Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
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

Method

Species / strain
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Additional strain characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9 mix prepared from the livers of male rats treated with phenobarbital and beta-naphthoflavone
Test concentrations with justification for top dose:
Pre-experiment: 3.16, 10, 31.6, 100, 316, 1000, 2500 and 5000 µg/plate (tested in strains TA98 and TA100)
Main experiment 1 and 2: 31.6, 100, 316, 1000, 2500 and 5000 µg/plate
Vehicle:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The solvent is compatible with the survival of the bacteria and the S9 activity
Controls
Negative controls:
yes
Solvent controls:
yes
Positive controls:
yes
Positive control substance:
sodium azide
methylmethanesulfonate
other: 4-nitro-o-phenyllene-diamine, 2-aminoanthracene
Details on test system and conditions:
Preparation of bacteria:
Samples of each tester strain were grown by culture for 12 hours at 38.5°C in Nutrient broth to the late exponential or early stationary phase of growth (approximately 10^9 cells/mL). A solution containing ampicillin was added in order to retain the phenotypic characteristics of the strain.

Pre-experiment: The toxicity of the test substance was determined with tester strains TA98 and TA100 in a pre-experiment. Eight concentrations were tested for toxicity and induction of mutations with three plates each. the experimental conditions in this pre-experiment were the same as the main experiment. Toxicity may be detected by a clearing or rather diminution of the background lawn or a reduction in the number of revertants down to a mutation factor of approximately < 0.5 in relation to the solvent control.

Main phase:
For the plate incorporation method the following materials were mixed in a test tube and poured over the surface of a minimal agar plate:
100 µL: test solution at each dose level, solvent control, negative control or reference mutagen solution.
500 µL: S9 mix (for testing with metabolic activation) or S9 mix substitution buffer (for testing with metabolic activation).

Counting:
The colonies were counted using a Protocol counter. If precipiation of the test substance precluded automatic counting the revertant colonies were counted by hand. In addition, tester strains with a low spontaneous mutation frequency like TA1535 and TA1537 were counted manually.

Cytotoxicity:
Assessed as per the preliminary test.

Rationale for test conditions:
Standard as per OECD guideline.
Evaluation criteria:
A test substance is considered mutagenic if:
- a clear and dose related increase in the number of revertants occurs and/or
- a biologically relevan positive response for at least one of the dose groups occurs in at least one tester strain with or without metabolic activation.
A biologically relevant increase is as follows:
- If in tester strains TA98, T100 and TA102 the number of reversions is at least twice as high.
- If in test strains TA1535 and TA 1537 the number of reversions is at least three times higher than the reversion rate of the solvent control.

A test substance producing neither a dose related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups is considered to be non-mutagenic in this system.
Statistics:
Not required or conducted.

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH:
None noted
- Effects of osmolality:
None noted
- Evaporation from medium:
None noted
- Water solubility:
None noted
- Precipitation:
None noted

Applicant's summary and conclusion

Conclusions:
The test substance was deemed not mutagenic in any of the strains tested both with and without metabolic activation under the conditions of the test.