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Administrative data

Key value for chemical safety assessment

Additional information

in vitro

Ames Test:

The test substance N,N’’-Hexane-1,6-diylbis[N’-benzylurea] was tested for its mutagenic potential based on the ability to induce point mutations in selected loci of several bacterial strains, i.e. Salmonella typhimurium and Escherichia coli, in a reverse mutation assay according to OECD guideline 471 and GLP (BASF, 2012).

STRAINS: TA 1535, TA 100, TA 1537, TA 98 and E. coli WP2 uvrA

DOSE RANGE: 33 μg - 5 000 μg/plate (SPT); 33 μg - 5 000 μg/plate (PIT)

TEST CONDITIONS: Standard plate test (SPT) and preincubation test (PIT) both with and without metabolic activation (liver S9 mix from induced rats).

SOLUBILITY: Precipitation of the test substance was found from 333 μg/plate onward with and without S9 mix.

TOXICITY: A bacteriotoxic effect was observed depending on the strain and test conditions from about 2 500 μg/plate onward.

MUTAGENICITY: A relevant increase in the number of his+ or trp+ revertants was not observed in the standard plate test or in the preincubation test either without S9 mix or after the addition of a metabolizing system.

CONCLUSION: Thus, under the experimental conditions of this study, the test substance N,N’’-Hexane-1,6- diylbis[N’-benzylurea] is not mutagenic in the Salmonella typhimurium/Escherichia coli reverse mutation assay in the absence and the presence of metabolic activation.

In vitro chromosome aberration test:

The test item N,N“-Hexane-1,6-diylbis[N’-benzylurea], suspended in DMSO, was assessed for its potential to induce structural chromosome aberrations in V79 cells of the Chinese hamster in vitro in two independent experiments according to OECD 473 guideline and GLP (Harlan, 2012).

In each experimental group two parallel cultures were set up. 100 metaphases per culture were evaluated for structural chromosome aberrations, except for the positive control in Experiment II after 28 hours continuous treatment without metabolic activation, where only 50 metaphases were evaluated.

The highest applied concentration (718.3 ug/mL; approx. 1.9 mM) was chosen with regard to the solubility properties of the test item and with respect to the current OECD Guideline 473.

Dose selection for the cytogenetic experiments was performed considering the occurrence of test item precipitation.

In the absence and presence of S9 mix no cytotoxicity was observed up to the highest applied concentration.

No clastogenicity was observed at the concentrations evaluated either with or without metabolic activation. However, in Experiment II after 18 hours treatment with 5.6 ug/mL in the absence of S9 mix, one statistically significant increase in chromosomal aberrations

occurred (3.5 % aberrant cells, excluding gaps). This value was in the range of the historical solvent control data (0.0 - 4.0 % aberrant cells, excluding gaps) and is therefore regarded as biologically irrelevant.

No evidence of an increase in polyploid metaphases was found after treatment with the test item as compared to the frequencies of the control cultures.

Appropriate mutagens (EMS and CPA) were used as positive controls. They induced statistically significant increases in cells with structural chromosome aberrations.

In conclusion, it can be stated that under the experimental conditions reported, the test item did not induce structural chromosome aberrations in V79 cells (Chinese hamster cell line) in vitro.

Therefore, N,N“-Hexane-1,6-diylbis[N’-benzylurea] is considered to be non-clastogenic in this chromosome aberration test in the absence and presence of metabolic activation, when tested up to precipitating concentrations.

HPRT-Test:

The study was performed to investigate the potential of N,N“-Hexane-1,6-diylbis[N’-benzylurea] to induce gene mutations at the HPRT locus in V79 cells of the Chinese hamster according to OECD 476 guideline and GLP (Harlan, 2013).

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 highest concentration of 720 µg/mL used in the pre-experiment was limited by the solubility properties of the test item. The concentration range of the main experiments was limited by precipitation of the test item. The test item was suspended in DMSO.

No substantial and reproducible dose dependent increase of the mutation frequency was observed up to the maximum concentration with and without metabolic activation.

Appropriate reference mutagens (and DMBA), used as positive controls, induced a distinct increase in mutant colonies and thus, showed the sensitivity of the test system and the activity of the metabolic activation system.

Conclusion

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, N,N“-Hexane-1,6-diylbis[N’-benzylurea] is considered to be non-mutagenic in this HPRT assay.


Short description of key information:
Ames Test (BASF, 2012): negative
in vitro chromosome aberration test (Harlan, 2012): negative
HPRT-Test (Harlan, 2013): negative

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Based on the available data, no classification and labeling is required (according to Directive 67/548/EEC and according to CLP) for genetic toxicity.