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Genetic toxicity: in vitro

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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
Study period:
19 July 2006 to 27 July 2006
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2006
Report date:
2006

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
Qualifier:
according to guideline
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
- Physical state: solid
- Appearance: grey powder with lumps
- Storage condition of test material: room temperature in the dark

Method

Target gene:
Salmonella typhimurium: histidine
Escherichia coli: tryptophan
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
not applicable
Species / strain / cell type:
E. coli WP2 uvr A
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Rat liver S9 mix induced by phenobarbital/ ß-naphthoflavone
Test concentrations with justification for top dose:
Experiment 1: 10, 33, 100, 333, 1000, 3330, 5000 µg/plate (with and without S9 mix)

Experiment 2: 100, 333, 1000, 3330, 5000 µg/plate (with and without S9 mix)
Vehicle / solvent:
Solvent: Dimethyl sulfoxide
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
dimethyl sulfoxide
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
2-nitrofluorene
sodium azide
methylmethanesulfonate
Details on test system and experimental conditions:
Concentration of the test substance resulting in precipitation: 5000 µg/plate
Evaluation criteria:
Acceptability of the assay: The assay was considered acceptable if the following criteria were met:
a) The negative control data (number of spontaneous revertants per plate) were within the laboratory historical range for each tester strain
b) The positive control chemicals produced responses in all tester strains, which were within the laboratory historical range documented for each positive control substance. Furthermore, the mean plate count was at least three times the concurrent vehicle control group mean
c) The selected dose range included a clearly toxic concentration or exhibited limited solubility as demonstrated by a preliminary toxicity range-finding test or extended to 5 mg/plate

Data evaluation and statistical procedures:

A test material is considered negative (not mutagenic) in the test if:
a) The total number of revertants in tester strain TA 100 is not greater than 2 times the concurrent control, and the total number of revertants in any tester strain is not greater than 3 times the concurrent control
b) The negative response should be reproducible in at least one independently repeated experiment

A test material is considered positive (mutagenic) in the test if:
a) The total number of revertants in tester strain TA 100 is greater than 2 times the concurrent control, or the total number of revertants in any tester strain is greater than 3 times the concurrent control
b) The positive response should be reproducible in at least one independently repeated experiment

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
Remarks:
(> 5000 µg/plate)
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
(> 5000 µg/plate)
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
EXPERIMENT 1
Precipitate
Test material precipitate was observed on the plates at the start of the incubation period at the concentrations of 3330 and 5000 µg/plate and at the end of the incubation period at the concentration of 5000 µg/plate.

Toxicity
No reduction of the bacterial background lawn and no biologically relevant decrease in the number of revertants were observed.

Mutagenicity
No biologically relevant increase in the number of revertants was observed upon treatment witht the test material under all conditions tested.

EXPERIMENT 2
Precipitate
Test material precipitate was observed on the plates at the start of the incubation period at the concentrations of 3330 and 5000 µg/plate and at the end of the incubation period at the concentration of 5000 µg/plate.

Toxicity
The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed.

Mutagenicity
No increase in the number of revertants was observed upon treatment with the test material under all conditions tested.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'. Remarks: (Experiment 1)

Any other information on results incl. tables

Controls

The negative and strain-specific positive control values were within the laboratory background historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative (with and without metabolic activation)

Under the conditions of the study all bacterial strains showed negative responses over the entire dose range since there were no significant dose-related increase in the number of revertants in two independently repeated experiments.
Based on the findings of this study it is concluded that the test material is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.
Executive summary:

The mutagenic activity of the test material was investigated in a study which was conducted under GLP conditions and in accordance with the standardised guidelines OECD 471 and EU Method B.13/14.

During the study the test material was tested in the Salmonella typhimurium reverse mutation assay with four histidine-requiring strains of Salmonella typhimurium (TA1535, TA1537, TA100 and TA98) and in the Escherichia coli reverse mutation assay with a tryptophan-requiring strain of Escherichia coli (WP2uvrA). The test was performed in two independent experiments in the

presence and absence of S9-mix (rat liver S9-mix induced by a combination of phenobarbital and ß-naphthoflavone).

The test material was suspended in dimethyl sulfoxide at concentrations of 0.1 mg/mL and above. In the first mutation assay, the test material was tested up to the concentration of 5000 µg/plate in the absence and presence of 5% (v/v) S9-mix. Test material precipitated on the plates at the top dose level of 5000 µg/plate. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed.

In the second mutation assay, the test material was tested up to the concentration of 5000 µg/plate in the absence and presence of 10% (v/v) S9-mix. The test material precipitated on the plates at the top dose of 5000 µg/plate. The bacterial background lawn was

not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed.

The test material did not induce a significant dose-related increase in the number of revertant (His+) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in tester strain WP2uvrA both in the absence and presence of S9-metabolic activation. These results were confirmed in an independently repeated experiment.

In this study, the negative and strain-specific positive control values were within our laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

Based on the results of this study it is concluded that the test material is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

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