Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 16 Feb to 19 Mar 1998
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study run to a method comparable with current guidelines and to GLP.

Data source

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

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Batch No.: 124114/A/19/1
Purity: Not specified

Method

Target gene:
Histidine gene in S. typhimurium, tryptophan gene in E. coli
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:
S9 mix
Test concentrations with justification for top dose:
Preliminary Toxicity Study: 50, 150, 500, 1500 and 5000 μg/plate
Experiment 1: 50, 150, 500, 1500 and 5000 μg/plate
Experiment 2: 50, 150, 500, 1500 and 5000 μg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: acetone
Controlsopen allclose all
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
N-ethyl-N-nitro-N-nitrosoguanidine
Remarks:
Without S9-mix for TA100, TA1535 and WP2uvrA strains.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
Without S9-mix for TA1537 strain.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 4-Nitroquinoline-1-oxide (4NQO)
Remarks:
Without S9-mix for TA98 strain.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
With S9-mix for TA 1535, TA 1537, TA 98, TA 100 and WP2uvrA strains.
Details on test system and experimental conditions:
METHOD OF APPLICATION:in agar (plate incorporation)
- Preliminary Toxicity Study: In order to select appropriate dose levels for use in the main study, a preliminary test was carried out to determine the toxicity material. Five concentrations of the test material formulation and a vehicle control (acetone) were tested in duplicate.
- Experiment 1: Five concentrations of the test material were assayed in triplicate against each tester strain, using the direct plate incorporation method. Measured aliquots (0.1 mL) of one of the bacterial cultures were dispensed into sets of test tubes followed by 2.0 mL of molten, trace histidine or tryptophan supplemented, top agar, 0.1 mL of the test material formulation, vehicle or positive control and either 0.5 mL of S9-mix or phosphate buffer. The contents of each test tube were mixed and equally distributed onto the surface of Vogel-Bonner Minimal agar plates (one tube per plate). This procedure was repeated, in triplicate, for each bacterial strain and for each concentration of test material both with and without S9-mix. All of the plates were incubated at 37℃ for approximately 48 hours and the frequency of revertant colonies assessed using a Domino colony counter. Manual counts were performed at 5000 μg/plate because of a test material induced particulate precipitate.
- Experiment 2: The second experiment was performed using methodology as described for experiment 1, using fresh bacterial cultures, test material and control solutions.
Evaluation criteria:
The test material may be considered to be positive in this test system if the following criteria are met:
The test material should have induced a reproducible, dose-related and statistically (Dunnett's method of linear regression) significant increase in the revertant count in at least one strain of bacteria. If a greater than twofold increase in revertant count is observed in two experiments then this is taken as evidence of a positive response.

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Preliminary Toxicity Study
The dose range of the test material used in the preliminary toxicity study was 0, 50, 150, 500, 1500 and 5000 μg/plate. The test material was non-toxic to the strains of bacteria used (TA100 and WP2uvrA).

Mutation Study (Experiment 1 and Experiment 2)
Results for the negative controls (spontaneous mutation rates) were considered to be acceptable.
No toxicity was exhibited to any of the strains of bacteria used. A white, particulate precipitate was observed at and above 1500 μg/plate, this did not prevent the scoring of revertant colonies.
No significant increases in the frequency of revertant colonies were recorded for any of the strains of bacteria, at any dose level either with or without metabolic activation.
All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies thus confirming the activity of the S9-mix and the sensitivity of the bacterial strains.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

The test material was considered to be non-mutagenic under the conditions of this test.