[Name confidential or not available]

Genetic toxicity: in vitro

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:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

of 1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Version / remarks:

of 2008

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Version / remarks:

of 1998

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Details on test material:

- Name of test material (as cited in study report): WS400506

Method

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Additional strain / cell type characteristics:

other: essential amino acid requiring strains

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Additional strain / cell type characteristics:

other: essential amino acid requiring strain

Metabolic activation:

with and without

Metabolic activation system:

liver S9 mix from male Sprague Dawley rats treated with phenobarbital and 5,6-benzoflavone for enzyme induction.

Test concentrations with justification for top dose:

Experiment 1: Doses: 0; 5; 15; 50; 150; 500; 1500 and 5000 μg/plate
Experiment 2: Doses: 0; 50; 150; 500; 1500 and 5000 μg/plate

Vehicle / solvent:

Ethanol
Justification for choice of solvent/vehicle:
Ethanol was a suitable vehicle for exposure to the test substance up to the maximum guideline recommended test substance concentration of of 5000 μg/plate.

Controls

Controls 1

Untreated negative controls:

other: Sterility controls were included, i.e. tester strain free plates with soft agar, S9 mix, buffer, vehicle and/or test substance.

Negative solvent / vehicle controls:

yes

Remarks:

without and with S9 mix

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: Sodium azide; 9-aminoacridine; 2-nitrofluorene; 4-nitroquinoline-1-oxide.

Remarks:

Positive control substances for tests without metabolic activation (S9 mix). All of them are well established reference mutagens.

Controls 2

Untreated negative controls:

other: Sterility controls were included, i.e. tester strain free plates with soft agar, S9 mix, buffer, vehicle and/or test substance.

Negative solvent / vehicle controls:

yes

Remarks:

without and with S9 mix

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene; Benzo[a]pyrene

Remarks:

Positive control substances for tests with metabolic activation (S9 mix). All of them are well established reference mutagens.

Details on test system and experimental conditions:

Standard Plate Incorporation Tests were performed in both experiments (Experiments 1 and 2) and both experiments were conducted without and with metabolic activation (S9 mix). Test procedures varied in that the proportion of S9 fraction in the S9 mix was 10% in Experiment 1 whereas 20% in Experiment 2.

In both experiments, precipitate was observed on all plates containing WS400506 at 5000 μg/plate.

The following positive controls were used to check mutability of the bacteria and activity of the S9 mix:

Without metabolic activation (S9 mix):

Sodium azide (CAS No. 26628-22-8):
- 2 μg/plate, dissolved in DMSO: - strains: TA 1535, TA 100

9-Aminoacridine (CAS No. 90-45-9):
- 50 μg/plate, dissolved in DMSO: - strain: TA 1537

2-Nitrofluorene (CAS No. 607-57-8):
- 2 μg/plate, dissolved in DMSO: - strain: TA 98

4-Nitroquinoline-1-oxide (CAS No. 56-57-5):
- 2 μg/plate, dissolved in DMSO: - strain: WP2 uvrA

With metabolic activation (S9 mix):

2-Aminoanthracene (CAS No. 613-13-8):
- 5 μg/plate, dissolved in DMSO: - strains: TA 1535, TA 100
- 10 μg/plate, dissolved in DMSO: - strain: WP2 uvrA

Benzo[a]pyrene (CAS No. 50-32-8):
- 5 μg/plate, dissolved in DMSO: - strains: TA 1537, TA 98

Evaluation criteria:

The test substance is considered to exhibit mutagenic activity in this assay if the following criteria are met:
A reproducible increase in revertant colony number, (i.e. at least twice for strains TA 100, TA 98 and WP2 uvrA and at least three times for strains TA 1535 and TA 1537 the concurrent vehicle controls), with some evidence of a positive dose-response relationship. Such positive response in at least one tester strain without or with metabolic activation (S9 mix.) is sufficient for concluding mutagenic activity.

A test substance is considered non-mutagenic in this test if:
Exposure to a test substance does not produce a reproducible increase in revertant colony numbers.

Statistics:

The data were not statistically analysed. The study result was unequivocal.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

in both experiments (Experiment 1 and 2)

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

tested up to the limit concentration of 5000 µg/plate, the only concentration with precipitate

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

in both experiments (Experiment 1 and 2)

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

tested up to the limit concentration of 5000 µg/plate, the only concentration with precipitate

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

in both experiments (Experiment 1 and 2)

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

tested up to the limit concentration of 5000 µg/plate, the only concentration with precipitate

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

in both experiments (Experiment 1 and 2)

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

tested up to the limit concentration of 5000 µg/plate, the only concentration with precipitate

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

in both experiments (Experiment 1 and 2)

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

tested up to the limit concentration of 5000 µg/plate, the only concentration with precipitate

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

All sterility control plates were colony free. Hence the absence of microbial contamination of the S9 mix, buffer and test substance formulation was confirmed. Viability counts were satisfactory meeting the acceptance criteria. See tables on results and viability attached as background material.

Applicant's summary and conclusion

Conclusions:

negative without and with metabolic activation (S9 mix)