A mixture of: 3,3'-dicyclohexyl-1,1'-methylenebis(4,1-phenylene)diurea; 3-cyclohexyl-1-(4-(4-(3-octadecylureido)benzyl)phenyl)urea; 3,3'-dioctadecyl-1,1'-methylenebis(4,1-phenylene)diurea

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Two AMES tests (OECD 471), one chromosome aberration test (OECD 473) and a MLA assay (OECD 476) were performed with the substance. MDI/CHA/ODA was shown to be negative with and without metabolic activation in all these in vitro tests.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

In a micronucleus assay in the bone marrow of the mouse, performed according to OECD 474 guideline and GLP principles, MDI/CHA/ODA was found not to be mutagenic.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Ames test 1:

The genetic toxicity of MDI/CHA/ODA was assessed using Salmonella typhimurium TA98, TA100, TA1535 and TA1537 strains, and Escherichia coli WP2uvrA strain, in accordance with OECD 471 guideline and GLP principles. Precipitation was observed at the top dose of 100 µg/plate. No toxicity was observed in all strains. All bacterial strains showed negative responses over the entire dose range, i.e. no biologically significant dose-related increase in the number of revertants in two independently repeated experiments with and without metabolic activation up to the highest concentration of 100 µg/plate. Based on the results it is concluded that the substance is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

Ames test 2: 

A Salmonella typhimurium reverse mutation assay was performed according to OECD 471 guideline and GLP principles. All bacterial strains (TA98, TA100, TA1535, TA1537 and TA1538) showed negative responses up to and including 5000 μg/plate, i.e. no significant dose-related increase in the number of revertants with or without metabolic activation was seen. The plates incubated with the test substance showed normal background growth up to 5000 μg/plate with and without S9-mix in all five strains used. In strains TA98, TA1535, TA1537 and TA1538 a reduction in the number of revertants was observed at 5000 μg/plate with and without S9. Negative controls, solvent controls and positive controls (appropriate reference mutagens) were included and showed expected results 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 MDI/CHA/ODA is not mutagenic in the Salmonella typhimurium reverse mutation assay with or without metabolic activation.

Chromosome aberration test:

A chromosome aberration study with MDI/CHA/ODA was performed according to OECD 473 guideline and GLP principles, in cultured peripheral human lymphocytes in two independent experiments. No toxicity was observed at the highest tested precipitating concentration of 100 mg/mL. MDI/CHA/ODA did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in the absence and presence of S9-mix, in two independently repeated experiments. No effects of MDI/CHA/ODA on the number of polyploid cells and cells with endoreduplicated chromosomes were observed both in the absence and presence of S9-mix. Therefore it can be concluded that MDI/CHA/ODA does not disturb mitotic processes and cell cycle progression and does not induce numerical chromosome aberrations under the experimental conditions described in this report. Based on the results it can be concluded that MDI/CHA/ODA is not clastogenic in human lymphocytes.

Mouse lymphoma assay:

The mutagenic activity of MDI/CHA/ODA was tested in the mouse lymphoma assay conducted according to OECD 476 guideline and GLP principles.MDI/CHA/ODA was tested for 3 hours up to concentrations of 164 µg/ml in the absence and presence of S9-mix in the first experiment. In the second experimentMDI/CHA/ODA was tested for 24 hours up to concentrations of 164 µg/ml in the absence of S9-mix.No toxicity was observed and all dose levels were evaluated in the absence and presence of S9-mix in both experiments. In the absence and presence of S9-mix, MDI/CHA/ODA did not induce a significant increase in the mutation frequency. It is concluded that MDI/CHA/ODA is not mutagenic in the mouse lymphoma L5178Y test system.

Micronucleus assay:

A micronucleus assay in the bone marrow of male and female mouse with MDI/CHA/ODA was performed according to OECD 474 guideline and GLP principles. Bone marrow cells were collected for micronuclei analysis 24 h, 48 h and 72 h after a single application of 5000 mg/kg bw of the test article. In a pre-experiment, this dose was determined to be the maximum attainable dose. The animals expressed slight toxic reactions (apathy and reduction of spontaneous activity). After treatment with the test substance the ratio between polychromatic erythrocytes and normochromatic erythrocytes was not affected indicating no cytotoxic effects. There was no significant enhancement in the frequency of detected micronuclei at any preparation interval. Appropriate negative and positive controls were included. Based on the results of this study it is concluded that MDI/CHA/ODA is not clastogenic in the micronucleus assay in the bone marrow of the mouse.

Justification for classification or non-classification

Based on the available data, MDI/CHA/ODA is not classified for genotoxicity according to Regulation (EC) No. 1272/2008.