[No public or meaningful name is available]

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2007-10-10 to 2007-10-29

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted 21st July, 1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

dated May 19, 2000

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

August 1998

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

477-470-2

EC Name:

-

Molecular formula:

UVCB substance

IUPAC Name:

Reaction product of tetraethylsilicate, ethylpolysilicate, dioctyltinoxide, methanol and benzoic acid

Test material form:

liquid

Method

Target gene:

histidine gene (his)

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Additional strain / cell type characteristics:

other: rfa-, uvrB- (all strains); R-factor (TA 98, TA 100, TA 102)

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9:
Male Wistar rats were induced with Phenobarbital (80 mg/kg bw) and ß-Naphtoflavone (100 mg/kg bw) for three consecutive days by the oral route. BSL BIOSERVICE GmbH prepared a S9 liver microsomal fraction.

- method of preparation of S9 mix:
A stock of the supematant containing the microsomes was frozen in ampoules of 2 and 4.5 mL and stored at :>-75 °C. The protein concentration in the S9 preparation (Lot: 140607) was 34 mg/mL. The S9 mix preparation was performed according to Ames et al. (1973).

- volume of S9 mix and S9 in the final culture medium: 500 µL

- quality controls of S9: biological activity and sterility

Test concentrations with justification for top dose:

0.0316, 0.100, 0.316, 1.0, 2.5 and 5.0 µL/plate (top dose according to guideline)

Vehicle / solvent:

DMSO

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO and Aqua dest.

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

methylmethanesulfonate

other: see "Remark"

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicate
- Number of independent experiments: 2

METHOD OF EXPOSURE:
- Test substance added in agar: plate incorporation (experiment I), preincubation (experiment II)

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period: 60 min (experiment II)
- Exposure duration: 48 h

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: 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.

METHODS FOR MEASUREMENTS OF GENOTOXICITY
The colonies were counted using a ProtoCOL counter (Meintrup DWS Laborgeräte GmbH). If precipitation of the test item precluded automatic counting the revertant colonies were counted by hand. In addition, tester strains with a low spontaneous mutation frequency like TA 1535 and TA 1537 were counted manually.

VALIDITY CRITERIA
A test is considered acceptable if for each strain:
- the bacteria demonstrate their typical responses to ampicillin (TA 98, TA 100, TA 102)
- the control plates with and without S9 mix are within the following ranges (mean values of the spontaneous reversion frequency are within the historical control data range):
-S9 +S9
TA 98: 18 - 54 16 - 71
TA 100: 75 - 171 83 - 168
TA 1535: 6 - 30 6 - 31
TA 1537: 5 - 31 6 - 36
TA 102: 166 - 394 153 - 594

- corresponding background growth on negative control, solvent control and test plates is observed.
- the positive controls show a distinct enhancement of revertant rates over the control plate.

Evaluation criteria:

The Mutation Factor is calculated by dividing the mean value of the revertant counts through the mean values of the solvent control (the exact and not the rounded values are used for calculation).

A test item is considered as mutagenic if:
- a clear and dose-related increase in the number of revertants occurs and/or
- a biologically relevant 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 described as folIows:
- if in tester strains TA 100 and TA 102 the number of reversions is at least twice as high if in tester strains TA 98, TA 1535 and TA 1537 the number of reversions is at least three times higher than the reversion rate ofthe solvent control.

According to OECD guidelines, the biological relevance of the results is the criterion for the interpretation of results, a statistical evaluation of the results is not regarded as necessary.

A test item 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.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 5 µL/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Applicant's summary and conclusion

Conclusions:

The test substance was concluded to be non-mutagenic in the bacterial reverse mutation assay under the experimental conditions reported.

Executive summary:

In order to investigate the potential of the test item for its ability to induce gene mutations, the plate incorporation test (experiment I) and the pre-ineubation test (experiment II) were performed with the Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and TA 102.
In two independent experiments several concentrations of the test item were used. Each assay was conducted with and without metabolic activation. The concentrations, including the controls, were tested in triplicate. The following concentrations of the test item were prepared and used in the experiments: 0.0316, 0.100, 0.316, 1.0, 2.5 and 5.0 µL/plate (experiment I and experiment II).
No precipitation of the test item was observed in any tester strain used in experiment I and II (with and without metabolic activation). No toxic effects of the test item were noted in any of the five tester strains used up to the highest dose group evaluated (with and without metabolic activation) in experiment I and II except for cytotoxic effects observed in tester strain TA 1537 at a dose of 5.0 µL/plate (with and without metabolic activation) in experiment II. No biologically relevant inereases in revertant colony numbers of any of the five tester strains were observed following treatment with Beta 30 at any concentration level, neither in the presence nor absence of metabolic activation in experiment I and II. The reference mutagens induced a distinct increase of revertant colonies indicating the validity of the experiments.
In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, Beta 30 did not cause gene mutations by base pair changes or frameshifts in the genome of the tester strains used.
Therefore, the test item is considered to be non-mutagenic in this bacterial reverse mutation assay.