Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
02 October 2013 - 30 October 2013
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 / 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
Test concentrations with justification for top dose:
Initial toxicity-mutation assay (with and without S9): 1.5, 5.0, 15, 50, 150, 500, 1500 and 5000 μg/plate
Confirmatory mutagenicity assay (with and without S9): 15, 50, 150, 500, 1500 and 5000 μg/plate
The top dose was according to OECD guideline 471
Vehicle / solvent:
- Solvent used: DMSO
- Justification for choice of solvent: a solubility test was performed to determine the appropriate solvent that permitted preparation of the highest soluble or workable stock concentration. The substance formed a clear solution in DMSO at approximately 500 mg/mL.
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
methylmethanesulfonate
Remarks:
For details on positive controls, see table 1
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar

DURATION
- Exposure duration: 48-72 hours

NUMBER OF REPLICATIONS: 2 in the intital assay, 3 in the confirmatory assay

DETERMINATION OF CYTOTOXICITY
- Method: The condition of the bacterial background lawn was evaluated for evidence of test article toxicity by using a dissecting microscope.

OTHER EXAMINATIONS:
- Precipitate was evaluated by visual examination without magnification.
Evaluation criteria:
Evaluation of results:
- For each replicate plating, the mean and standard deviation of the number of revertants per plate were calculated.
- For the test article to evaluated positive, it must cause a dose-related increase in the mean revertants per plate of at least one tester strain with a minimum of two increasing concentrations of test substance.
- Data sets for tester strains TA1535 and TA1537 were judged positive if the increase in mean revertants at the peak of the dose response was greater than or equal to 3.0-times the mean vehicle control value. Data sets for tester strains TA98, TA100 and WP2 uvrA were judged positive if the increase in mean revertants at the peak of the dose response was greater than or equal to 2.0-times the mean vehicle control value.
- An equivocal response is a biologically relevant increase in a revertant count that partially meets the criteria for evaluation as positive. This could be a dose-responsive increase that does not achieve the respective threshold cited above or a non-dose responsive increase that is equal to or greater than the respective threshold cited. A response was evaluated as negative, if it was neither positive nor equivocal.

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:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
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:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No precipitation was observed.

HISTORICAL CONTROL DATA: see the attached illustration.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Initial toxicity-mutation assay: No background lawn toxicity was observed; however, reductions in revertant counts were observed at 5000 μg per plate with tester strains TA1535 and WP2 uvrA in the absence of S9 activation.
- Confirmatory mutagenicity assay: No background lawn toxicity was observed; however, reductions in revertant counts were observed at 5000 μg per plate with tester strain WP2 uvrA in the presence and absence of S9 activation and tester strain TA1535 in the presence of S9 activation.
Remarks on result:
other: Reductions in revertant counts were observed at 5000 μg per plate without S9 (initial assay) and with S9 (confirmatory assay)

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 guideline 471 and GLP principles.
Executive summary:

The mutagenic activity of the substance was evaluated according to OECD guideline 471 and GLP principles. The test was performed in two independent plate incorporation assays: one initial test to establish the dose-range and one confirmatory test to evaluate and confirm the mutagenic potential of the substance. The test was performed with and without metabolic activation (S9 -mix) up to and including a concentration of 5000 μg substance/plate. Adequate negative and positive controls were included. 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 (Tryp+) colonies in E.coli WP2uvrA, both in the absence and presence of S9-metabolic activation. No precipitation or background lawn toxicity was observed. Reduction in revertant counts was observed in both assays in test strains TA1535 (without S9 in the initial assay and with S9 in the confirmatory assay) and WP2 uvrA (without S9 in the initial assay and with and without S9 in the confirmatory assay). Based on the results of this study it is concluded that the substance is not mutagenic in the Salmonella typhimurium reverse mutation assay and not mutagenic in the Escherichia coli reverse mutation assay.