Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2015-08-24 to 2015-09-14
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report Date:
2015

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
Adopted July 21, 1997
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
31 May 2008
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
- Physical state: powder
- Appearance: white powder
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 0020726249
- Expiration date of the lot/batch: 2016-11-13 (retest date)
- Purity test date: 2015-10-12
- Manufacture date: 2014-11-13

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature
- Stability under test conditions: not indicated
- Solubility and stability of the test substance in the solvent/vehicle: 62-140 g/L
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: not indicated

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test item was dissolved in Milli-Q water. The test item concentations were used within approximately 2 hours of preparation.

OTHER SPECIFICS: A correction factor of 1.23 for the purity/composition of the test item was applied

Method

Target gene:
Histidine locus (S. typhimurium histidine-dependent strains TA98, TA100, TA102, TA1535 and TA1537)
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9-mix (Aroclor 1254 induced rat liver metabolic activation system)
Test concentrations with justification for top dose:
5000 μg/plate was selected as the maximum final concentration for the dose range finding test based on the solubility of the test item in solvent (maximum recommended concnetration).
Dose-range finding test: 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 μg/plate.

No precipitation and no cytotoxicity was observed in the dose range finding test; therefore 5000 µg/plate was selected as the maximal dose in the main experiments.
Mutation experiment I: 52, 164, 512, 1600 and 5000 μg/plate
Mutation experiment II: 492, 878, 1568, 2800 and 5000 μg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle: The test item was observed to be soluble in Milli-Q water up to a concentration of 50 mg/ml. Therefore, Milli-Q water was selected as vehicle.
Controlsopen allclose all
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
Without metabolic activation, 5 µg/plate (TA1535)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: ICR-191
Remarks:
Without metabolic activation, 2.5 μg/plate (TA1537)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
2-nitrofluorene
Remarks:
Without metabolic activation, 10 μg/plate (TA98)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
Without metabolic activation, 650 μg/plate (TA100)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Remarks:
TBH
Positive control substance:
other: tert-butyl hydroperoxide
Remarks:
Without metabolic activation, 250 μg/plate (TA102)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Remarks:
2-AA
Positive control substance:
other: 2-aminoanthracene
Remarks:
With metabolic activation, 2.5 μg/plate (TA1535 at 5 and 10% S9; TA1537 at 5% S9), 5 μg/plate (TA153 7 at 10% S9), 1 μg/plate (TA98 at 5 and 10% S9; TA100 at 5% S9), 2 μg/plate (TA100 at 10% S9), 10 μ g/plate (TA102 at 5 and 10% S9)
Details on test system and experimental conditions:
NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicate
- Number of independent experiments: 2

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): 0.1 ml of a fresh bacterial culture (10^9 cells/ml) added to molten agar
- Test substance added in medium; in agar (plate incorporation)

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method, e.g.: the reduction of the bacterial background lawn; the increase in the size of the microcolonies; the reduction of the revertant colonies

METHODS FOR MEASUREMENTS OF GENOTOXICIY
increase in the number of revertant colonies

Evaluation criteria:
In addition to the criteria stated below, any increase in the total number of revertants was evaluated for its biological relevance including a comparison of the results with the historical control data range.

A test item is considered negative (not mutagenic) in the test if:
a) The total number of revertants in the tester strain TA100 or TA102 is not greater than two (2) times the concurrent control, and the total number of revertants in tester strains TA1535, TA1537 or TA98 is not greater than three (3) times the concurrent vehicle control.
b) The negative response should be reproducible in at least one follow-up experiment.

A test item is considered positive (mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 or TA102 is greater than two (2) times the concurrent control, or the total number of revertants in tester strain TA1535, TA1537 or TA98 is greater than three (3) times the concurrent control.
b) A concentration related effect is observed.
c) In case a follow up experiment is performed when a positive response is observed in one of the tester strains, the positive response should be reproducible in at least one follow up experiment.
Statistics:
No formal hypothesis testing was done.

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
True negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
True negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
True negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
True negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
True negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: The test item was observed to be soluble in Milli-Q water up to a concentration of 50 mg/mL.
- Precipitation: Precipitation of the test item on the plates was not observed at the start or at the end of the incubation period in any tester strain in any experiment.

RANGE-FINDING/SCREENING STUDIES: The test item was tested in tester strain TA100 at concentrations of 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 μg/plate in the absence and presence of S9-mix. Normal bacterial background lawn and no precipitate were observed up to the highest dose. Based on the results of the dose range finding test, the dose ranges for experiment I for TA1535, TA1537, TA98 and TA102 were selected.

STUDY RESULTS
- Positive historical control data: The strain-specific positive control values were within the laboratory historical control data ranges
- Negative (solvent/vehicle) historical control data: The negative control values were within the laboratory historical control data ranges
- Signs of toxicity: Fluctuations in the number of revertant colonies below the historical control data range in tester strain TA102 (with S9-mix) were observed at 5000 μg/plate. The reduction in the mean number of revertant colonies was only minor when compared against relevant historical control data and no toxicity was observed in any of the other tester strains in both experiments, indicating the reduction is caused by incidental fluctuations in the number of revertant colonies
- Mutagenicity: No increase in the number of revertants was observed upon treatment with the test item under all conditions tested.

Applicant's summary and conclusion

Conclusions:
Based on the results of this study it is concluded that the test item is not mutagenic in the Salmonella typhimurium reverse mutation assay.