Registration Dossier

Toxicological information

Genetic toxicity: in vitro

Currently viewing:

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
1995

Materials and methods

Principles of method if other than guideline:
The Ames fluctuation test, an in vitro test detecting point mutation on Salmonella typhimurium. This assay is the modification, in liquid medium, of the widely used Ames/Salmonella assay. Thanks to its better sensitivity, the fluctuation test is very well suited to the search for mutagenicity in water samples (Wilcox and Denny, 1985; Monarca et al., 1985).
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid

Method

Species / strain
Species / strain / cell type:
S. typhimurium TA 100
Details on mammalian cell type (if applicable):
Source of cells: B.N. Ames (University of California, Berkeley, USA)
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9 (rat)
Test concentrations with justification for top dose:
3-300 µg/ml without metabolic activation, 1-1000 µg/ml with metabolic activation
Vehicle / solvent:
DMSO
Controls
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Remarks:
1 ng/ml 4-NQO without the metabolic system and 60 µg/ml CPA with metabolic activation
Details on test system and experimental conditions:
The Ames-fluctuation test was performed as described by Hubbard et al. (1984). This assay is a modification of the Ames test: the compound under study is exposed to bacteria in a liquid medium in many replicate cultures (96-well microplate) instead of the agar plate used in the Ames assay.
After the 3-day incubation, bromothymol blue (600 µg/ml) was added. Positive wells (containing prototrophic mutants) turned yellow whereas negative wells remained green. For each experiment, spontaneous reversion in response to the solvent used (DMSO) was included. Compounds were tested at least twice (two independent assays) using 3 experimental points for each concentration.
Rationale for test conditions:
Strain TA100 was used during all the experiment because it is considered as the most sensitive strain to water mutagens and particularly to chlorinated by-products (Loper, 1980; Forster et al., 1983; Harrington et al., 1983; Vartiainen and Liimatainen, 1986; Fielding and Horth, 1987; Meier, 1988).
Evaluation criteria:
A chemical is considered as toxic when it produces a significant decrease in the number of positive wells compared to the solvent control.
A compound is considered as mutagenic:
(1) if it induces a statistically significant increase in the number of positive wells compared to the solvent control,
(2) if a dose-effect relationship is noticeable and
(3) if the result is reproducible.
Statistics:
The statistical significance of the results was assessed with the X2 test: p < 0.05 for X2 > 3.84 and p < 0.01 for X2 > 6.63 (Green et al., 1976).

Results and discussion

Test results
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
positive
Remarks:
30 µg/ml (in the presence of S9-mix only)
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
300 µg/ml (threshold toxic concentration)

Applicant's summary and conclusion