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EC number: 229-713-7
CAS number: 6674-22-2
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.
The potential of the substance to cause genetic toxicity has been
investigated in an appropriate battery of assays in vitro.
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
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.
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.
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.
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