reaction product of diphenylmethanediisocyanate, octylamine, 4-ethoxyaniline and ethylenediamine (1:0,37:1,53:0,05)

Endpoint summary

Additional information

Bacterial reverse mutation test

The test item Urea 4 was investigated for its mutagenic potential in a bacterial reverse mutation test according to EU Method B.13/14, OECD Guideline 471 and EPA OPPTS 870.5265. Five S. typhimurium strains (TA 1535, TA 1537, TA 98, TA 100 and TA 102) were tested with and without metabolic activation. A plate incorporation test and a pre-incubation test were performed independently and the following concentrations of the test item, tested in triplicate, were used in both experiments: 33.3; 100.0; 333.3; 1000.0; 2500.0 and 5000.0 µg/plate.

Precipitation of the test item was observed on the agar plates at concentrations >= 333.3 µg/plate. Cytotoxic effects of the test item were not observed. Reference mutagens showed a distinct increase of induced revertant colonies.

No substantial increase in the revertant colony numbers of any of the five test strains was detected at any dose level of the test item either with or without metabolic activation in both independently performed experiments. Thus, Urea 4 was considered non-mutagenic in this bacterial reverse mutation assay.

In vitro mammalian cell chromosome aberration test

An in vitro mammalian chromosome aberration test was performed with the test item Urea 4 according to EU Method B.10, OECD Guideline 473 and EPA OPPTS 870.5375. The test item potential to induce structural chromosome aberrations in V79 Chinese hamster cells was investigated in two independent experiments. In the two independent experiments the chromosomes were prepared 20 h after start of treatment with the test item. Experiment I was performed with and without S9 mix using a treatment interval of 4 h and a preparation interval of 20 h. Experiment II was performed only without metabolic activation with a treatment and preparation interval of 20 h. Two parallel cultures were set up per test group. Per culture 100 metaphases were scored for structural chromosomal aberrations. The following concentrations were evaluated (higher concentrations could not be evaluated due to the solubility characteristics of the test item):

Experiment I: 4 h treatment, 20 h preparation interval:

with and without metabolic activation (S9 mix) and test item concentrations: 25, 250, 500 µg/mL

Experiment II: 20 h treatment and preparation interval:

without metabolic activation (S9 mix) and test item concentrations: 10, 25, 50 µg/mL

Reference mutagens as positive controls were tested in parallel to the test item and showed an expected distinct increase in cells with structural chromosome aberrations.

Treatment of the cells with the test item in both experiments did not lead to a relevant decrease of the relative mitotic index or of the cell density.

When compared to the corresponding solvent control no biologically relevant increase in aberrant cells was obtained with and without metabolic activation in both independent experiments. No biologically relevant increase in the frequencies of polyploidy metaphases was found after treatment with the test item if compared to the frequencies of the controls.

It was concluded that the test item did not induce structural chromosome aberrations with and without metabolic activation. Thus, Urea 4 was considered non-mutagenic in this chromosome aberration test.

The in vitro mammalian cell gene mutation test

A study according to OECD guideline 476 has been commissioned and the data will be submitted to ECHA as soon as the results of the study become available. Results of the HPRT study should be available in mid of 2016.

Justification for selection of genetic toxicity endpoint
Two GLP and guideline conform studies investigating gene mutations in bacteria and chromosome aberrations in vitro.

Short description of key information:
The test substance was tested for in vitro mutagenic/ genotoxic activity in a bacterial reverse mutation test (Ames test) and in an in vitro mammalian chromosome aberration test according to OECD Guideline 471 and OECD 473 Guideline, respectively. The in vitro mammalian cell gene mutation test (OECD 476) has bee commissioned.
The test item did not induce point or frameshift mutations in the bacterial reverse mutation test performed with five strains of S. typhimurium with and without metabolic activation. Therefore, the test item was considered non-mutagenic in the Ames test performed. The test item did not induce structural chromosome aberrations in the in vitro chromosome aberration test performed with Chinese hamster V79 cells in the presence and absence of metabolic activation. Therefore, the test item is not considered clastogenic in this chromosome aberration test.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Based on the available in vitro studies, the test item was not classified and labelled as mutagenic according to Regulation (EC) No 127272008 (CLP), as amended for the eighteenth time in Regulation (EU) 2022/692.