Aluminate(1-), tetrafluoro-, cesium (1:1), (T-4)-

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:

From 03 July 1998 (Study Plan completion) to 21 August 1998 (GLP compliance statement)

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study performed according to the OECD testing guideline and GLP.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1998

Report date:

1998

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Reference substance 001

EC Number:

604-086-6

Cas Number:

138577-01-2

Molecular formula:

Al.Cs.F

Test material form:

solid: particulate/powder

Details on test material:

- Name of test material: Cesium Fluoro Aluminate Complex
- Purity: 99.5%
- Batch: 604/013
- Physical state: White amorphous powder
- Storage condition of test material: At room temperature in the dark
- Expiration date of the lot/batch: 22 May 2001

Method

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Metabolic activation system:

Rat liver microsomal enzymes were routinely prepared from adult male Wistar rats, which were obtained from Charles River, Sulzfeld, Germany

Test concentrations with justification for top dose:

Dose range finding test with tester strain TA100 and WP2uvrA (both with and without S9-mix): 3, 10, 33, 100, 333, 1000, 3330 and 5000 µg/plate.
Mutation assay (with and without S9-mix): 100, 333, 1000, 3330 and 5000 µg/plate

Vehicle / solvent:

The test substance was suspended in dimethyl sulfoxide (DMSO) of spectroscopic quality (Merck). The stock solution was treated with ultra-sonication to obtain a homogeneous suspension. Test substance concentrations were prepared directly prior to use.

Controls

Controls 1

Untreated negative controls:

yes

Remarks:

DMSO (vehicle of the test article)

Negative solvent / vehicle controls:

yes

Remarks:

Saline

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

TA1535 without metabolic activation

Controls 2

Untreated negative controls:

yes

Remarks:

DMSO (vehicle of the test article)

Negative solvent / vehicle controls:

yes

Remarks:

Saline

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

TA1537 without metabolic activation

Controls 3

Untreated negative controls:

yes

Remarks:

DMSO (vehicle of the test article)

Negative solvent / vehicle controls:

yes

Remarks:

Saline

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: daunomycine

Remarks:

TA98 without metabolic activation

Controls 4

Untreated negative controls:

yes

Remarks:

DMSO (vehicle of the test article)

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

TA100 without metabolic activation

Controls 5

Untreated negative controls:

yes

Remarks:

DMSO (vehicle of the test article)

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

WP2uvrA without metabolic activation

Controls 6

Untreated negative controls:

yes

Remarks:

DMSO (vehicle of the test article)

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

TA98, TA100, TA1535 , TA1537 and WP2uvrA with metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Preincubation period: none
- Exposure duration: 48h in the dark at 37°C
- Expression time (cells in growth medium): cells were counted just after the exposure

NUMBER OF REPLICATIONS: triplicate

NUMBER OF CELLS EVALUATED: 0.1 mL of fresh bacterial culture (10E9 cells/mL)

DETERMINATION OF CYTOTOXICITY
- Method: To determine the toxicity of cesium fluoro aluminate complex, the reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies were examined.

OTHER EXAMINATIONS:
precipitation of the test substance

Evaluation criteria:

A test substance is considered to have mutagenic potential, if it induces at least a two-fold increase in the number of revertants with respect to the nunumber induced by the solvent control in any of the tester strains, either with or without metabolic activation. However, any mean plate count of less than 20 is considered to be not significant. The positive response should be reproducible in at least one independently repeated experiment.

Statistics:

No statistical analysis was performed.

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 1535

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 applicable

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 1537

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 applicable

Positive controls validity:

valid

Test results 3

Species / strain:

S. typhimurium TA 98

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 applicable

Positive controls validity:

valid

Test results 4

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Slight reduction of background lawn at 5000 µg/plate in the absence of S9

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 5

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 applicable

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING TEST:
Cesium fluoro aluminate complex was tested in tester strain TA100 and WP2uvrA with concentrations of 3, 10, 33, 100, 333, 1000, 3330 and 5000 µg/plate in the absence and presence of S9-mix. This dose range finding test is reported as a part of the first mutation experiment. Precipitation of the test substance on the plates was observed at the start of the incubation period at the concentration of 5000 µg/plate but not at the end of the incubation period. In strain WP2uvrA, no reduction of the bacterial background lawn and no decrease in the number of revertants was observed. In strain TA100, no reduction of the bacterial background lawn was observed and a slight decrease in the number of revertants was observed at the highest dose levels.

MUTATION EXPERIMENT 1 AND 2:
Based on the results of the dose range finding test, cesium fluoro aluminate complex was tested up to concentrations of 5000 µg/plate in the absence and presence of S9-mix in two mutation experiments. The first experiment was performed with the strains TA1535, TA1537 and TA98 and the second experiment was performed with the strains TA1535, TA1537, TA98, TA100 and WP2uvrA.
Precipitate: Precipitation of the test substance on the plates was observed at the start of the incubation period at the concentration of 5000 µg/plate but not at the end of the incubation period.
Toxicity: The bacterial background lawn was not reduced at all concentrations tested. In tester strain TA100, a slight reduction in the number of revertants was observed at the concentration of 5000 µg/plate in the absence of S9-mix in the second experiment. All other bacterial strains showed no reduction in the number of revertants which was below the minimal value of historical control data range.
Mutagenicity: All bacterial strains showed negative responses over the entire dose range, i.e. no dose-related, two-fold, increase in the number of revertants in two independently repeated experiments.
The negative and strain-specific positive control values were within the laboratory historical background data ranges.

Applicant's summary and conclusion

Conclusions:

Based on the results of this study it is concluded that cesium fluoro aluminate complex is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

Executive summary:

The mutagenic activity of cesium fluoro aluminate complex was investigated in the Salmonella typhimurium reverse mutation assay and the

Escherichia coli reverse mutation assay according to the OECD Testing Guideline 471 and under GLP.

Cesium fluoro aluminate complex 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 one tryptophan-requiring strain of Escherichia coli (WP2uvrA) in two independent experiments.

Cesium fluoro aluminate complex was tested up to concentrations of 5000 µg/plate in the absence and presence of S9-mix.

The test substance did not induce a 2-fold and/or 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.

The strain-specific positive control values were at least three times the concurrent vehicle control grop mean indicating that the test conditions were adequate. Based on the results of this study it is concluded that cesium fluoro aluminate complex is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.