Z-109

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

Study period:

2007

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted to GLP in accordance with recognised guideline

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2007

Report date:

2007

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

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:

- Physical state: Clear colorless visous liquid
- Storage condition of test material: At room temperature in the dark
- Other: Stavle under storage conditions.

Method

Target gene:

Not required

Species / strain

Species / strain 1

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

Non-mammalian study

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Details on mammalian cell type (if applicable):

Non-mammalian study

Metabolic activation:

with and without

Metabolic activation system:

phenobarbitone/β-naphthoflavone induced rat liver S9

Test concentrations with justification for top dose:

Experiment 1: Range-finding test
3, 10, 33, 100, 333, 1000, 3300, 5000 µg/plate


Main test
Experiment 1: TA 1535, TA1537, and TA98
10, 33, 100, 333, 1000 µg/plate

Experiment 2: TA 1535, TA1537, TA98 and TA100, and WP2uvrA.
10, 33, 100, 333, 1000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: ethanol

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Concurrent

True negative controls:

yes

Positive controls:

yes

Positive control substance:

other: Sodium azide, 9-aminoacridine, 2-nitrofluorene, Methylmethanesulfonate, 4-nitroquinoline-N-oxide

Remarks:

without metabolic activation

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Concurrent

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene (2AA)

Remarks:

with metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)
DURATION
- Preincubation period: not applicable.
- Exposure duration: approximately 48 hours incubation at 37°C
- Expression time (cells in growth medium): at 37°C for approximately 10 hours
- Selection time (if incubation with a selection agent): Not applicable.
- Fixation time (start of exposure up to fixation or harvest of cells): Not applicable.

SELECTION AGENT (mutation assays): Not applicable.
SPINDLE INHIBITOR (cytogenetic assays): Not applicable.
STAIN (for cytogenetic assays): Not applicable.

NUMBER OF REPLICATIONS: Triplicate.

NUMBER OF CELLS EVALUATED: 108.

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: the number of revertant colonies

OTHER EXAMINATIONS:
- Determination of polyploidy: not applicable.
- Determination of endoreplication: not applicable.
- Other:

Evaluation criteria:

A test substance is considered negative (not mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 is not greater than two (2) times the
concurrent control, and the total number of revertants in tester strains TA1535, TA1537, TA98 or WPzuvrA is not greater than three (3) times the concurrent control.
b) The negative response should be reproducible in at least one independently repeated experiment.
A test substance is considered positive (mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 is greater than two (2) times the concurrent control, or the total number of revertants in tester strains TA1535, TA1537, TA98 or WP,uvrA is greater than three (3) times the concurrent control.
b) In case a positive response will be repeated, the positive response should be reproducible in
at least one independently repeated experiment. The preceding criteria were not absolute and other modifying factors might enter into the final evaluation decision.

Statistics:

No formal hypothesis testing was done.

Results and discussion

Test results

Test results 1

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:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 2

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

The substance was considered to be non-mutagenic under the conditions of this test.

Executive summary:

Introduction.The method was designed to meet the requirements of themost recent OECD and EEC guidelines.

Methods.The test material was tested in the Salmonella typhimuriurn reverse mutation assay with four histidine- requiring strains of Salmonella typhimurium (TA1535, TA1537, TA98 and TA100) 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 R-naphthoflavone).

Results.In the dose range finding test, the test material was tested up to concentrations of 5000 ug/plate in the absence and presence of S9-mix in the strains TA100 and WP₂uvrA. The test material precipitated on the plates at dose levels of 1000 ug/plate and upwards. No biologically relevant decrease in the number of revertants was observed at all dose levels tested. No reduction of the bacterial background lawn was observed up to the dose level of 5000 ug/plate. Due to precipitate the bacterial background lawn, the test material was tested in the first mutation assay at a concentration range of 10 to 1000 ug/plate in the absence and presence of 5% (vlv)

S9-mix in tester strains TA1535, TA1537 and TA98. In an independent repeat of the assay, the test material was tested at the same concentration range as the first assay in the absence and presence of 10% (vlv) S9-mix in tester strains TA1535, TA1537, TA98, TA100 and WP2uvrA. The test material precipitated on the plates at the dose of 1000 ug/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 this study, the solvent and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

All bacterial strains showed negative responses over the entire dose range.

Conclusion. Based on the results of this study it is concluded that the test material is not mutagenic in any tester strains.