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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1998
Report Date:
1998

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid
Specific details on test material used for the study:
- Test substance storage: At room temperature in the dark
- Description: Off white solid
- Expiry date: 19 January 1999
- Stability in vehicle: at least 96 hours in ethanol

Method

Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254 induced rat liver S9 homogenate
Test concentrations with justification for top dose:
Experiment 1a: 3, 10, 33, 100, 333, 1000, 3330, 5000 µg/plate (TA100, WP2uvrA)
Experiment 1b: 10, 33, 100, 333, 1000 µg/plate (TA 1535, TA 1537, TA98)
Experiment 2: 10, 33, 100, 333, 1000 µg/plate (All strains)
Vehicle / solvent:
ethanol
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
sodium azide
methylmethanesulfonate
other: Daunomycine
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 hours

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies
Evaluation criteria:
Acceptability of the assay: A Salmonella typhimurium reverse mutation assay and/or Escherichia coli reverse mutation assay is considered acceptable if it meets the following criteria: a) The negative control data (number of spontaneous revertants per plate) should be within the laboratory historical range for each tester strain. b) The positive control chemicals should produce responses in all tester strains, which are within the laboratory historical range documented for each positive control substance. Furthermore, the mean plate count should be at least two times the concurrent vehicle control group mean. c) The selected dose range should include a clearly toxic concentration or should exhibit limited solubility as demonstrated by the preliminary toxicity range-finding test or should extend to 5 mg/plate.
Data evaluation: A test substance is considered negative (not mutagenic) in the test if: a) The total number of revertants in any tester strain is not greater than two (2) times the solvent control, with or without metabolic activation b) The negative response should be reproducible in at least one independently repeated experiment. A test substance is considered positive (mutagenic) in the test if: a) It induces at least 2-fold, dose related increase in the number of revertants with respect to the number induced by the solvent control in any of the tester strains, either with or without metabolic activation. However, any mean plate count of less than 20 is considered to be not significant. b) The positive response should be reproducible in at least one independently repeated experiment.

Results and discussion

Test results
Key result
Species / strain:
other: S. typhimurium TA 1535, TA 1537, TA 98 and TA 100 and E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
Dose range finding test:
- The test substance precipitated in the top agar at concentrations of 333 µg/plate and upwards. Precipitation of the test substance on the plates was observed at the start and at the end of the incubation period at concentrations of 1000 µg/plate and upwards in tester strain TA100 and WP2uvrA.
- In tester strain WP2uvrA, no reduction of the bacterial background lawn and no decrease in the number of revertants was observed. In tester strain TA100, no reduction of the bacterial background lawn was observed. In the absence of S9-mix, a reduction in the number of revertant colonies was observed at concentrations of 333 µg/plate and upwards, which was less than the minimal value of the historical control data range. In the presence of S9-mix, a reduction in the number of revertant colonies was observed at concentrations of 1000 µg/plate and upwards.

Mutation assay:
- The test substance precipitated in the top agar at concentrations of 333 and 1000 µg/plate. Precipitation of the test substance on the plates was observed at the start and at the end of the incubation period at the concentration of 1000 µg/plate in all tester strains.
- In tester strain TA100, a reduction in the number of revertant colonies was observed at the concentration of 1000 µg/plate, which was less than the minimal value of the historical control data range. In the other strains, no clear decrease in the number of revertants was observed. The bacterial background lawn was not reduced at all concentrations tested.

Number of revertants
- All bacterial strains showed negative responses over the entire dose range, i.e. no dose-related, two-fold, increase in the number of revertants in two independently repeated experiments. The strain-specific positive control values were within our laboratory background historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly. The negative control values were within our laboratory background historical control data ranges, except for WP2uvrA (first experiment). However, since these values were just outside the limit of the range, the validity of the test was considered not to be affected.

Applicant's summary and conclusion