytotoxic concentrations according to OECD 476.

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. Except the response of one of the solvent control cultures in the absence of S9-mix. However since this response was just below the lower limit of the range and clear positive results were obtained, the validity of the test was considered to be not affected. Mutation frequencies in cultures treated with positive control chemicals were appropriate.

In the absence of S9-mix, FAT 40854/A TE induced an up to 5.7-fold increase in the mutation frequency. The mutation frequencies were above the GEF + MF(controls)(174 per 106survivors), more than three-fold, outside the historical control data range and in a dose dependent manner, therefore, this increase is considered relevant and FAT 40854/A TE is considered mutagenic in the absence of S9-mix.

In the presence of S9-mix, FAT 40854/A TE induced an up to 11-fold increase in the mutation frequency. The mutation frequencies were above the GEF + MF(controls)(207 per 106survivors), more than three-fold, outside the historical control data range and in a dose dependent manner, therefore, this increase is considered relevant and FAT 40854/A TE is considered mutagenic in the presence of S9-mix.

Based on these results, FAT 40854/A TE is considered to be mutagenic.

In vivo micronucleus

In a micronucleus test rats were treated with 500, 1000 or 2000 mg/kg bw FAT 40854/A TE by gavage according to OECD 474. The incidence of micronucleated polychromatic erythrocytes in the bone marrow of the negative control animals was within the historical vehicle control range. Cyclophosphamide as positive control induced an appropriate response. No decrease of the ratio of polychromatic to normochromatic erythrocytes in FAT 40854/A TE or positive control treated animals was observed compared to vehicle control, indicating a lack of toxic effects of this test substance on erythropoiesis. None of the animals showed any clinical signs or mortality.

No increase in the mean frequency of micronucleated polychromatic erythrocytes was observed in the bone marrow of animals treated with FAT 40854/A TE. Therefore, FAT 40854/A TE can be considered to be not clastogenic or aneugenic.

Unscheduled DNA synthesis (UDS) assay (OECD 486)

The test article, FAT 40854, was tested in the unscheduled DNA synthesis (UDS) assay with rat liver cells in vivo. The UDS assay was used to evaluate the potential of the test article to induce unscheduled DNA synthesis in primary cultures obtained from test article-treated Sprague-Dawley rats. This experiment was done according to the OECD Guideline 486 (Unscheduled DNA Synthesis (UDS) Test with Mammalian Liver Cells in vivo).

In the UDS assay, FAT 40854 was administered to 4 male rats per dose at two time points (2 to 4 hours and 12 to 16 hours) in single oral doses of 500, 1000 and 2000 mg/kg bw. Two additional groups of 4 rats each received a single oral dose of 0.9% NaCl Injection, USP or 35 mg/kg dimethylnitrosamine (DMN) which served as the vehicle and positive control, respectively. The test and control articles were administered at a constant volume of 10 mL/kg by a single oral gavage. In the definitive assay, no mortality was observed. However, clinical signs were observed in the positive control group at the 2 to 4 hour harvest.

The animals (3/group) were euthanized 2 to 4 hours postdose and 12 to 16 hours postdose to harvest hepatocytes. Hepatocytes were exposed to culture media containing tritiated thymidine for 4 hours, washed and incubated another 17 to 20 hours, and then examined using a microscope and software to quantify the amount of tritiated thymidine incorporated into the cells, as measured by the mean net nuclear grain count (MNNGC). These data were compared between groups to determine if FAT 40854 increased incorporation of tritiated thymidine, which would be considered an indication that it caused DNA damage that induced DNA synthesis/repair.

Conclusion:

In the UDS assay, FAT 40854 did not cause a significant increase in average mean MNNGC at any dose level or harvest time. At both harvest times, the proportion of cells in repair in the vehicle control group was less than 15%, the average mean MNNGC of the vehicle control group was less than 1, and the average mean MNNGC of the positive control group was at least 5 counts over that of the vehicle control group.

Based on these results, all criteria for a valid study were met, and FAT 40854 was concluded to be negative for genotoxic potential in hepatocytes from male Sprague-Dawley rats given single oral dose at up to 2000 mg/kg bw.

Justification for selection of genetic toxicity endpoint
A reliable bacterial reverse mutation test, in vitro chromosome aberration and mouse lymphoma test, and in vivo micronucleus and unscheduled DNA synthesis (UDS) test, all performed according to OECD/EC guidelines, are available.

Short description of key information:
A bacterial reverse mutation test, in vitro chromosome aberration test and mouse lymphoma test, and in vivo micronucleus and unscheduled DNA synthesis (UDS) test performed according to OECD guidelines and GLP principles were available. FAT 40854/A TE was found to be mutagenic in the bacterial reverse mutation assay and in the mouse lymphoma assay in the absence and presence of S9-mix. FAT 40854/A TE was not found to be clastogenic in the chromosome aberration test with and without metabolic activation. In an in vivo micronucleus test FAT 40854/A TE was not clastogenic or aneugenic. In the UDS assay, FAT 40854 was concluded to be negative for genotoxic potential in hepatocytes from male Sprague-Dawley rats given single oral dose at up to 2000 mg/kg bw.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Based on the results above, classification for genotoxicity is not needed.