Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
19 June 2014 - 29 June 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report Date:
2014

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1997
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Test material form:
solid: crystalline
Details on test material:
- Storage condition of test material: Store in cool dark place at room temperature

Method

Target gene:
- S. typhimurium: Histidine gene
- Escherichia coli: Tryptophan gene
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:
Rat liver S9-mix induced by Aroclor 1254.
Test concentrations with justification for top dose:
- Experiment 1:
All five strains (without and with S9): 5, 16, 50, 160, 500, 1600 and 5000 μg/plate
- Experiment 2:
TA 1535, TA 1537, TA 98 and TA 100 (without S9): 1.6, 5, 16, 50, 160 and 500 μg/plate
WP2uvrA (without and with S9): 16, 50, 160, 500, 1600 and 5000 μg/plate
Vehicle / solvent:
- Vehicle used: DMSO
- Justification for choice of vehicle: In a solubility test using the vehicle selected for this study, DMSO, the test article formed a non-viscous, transparent, colorless solution at 222.4 mg/mL.
Controls
Negative solvent / vehicle controls:
yes
Remarks:
100 μL/plate DMSO
Positive controls:
yes
Positive control substance:
other: see section "Any other information on materials and methods incl. tables"
Details on test system and experimental conditions:
METHOD OF APPLICATION:
- Experiment 1 and 2: in agar (plate incorporation)

DURATION
- Exposure duration: 52 ± 4 hours at 37 ± 2°C

NUMBER OF REPLICATIONS:
- Doses of the test substance were tested in triplicate in each strain (in all experiments)

NUMBER OF CELLS EVALUATED: ≥ 1x10^9 cells/mL

DETERMINATION OF CYTOTOXICITY
- Method: The condition of the bacterial background lawn was evaluated macroscopically and microscopically (using a dissecting microscope) for indications of cytotoxicity and test article precipitate. Evidence of cytotoxicity was scored relative to the vehicle control plate and was recorded along with the revertant counts for all plates at that dose level.
Evaluation criteria:
A test article is considered to have produced a positive response if it induces a dosedependent increase in revertant frequency that is ≥ 2.0-fold vehicle control values for tester strains TA98, TA100, and WP2uvrA, or ≥ 3.0-fold vehicle control values for tester strains TA1535 and TA1537. In addition, any response should be reproducible.
A test article is considered to have produced a negative response if no dose-dependent, ≥ 2.0-fold or ≥ 3.0-fold increases are observed in tester strains TA98, TA100, and WP2uvrA, or TA1535 and TA1537, respectively.
Even after repeated trials, a test article may produce results that are neither clearly positive nor clearly negative (e.g., responses that do not meet the dose-dependency or fold increase requirements but are reproducible). In those rare instances, the test article may be considered to have produced an equivocal response.
Statistics:
Not performed.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Experiment 1: at ≥ 160 μg/plate without S9 and at ≥ 500 μg/plate with S9. Experiment 2: at ≥160 μg/plate without S9 and at 500 μg/plate with S9.
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Experiment 1: at ≥ 160 μg/plate without and with S9. Experiment 2: at ≥160 μg/plate without S9 and at 500 μg/plate with S9.
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Experiment 1: at ≥ 160 μg/plate without S9 and at ≥ 500 μg/plate with S9. Experiment 2: at ≥160 μg/plate without S9 and at 500 μg/plate with S9.
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Experiment 1: at ≥ 160 μg/plate without S9 and at ≥ 500 μg/plate with S9. Experiment 2: at ≥160 μg/plate without S9 and at 500 μg/plate with S9.
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 5000 μg/plate without and with S9.
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Precipitate was observed at 5000 μg/plate with and without S9.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
All positive and vehicle control values were within the acceptable ranges, and all criteria for a valid study were met.
- Negative (solvent/vehicle) historical control data:
TA98: 8 - 60
TA100: 60 - 240
TA1535: 4 - 45
TA1537: 2 - 25
WP2uvrA: 5 - 40

Applicant's summary and conclusion

Conclusions:
The substance is not mutagenic in the Salmonella typhimurium reverse mutation assay and Escherichia coli reverse mutation assay performed according to OECD 471 (1997) and GLP principles.
Executive summary:

The mutagenic activity of the substance was evaluated in accordance with OECD 471 (1997) guideline and according to GLP principles. The test was performed in two independent direct plate experiments in the absence and presence of S9-mix up to and including the precipitating concentration of 5000 μg/plate in the first experiment. Toxicity, as evident by the absence or reduction in the mean number of revertant colonies and/or absence or reduction of bacterial background lawn, was observed at ≥ 160 μg/plate in all tester strains without S9 and at ≥ 500 μg/plate with S9 except TA1537 and WP2uvrA, where toxicity was observed at ≥ 160 and 5000 μg/plate, respectively. In the second experiment, the substance was tested in the four Salmonella strains in the absence and presence of S9-mix up to and including 500 μg/plate and in strain WP2uvrA in the absence and presence of S9-mix up to and including the precipitating concentration of 5000 μg/plate. Toxicity, as evident by the absence or reduction in the mean number of revertant colonies and/or reduced bacterial background lawn, was observed at ≥ 160 μg/plate in all tester strains without S9 and at 500 μg/plate with S9 except WP2uvrA, where toxicity was observed at

5000 μg/plate.

No increase in the mean number of revertant colonies was observed at any tested concentration in any tester strain in the presence or absence of S9 in the initial or the confirmatory assay. All positive and vehicle control values were within the acceptable ranges, and all criteria for a valid study were met. Based on the results of this study it is concluded that the substance is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.