Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

A weight of evidence approach is used for this endpoint, based on negative results obtained in vitro in studies of bacterial reverse mutation and studies of forward mutation, DNA damage and micronucleus formation in mammalian cells.

Additional information

The potential of the substance to cause genetic toxicity has been investigated in an appropriate battery of assays in vitro.

Bacterial mutation

In a guideline- and GLP-compliant Ames test (BASF, 1999), DBU was investigated for the ability to induce reverse mutation in Salmonella typhimurium strains TA98, TA100, TA 1535 and TA 1535 and in Eschericia coli strain WP2uvrA. Strains were exposed to concentrations of DBU up to the limit concentration of 5000 ug/plate in the absence and presence of an exogenous metabolic activation system (Aroclor 1254 -induce rat liver S9 fraction) in an initial plate-incorporation assay and at concentrations of up to 2000 ug/plate in a confirmatory pre-incubation assay. Cytotoxicity was apparent at the highest concentrations used in both assays. No evidence of any induction of reverse mutation was seen under the conditions of this study; the sensitivity of the assay was confirmed by appropriate responses to positive control compounds.

Mammalian cell mutation

In a modern, guideline- and GLP-compliant HPRT assay (BASF, 2012), the ability of DBU to induce forward gene mutations was tested in Chinese hamster ovary (CHO) cells. Two independent experiments were carried out, both with and without the addition of metabolic activation (S9 mix) and with exposure periods of 4 and 24 hours. Appropriate vehicle controls and positive control substances were also used. No cytotoxicity was observed up to the highest required concentration evaluated for gene mutations. The substance did not cause any relevant increase in the mutant frequencies either without S9 mix or after the addition of a metabolising system in two experiments performed independently of each other.

Micronucleus assay

In a modern, guideline- and GLP-compliant study (BASF, 2013), the ability of DBU to induce the formation of micronuclei was assessed in cultured V79 cells in vitro. Two independent experiments were carried out, both with and without the addition of metabolic activation. In the first experiment (4 hour exposure, 24 hour harvest), cells were exposed to concentrations of up to 1600 μg/mL DBU in the absence and presence of metabolic activation. In the second experiment, cells were exposed to concentrations of up to 1600 μg/mL DBU in the absence of metabolic activation (24 hour exposure, 24 hour harvest) and up to 1600 μg/mL DBU in the presence of metabolic activation (4 hour exposure, 24 hour harvest. A sample of at least 1000 cells from each culture was analysed for the presence of micronuclei. No cytotoxicity (as indicated by reduced relative increase in cell count or proliferation index was observed in with 4-hour treatment; cytotoxicity was observed at the two highest concentrations in the second experiment in the absence of metabolic activation after 24 hours continuous treatment.  DBU did not cause any biologically relevant increase in the number of cells containing micronuclei with or without metabolic activation in two experiments carried out independently of each other.

Comet assay

DBU was additionally tested for the ability to cause DNA damage in a Comet assay using cultured primary human lymphocytes and chondrocytes. Cells were exposed to 0 and 0.5 mg/mL DBU for 60 minutes according to standard protocols for this assay. Cytotoxicity was assessed using staining with propidium iodide and fluorescein diacetate and the EZ4U proliferation assay in human chondrocytes. Cytotoxicity in lymphocytes was determined by visual observation using trypan blue staining. Genotoxicity in lymphocytes was assessed using the alkaline single cell microgel electrophoresis (Comet) assay. The test material was cytotoxic at concentration greater than 0.5 mg/mL and did not induce DNA damage under the test conditions.

The results of this battery of studies in vitro demonstrate that DBU does not cause DNA damage, gene mutations or chromosomal damage. No further testing for genotoxicity is therefore required.

Justification for classification or non-classification

The results of in vitro studies of genetic toxicity are negative; therefore no classifcation is proposed for genetic toxicity according to Regulation (EC) No. 122/2008.