Reaction mass of 3,6-Bis(3-chlorophenyl)-2,5-dihydro-pyrrolo[3,4-c]pyrrole-1,4-dione, 3-(3-Chlorophenyl)-6-(4-chlorophenyl)-2,5-dihydro- pyrrolo[3,4-c]pyrrole-1,4-dione and 3,6-Bis(4-chlorophenyl)-2,5-dihydro-pyrrolo[3,4-c]pyrrole-1,4-dione

Endpoint summary

Additional information

Ames test:

In a GLP-compliant study tested, performed according to OECD guideline 471, the mutagenic activity of the test substance was evaluated (Notox 2005). The test substance was tested in the Salmonella typhimurium reverse mutation assay with four histidine-requiring strains of Salmonella typhimurium (TA 1535, TA 1537, TA 100 and TA 98) and in the Escherichia coli reverse mutation assay with a tryptophan-requiring strain of Escherichia coli (WP2uvrA). The test was performed in two independent experiments in the presence and absence of S9-mix (rat liver S9-mix induced by a combination of phenobarbital and ß-naphthoflavone). In the first mutation assay, the test substance was tested up to concentrations of 5000 µg/plate and precipitation was observed on the plates at dose levels of 100 µg/plate and upwards. In the second mutation assay, the test substance was tested up to concentrations of 100 µg/plate and precipitation on the plates at the top dose of 100 µg/plate was observed. In both experiments, the bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed. Furthemore, no increase in the number of revertants was observed under all conditions tested. In this study, 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. Based on the results of this study it is concluded that the test substance is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

Chromosomal aberration test:

In a GLP compliant study, performed according to OECD guideline 473, the ability of the test substance to induce chromosome aberrations in cultured peripheral human lymphocytes was investigated. (Notox 2006) The possible clastogenicity was tested in two independent experiments, in the presence and absence of a metabolic activation system (phenobarbital and ß-naphthoflavone induced rat liver S9-mix). The test substance was suspended in dimethyl sulfoxide. In the first cytogenetic assay, the test substance was tested up to 10 µg/mL for a 3 h exposure time with a 24 h fixation time in the absence and presence of 1.8% (v/v) S9-fraction. In the second cytogenetic assay, the test substance was tested up to 33 µg/mL for a 24 h and 48 h continuous exposure time with a 24 h and 48 h fixation time in the absence of S9-mix. In the presence of S9-mix the test substance was tested up to 10 µg/mL for a 3 h exposure time with a 48 h fixation time. Positive control chemicals, mitomycin C and cyclophosphamide, both produced a statistically significant increase in the incidence of cells with chromosome aberrations, indicating that the test conditions were adequate and that the metabolic activation system functioned properly. In the first cytogenetic assay in the absence of S9-mix a statistically significant increase in the number of cells with chromosome aberrations at the lowest tested concentration only, when gaps were included, was observed. Since the type of aberrations observed were only breaks and gaps, the increase was not dose related and moreover the number of cells with chromosome aberrations was well within the historical control data range. Therfore, the increase was considered not biologically relevant. In the presence of S9-mix, no statistically significant or biologically relevant increase in the number of cells with chromosome aberrations was observed. In the second cytogenetic assay, both in the absence and presence of S9-mix no statistically significant or biologically relevant increase in the number of cells with chromosome aberrations was observed. No effects 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 the test substance does not disturb mitotic processes and cell cycle progression and does not induce numerical chromosome aberrations under the experimental conditions described in this report.

Short description of key information:
The test substance is not mutagenic in the Salmonella typhimurium and Escherichia coli reverse mutation assay and is not clastogenic in human lymphocytes.

Endpoint Conclusion:

Justification for classification or non-classification

As only results of a reverse mutation assay and an in vitro chromosome aberration test is available, a conclusion regarding classification according to Directive 67/548/EEC and according to the EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008 cannot be drawn.