Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
30 November - 19 December 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
21 July 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:
Rat liver S9-mix from male Sprague Dawley rats injected intraperitoneally with Aroclor 1254 (500 mg/kg body weight).
Test concentrations with justification for top dose:
Initial mutagenicity assay: 1.50, 5.00, 15.0, 50.0, 150, 500, 1500 and 5000 μg per plate
Confirmatory mutagenicity assay: 15.0, 50.0, 150, 500, 1500 and 5000 μg per plate
The substance was tested up to and including the highest dose level as recommended in the OECD guideline. Dose levels for the confirmatory assay were based on the results of the initial assay.
Vehicle / solvent:
- Vehicle used: DMSO, purity 99.96%
- Justification for choice of vehicle: DMSO was the vehicle of choice based on the solubility of the test substance and compatibility with the target cells. The test substance formed a clear solution in DMSO at a concentration of approximately 500 mg/mL in the solubility test conducted at BioReliance.
- Test substance dilutions were prepared immediately before use and delivered to the test system at room temperature under filtered light.
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
other: 2-aminoanthracene
Remarks:
For more details see table 1 in 'any other information on materials and methods'.
Details on test system and experimental conditions:
Two independent experiments were performed using the plate incorporation method; an initial mutagenicity assay to determine the dose-range and to provide a preliminary mutagenicity evaluation followed by a confirmatory mutagenicity assay.

METHOD OF APPLICATION: in agar

DURATION
- Exposure duration (both experiments): 48 - 72 hours at 37±2°C

NUMBER OF REPLICATIONS:
- Initial assay: 2 replicates; confirmatory assay: 3 replicates

DETERMINATION OF CYTOTOXICITY
- Method: condition of the bacterial background lawn by dissecting microscope; precipitation by visual examination without magnification; colonies were enumerated either by hand or by machine.
Evaluation criteria:
INTERPRETATION:
A test item is considered positive (mutagenic) in the test if:
a) The total number of revertants in tester strain TA100, TA98 or WP2uvrA is equal to or greater than two (2) times the mean vehicle control and above the corresponding acceptable vehicle control range.
b) The total number of revertants in tester strains TA1535 or TA1537 is greater than or equal to three (3) times the mean vehicle controland above the corresponding acceptable vehicle control range.

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.

ACCEPTABILITY CRITERIA:
- All Salmonella tester strain cultures must demonstrate the presence of the deep rough mutation (rfa) and the deletion in the uvrB gene. Cultures of tester strains TA98 and TA100 must demonstrate the presence of the pKM101 plasmid R-factor. All WP2 uvrA cultures must demonstrate the deletion in the uvrA gene.
- Based on historical control data (95% control limits), all tester strain cultures must exhibit characteristic numbers of spontaneous revertants per plate with the vehicle controls. The mean revertants per plate of all tester strain cultures and untreated controls must be within the ranges shown in table 2 in 'any other information on materials and methods'.
- To ensure that appropriate numbers of bacteria are plated, tester strain culture titers must be greater than or equal to 0.3x10^9 cells/mL.
- The mean of each positive control must exhibit at least a 3.0-fold increase in the number of revertants over the mean value of the respective vehicle control.
- A minimum of four non-toxic dose levels is required to evaluate assay data.

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:
- No contaminant colonies were observed on the sterility plates for the vehicle control, the test substance dilutions or the S9 and Sham mixes.
- In both the initial and the confirmatory mutagenicity assay neither precipitate nor toxicity was observed. No positive mutagenic responses were observed with any of the tester strains in either the presence or absence of S9 mix.
- Results of the solvent controls and the positive controls were within the historical data range.

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 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 experiments, one initial mutagenicity assay followed by a confirmatory mutagenicity assay. Both experiments were performed using the plate incorporation method with Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2 uvrA. Dose levels up to and including 5000 µg/plate were tested in the absence and presence of S9-mix. Adequate positive controls and a solvent control (DMSO) were included.

In both the initial and the confirmatory mutagenicity assay neither precipitate nor toxicity was observed. All validity criteria were met and the study was considered to be valid. No positive mutagenic responses were observed with any of the tester strains in either the presence or absence of S9 mix. 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.