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:
16 December 2013 - 27 February 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
Type:
Constituent
Test material form:
liquid

Method

Target gene:
- S. typhimurium: Histidine gene
- E. coli: Tryptophan gene
Species / strainopen allclose all
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Metabolic activation system:
Rat liver S9-mix induced with Aroclor 1254
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
Rat liver S9-mix induced with Aroclor 1254
Test concentrations with justification for top dose:
- Experiment 1:
TA 1535, TA 1537, TA 98, TA 100 and WP2uvrA (without and with S9): 5, 16, 50, 160, 500, 1600 and 5000 µg/plate

- Experiment 2:
TA 1535, TA 1537, TA 98, TA 100 and WP2uvrA (without and with S9): 16, 50, 160, 500, 1600 and 5000 µg/plate
Vehicle:
- Solvent used: DMSO
- Justification for choice of solvent: In a solubility test using the vehicle selected for this study, DMSO, the test article formed a non-viscous, transparent, colorless solution at 167 mg/mL.
Controls
Solvent controls:
yes
Remarks:
DMSO
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
2-nitrofluorene
sodium azide
benzo(a)pyrene
other: See remarks
Remarks:
For further information on positive controls see Any other information on materials and methods incl. tables.
Details on test system and 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)

DETERMINATION OF CYTOTOXICITY
- Method: on the basis of a decline in the number of revertants and/or a thinning of the background lawn.
Evaluation criteria:
Criteria for a Positive Response:
A test article is considered to have produced a positive response if it induces a dose dependent 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.

Criteria for a Negative Response:
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.

Criteria for an Equivocal Response:
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.
Other criteria also may be used in reaching a conclusion about the study results (e.g., comparison to historical control values, biological significance, etc.). In such cases, the Study Director will use sound scientific judgment and clearly report and describe any such considerations.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
yes
Remarks:
at 5000 μg/plate: exp 1 with S9 and exp 2 with and without S9; no cytotoxicity, but tested up to limit concentration: exp 1 without S9
Vehicle controls valid:
yes
Positive controls valid:
yes
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
yes
Remarks:
at 5000 μg/plate: exp 1 and 2, with and without S9
Vehicle controls valid:
yes
Positive controls valid:
yes
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
yes
Remarks:
at 5000 μg/plate: exp 1 and 2, with and without S9
Vehicle controls valid:
yes
Positive controls valid:
yes
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
yes
Remarks:
at 5000 μg/plate: exp 1 and 2, with and without S9
Vehicle controls valid:
yes
Positive controls valid:
yes
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
yes
Remarks:
at 5000 μg/plate: exp 1 and 2, with and without S9
Vehicle controls valid:
yes
Positive controls valid:
yes
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No precipitation was observed up to and including the top dose of 5000 µg/plate.

COMPARISON WITH HISTORICAL CONTROL DATA:
- Exept for the solvent control in strain TA 100 tested with S9 in the second experiment, the negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly. Although outside the laboratory historical control data range, the solvent control in strain TA 100 tested with S9 in the second experiment was within the acceptance limits reported in this study, and therefore this control is also considered valid.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Toxicity, as evident by the reduction in the mean number of revertant colonies and/or reduced bacterial background lawn, was observed at 5000 μg/plate in all tester strains in the presence and absence of S9, exept for strain TA1535 in the absence of S9 which did not show toxicity when tested up to 5000 μg/plate in the first experiment.

Applicant's summary and conclusion

Conclusions:
The substance is negative in the Salmonella typhimurium reverse mutation assay and Escherichia coli reverse mutation assay performed according to OECD 471 guideline and GLP principles.
Executive summary:

The mutagenic activity of the substance was evaluated in accordance with OECD 471 guideline and according to GLP principles. The test was performed in two independent plate incorporation experiments up to and including 5000 μg/plate, in the absence and presence of S9-mix. Adequate negative and positive controls were included. Toxicity, as evident by the reduction in the mean number of revertant colonies and/or reduced bacterial background lawn, was observed at 5000 μg/plate in all tester strains in the presence and absence of S9 in both experiment 1 and 2, except for strain TA1535 in the absence of S9 which did not show toxicity when tested up to 5000 μg/plate in the first experiment. The substance did not induce a significant dose related increase in the number of revertant (His+) colonies in each of the four S. typhimurium tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in E.coli WP2uvrA, both in the absence and presence of S9-metabolic activation in both experiment 1 and 2. Based on the results of this study it is concluded that the substance is negative in the Salmonella typhimurium reverse mutation assay and negative in the Escherichia coli reverse mutation assay.