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Endpoint summary

Additional information

Genetic toxicity in vitro:

Gene mutation in bacteria / Bacterial reverse mutation assay:

In a GLP-compliant bacterial reverse mutation assay, according to OECD guideline 471, the test substance was tested for its mutagenic potential based on the ability to induce point mutations in selected loci of several bacterial strains, (Salmonella typhimurium strains: TA 1535, TA 100, TA 1537, TA 98 and Escherichia coli WP2 uvrA) with and without metabolic activation (Aroclor-induced rat liver S-9 mix) (BASF, 2006). Three experiments were carried out independently (two standard plate tests and a preincubation assay). In the first standard plate test all strains were tested with and without S-9 mix at a dose range of 20.0 µg - 5000 µg/plate and in the second standard plate test all strains were tested without S-9 mix at a dose range of 0.8 µg - 500 µg/plate. In the plate incorporation test Salmonella strains without S-9 mix (0.8 µg - 500 µg/plate) and with S-9 mix (4.0 µg - 2 500 µg/plate) were tested as well as the E. coli strain with and without S-9 mix (4.0 µg - 2 500 µg/plate). Precipitation of the test substance was found from about 2500 µg/plate onward. A bacteriotoxic effect was observed depending on the strain and test conditions from about 500 µg - 2500 µg/plate onward. An increase in the number of his+ or trp+ revertants was not observed in the standard plate test or in the preincubation test either without S-9 mix or after the addition of a metabolizing system. According to the results of the present study, the test substance is not mutagenic in the Salmonella typhimurium/Escherichia coli reverse mutation assay under the experimental conditions chosen.

Cytogenicity in mammalian cells / Micronucleus test:

Two in vitro micronucleus assays were performed with the allophanate-type HDI oligomers. In principle, both assays solely vary by the use of the solvent applied (EGDE vs. DMSO). Diisocyanates have been shown to derivatize with polar solvents such as commercially available dimethylsulfoxide (DMSO; Herbold et al., 1998). The use of a less polar solvent such as ethyleneglycoldimethylether (EGDE) has been shown to effectively prevent such derivatization. This experimental adaptation can therefore guarantee the stability of the test substance ahead of its application into the test system. The micronucleus assay performed with EGDE (Bayer, 2012) can therefore be validated to be more reliable to assess the in vitro cytogenicity of the allophanate-type HDI oligomers in mammalian cells compared to the one performed with DMSO (BASF SE, 2006).

In this GLP-compliant study, performed according to OECD guideline 487, the test substance was examined for mutagenic activity in the micronucleus test in vitro, in V79 cells (Bayer, 2012). In this study, two independent assays were performed, the first assay conducted with a treatment time of 4 hours (pulse treatment), consisting of one experiment in the absence and one experiment in the presence of an extrinsic metabolizing system (Aroclor 1254 incuced S9 mix). In the second assay, one experiment without S9 mix was performed with treatment time extended to 24 hours (continuous treatment) and one experiment in the presence of S9 mix with a treatment time of 4 hours. Ethylene glycol dimethylether (EGDE, dried with a molecular sieve, 0.3 nm) was selected as solvent. The negative control (dried ethylene glycol dimethylether) and appropriate positive controls with known mutagens (mitomycin C, vinblastine sulfate, cyclophosphamide) demonstrated the suitability and sensitivity of the test system. The micronucleus test showed no biologically relevant increase in the frequencies of micronucleus containing V79 cells treated with the test item in the absence (4 hours or 24 hours treatment) or in the presence of S9 mix. Evaluation of the data indicates that the test substance is no mutagen in the micronucleus test in vitro, when tested in the absence or presence of metabolic activation up to clearly cytotoxic or precipitating concentrations.

Gene mutation in mammalian cells / HPRT mutation assay:

A GLP-compliant gene mutation assay, tested according to OECD guideline 476, was performed to investigate the potential of the test substance to induce gene mutations at the HPRT locus in V79 cells of the Chinese hamster (Harlan 2012). The assay was performed in two independent experiments, using two parallel cultures each. The first main experiment was performed with and without liver microsomal activation and a treatment period of 4 hours. The second experiment was performed with a treatment time of 4 hours with and 24 hours without metabolic activation. The test item was dissolved in ethylene glycol dimethyl ether (EGDE, dry). The concentration range of the main experiments was limited by cytotoxic effects observed in the pre-experiment. No substantial and reproducible dose dependent increase of the mutation frequency was observed up to the maximum concentration with and without metabolic activation. In conclusion it can be stated that under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells.Therefore, the test substance is considered to be non-mutagenic in this HPRT assay.

 

Genetic toxicity in vivo:

Micronucleus assay:

In a GLP-compliant study, according to OECD guideline 474, the test substance was tested for chromosomal damage (clastogenicity) and for its ability to induce spindle poison effects (aneugenic activity) in NMRI mice using the micronucleus test method (BASF 2006). For this purpose, the test substance, dissolved in corn oil, was administered twice orally, with a 24-hour interval between administrations, to male animals at dose levels of 500, 1000 mg/kg, and 2000 mg/kg body weight in a volume of 10 mL/kg body weight. The animals were sacrificed and the bone marrow of the two femora was prepared 24 hours after the second administration. After staining of the preparations, 2000 polychromatic erythrocytes were evaluated per animal and investigated for micronuclei. The normocytes with and without micronuclei occurring per 2000 polychromatic erythrocytes were also recorded. The two oral administrations of the test substance did not lead to any increase in the number of polychromatic erythrocytes containing either small or large micronuclei. The rate of micronuclei was always close to the same range as that of the concurrent negative control in all dose groups and within the range of the historical control data. A slight inhibition of erythropoiesis, determined from the ratio of polychromatic to normochromatic erythrocytes, was detected all doses administered. Thus, under the experimental conditions chosen here, the test substance does not have any chromosome-damaging (clastogenic) effect, and there were no indications of any impairment of chromosome distribution in the course of mitosis (aneugenic activity) in bone marrow cells in vivo. With the limitations of testing hydrolytically unstable substances on the oral route of exposure, no indication for a genotoxic mode of action was identified in this assay.

Herbold B. et al. (1998), Studies on the effect of the solvents dimethylsulfoxide and ethyleneglycoldimethylether on the mutagenicity of four types of diisocyanates in the Salmonella/microsome test, Mutation Research 412 (1998) 167-175.

Short description of key information:
The test substance is not mutagenic in the Salmonella typhimurium/Escherichia coli reverse mutation assay (BASF SE, 2006), and in the HPRT assay (Harlan, 2013).
The test substance is not genotoxic in the micronucleus test in vitro in V79 cells (BASF SE, 2006).
All assays were performed with and without the presence of a metabolozing system.
In a mouse miconucleus assay two oral applications of up to 2000 mg/kg bw did not result in an increase of polychromatic erythrocytes containing micronuclei (BASF SE, 2006).

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Based on the available genotoxicity tests, the test substance does not need to be classified for genotoxicity according to the Directive 67/548/EEC and according to the EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.