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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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

Data source

Reference
Reference Type:
other: Draft study report
Title:
Unnamed
Year:
2012
Report date:
2012

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Details on test material:
- CAS No.: 1312296-85-7
- Purity/composition: 100.4 g/100 g
- Homogeneity: The homogeneity of the test substance was ensured by mixing before preparation of the test substance solutions.
- Storage stability: The stability of the test substance under storage conditions throughout the study period was guaranteed until 22 Feb 2012 as indicated by the sponsor, and the sponsor holds this responsibility.
- Date of production: 22 Aug 2011
- Physical state, appearance: Solid, beige
- Storage conditions: Room temperature (under N2 conditions)

Method

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:
Phenobarbital/β-naphthoflavone-induced rat liver S9 mix
Test concentrations with justification for top dose:
0; 33; 100; 333; 1000; 2500 and 5000 μg/plate
Vehicle / solvent:
- Vehicle/solvent used: water
- Justification for choice of solvent/vehicle: due to the good solubility of the test substance in water.
Controls
Untreated negative controls:
yes
Remarks:
Sterility control
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Remarks:
W/ S-9: 2-AA (2.5 µg/plate for all S. Typhimurium strains; 60 µg/plate for E. coli WP2 uvrA). W/o S-9: MNNG (5 µg/plate for TA 1535, TA 100), NOPD (10 µg/plate for TA98), AAC (100 µg/plate for TA1537), 4-NQO (5 µg/plate for E. coli WP2 uvrA).
Details on test system and experimental conditions:
METHOD OF APPLICATION: Standard plate test (SPT) and Preincubation test (PIT)

NUMBER OF REPLICATIONS: 2 individual experiments (1st experiment SPT, 2nd experiment PIT) with 3 test plates per dose or per control

DETERMINATION OF CYTOTOXICITY
- Method: decrease in the number of revertants, clearing or diminution of the background lawn (= reduced his- or trp- background growth), and reduction in the titer

OTHER EXAMINATIONS:
- Precipitation of the test substance
Evaluation criteria:
Generally, the experiment is considered valid if the following criteria are met:
- The number of revertant colonies in the negative controls was within the range of the historical negative control data for each tester strain
- The sterility controls revealed no indication of bacterial contamination
- The positive control substances both with and without S9 mix induced a distinct increase in the number of revertant colonies within the range of the historical positive control data or above
- The titer of viable bacteria was ≥ 10E+08/mL

The test substance is considered positive in this assay if the following criteria are met:
- A dose-related and reproducible increase in the number of revertant colonies, i.e. about doubling of the spontaneous mutation rate in at least one tester strain either without S9 mix or after adding a metabolizing system.
A test substance is generally considered non-mutagenic in this test if:
- The number of revertants for all tester strains were within the historical negative control range under all experimental conditions in at least two experiments carried out independently of each other.

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS:
- Precipitation: No test substance precipitation was found with and without S9 mix.

COMPARISON WITH HISTORICAL CONTROL DATA:
In this study with and without S9 mix, the number of revertant colonies in the negative controls was within the range of the historical negative control data for each tester strain. In addition, the positive control substances both with and without S9 mix induced a significant increase in the number of revertant colonies within the range of the historical positive control data or above.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
A weak bacteriotoxic effect (slight decrease in the number of his+ revertants) was occasionally observed in the standard plate test depending on the strain and test conditions at 5000 μg/plate. In the preincubation assay bacteriotoxicity (reduced his- or trp- background growth, decrease in the number of his+ or trp+ revertants, reduction in the titer) was observed depending on the strain and test conditions from about 2500 μg/plate onward.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

The test substance was not mutagenic in the Salmonella typhimurium/Escherichia coli reverse mutation test in the absence and the presence of metabolic activation.
Executive summary:

The study was performed according to OCD guideline 471 in compliance with GLP.

The test substance was tested for mutagenicity in the Salmonella typhimurium / Escherichia coli reverse mutation assay both in the standard plate test and in the preincubation test with and without the addition of a metabolizing system (S9 mix) obtained from rat liver using the Salmonella strains TA 1535, TA 100, TA 1537, TA 98 and Escherichia coli WP2 uvrA in a dose range of 33 - 5000 µg/plate.

The test substance did not lead to a biologically relevant increase in the number of revertant colonies either without S9 mix or after adding a metabolizing system in two experiments carried out independently of each other (standard plate test and preincubation assay). A bacteriotoxic effect was observed depending on the strain and test conditions from about 2500 μg/plate onward. Besides, the results of the negative as well as the positive controls performed in parallel corroborated the validity of this study, since the values fulfilled the acceptance criteria of this study.

Conclusion: Under the experimental conditions of this study, the test substance was not mutagenic in the Salmonella typhimurium/Escherichia coli reverse mutation assay in the absence and the presence of metabolic activation.