Cesium potassium fluoroaluminate

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 14 Oct 2003 to 18 Nov 2003

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2003

Report date:

2003

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Test material form:

solid: particulate/powder

Details on test material:

Cesium potassium fluoroaluminate, purity 100%

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:

S9 mix from rat liver cells

Test concentrations with justification for top dose:

Experiment 1: 0, 3, 10, 33, 100, 333, 1000, 3330 and 5000 µg/plate
Experiment 2: 0, 100, 333, 1000, 3330 and 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used:ethanol
- Justification for choice of solvent/vehicle: one of those recommended by guidelines

Controls

Untreated negative controls:

yes

Remarks:

solvent served as control

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

sodium azide

methylmethanesulfonate

other: daunomycin

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

CELL CULTURE
Preparation of bacterial cultures
Samples of frozen stock cultures of bacteria were transferred into enriched nutrient broth (Oxoid no.2) and incubated in a shaking incubator (37°C, 150 spm), until the cultures reached an optical density of 1.0 ± 0.1 at 700 nm (109 cells/ml). Freshly grown cultures of each strain were used for a test.

Permeabilization of the Escherichia coli strain
WP2uvrA bacteria were washed twice in 0.25 the original volume of ice-cold 0.12 M Tris-HCI buffer pH 8.0, then gently resuspended in 0.2 vol. 0.12 M Tris-HCI, 0.5 mM EDTA pH 8.0, and shaken for 2.5 min at 37°C. MgCl2 was then added to a final concentration of 10 mM. The cells were centrifuged and resuspended in the original volume of nutrient broth.

Agar plates
Agar plates (0 9 cm) contained 25 ml glucose agar medium. Glucose agar medium contained per liter: 18 g purified agar (Oxoid, code L28) in Vogel-Bonner Medium E, 20 g glucose.
N.B. The agar plates for the test with the Salmonella typhimurium strains also contained 12.5 µg/plate biotin and 15 µg/plate histidine and the agar plates for the test with the Escherichia coli strain contained 15 µg/plate tryptophan.

Top agar
Milli-Q water containing 0.6% (w/v) bacteriological agar (Oxoid, code L11) and 0.5% (w/v) Sodium Chloride was heated to dissolve the agar. Samples of 3 ml top agar were transferred into 10 ml glass tubes with metal caps. Top agar tubes were autoclaved for 20 min at 121 ± 3°C.

Environmental conditions
All incubations were carried out in the dark at 37 ± 1°C. The temperature was monitored during the experiment.

DOSE RANGE-FINDING TEST
Selection of an adequate range of doses was based on a dose range finding test with strain TA100 and the WP2uvrA strain, both with and without S9-mix. Eight concentrations, 3, 10, 33, 100, 333, 1000, 3330 and 5000 µg/plate were tested in triplicate. This dose range finding test was reported as a part of the first experiment of the mutation assay. The highest concentration of Cesium potassium fluoroaluminate (Cesium 5.64% w/w) used in the subsequent mutation assay was 5 mg/plate.

MUTATION ASSAY
At least five different doses (increasing with approximately half-log steps) of the test substance were tested in triplicate in each strain.
The test substance was tested both in the absence and presence of S9-mix in each strain, in two independent experiments.
Top agar in top agar tubes was molten and heated to 45°C. The following solutions were successively added to 3 ml molten top agar: 0.1 ml of a fresh bacterial culture {109 cells/ml) of one of the tester strains, 0.1 ml of a dilution of the test substance in ethanol and either 0.5 ml S9-mix (in case of activation assays) or 0.5 ml 0.1 M phosphate buffer (in case of non-activation assays). The ingredients were mixed on a Vortex and the content of the top agar tube was poured onto a selective agar plate. After solidification of the top agar, the plates were turned and incubated in the dark at 37 ± 1°C for 48 h. After this period revertant colonies (histidine independent (His+) for Salmonella typhimurium bacteria and tryptophan independent (Trp+) for Escherichia coli) were counted.

COLONY COUNTING
The revertant colonies (histidine independent c.q. tryptophan independent) were counted automatically with a Protes model 50000 colony counter or manually, if less than 40 colonies per plate were present.

Evaluation criteria:

No formal hypothesis testing was done.
A test substance is considered negative (not mutagenic) in the test if:
a) The total number of revertants in any tester strain at any concentration is not greater than two times the solvent control value, with or without metabolic activation.
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) lt induces a number of revertant colonies, dose related, greater than two-times the number of revertants 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.
b) 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.

Results and discussion

Test results

Key result

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES:
Cesium potassium fluoro aluminate (Cesium 5.64% w/w) was tested in the tester strains 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 experiment of the mutation test.

Precipitate
The test substance precipitated in the top agar at concentrations of 1000 µg/plate and upwards. Precipitation of Cesium potassium fluoro aluminate on the plates was observed at the start of the incubation period at concentrations of 1000 µg/plate and upwards and no precipitate was observed at the end of the incubation period in all tester strains.

Toxicity
To determine the toxicity of Cesium potassium fluoro aluminate, the reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies were examined.
No reduction of the bacterial background lawn and no biologically relevant decrease in the number of revertants were observed.

COMPARISON WITH HISTORICAL CONTROL DATA:
The negative 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.

Any other information on results incl. tables

All results are expressed as mean number of revertant colonies/3 replicate plates (± S.D.) with different strains of Salmonella typhimurium and one Escherichia coli strain.

Experiment 1: Mutagenic response of Cesium potassium fluoroaluminate in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay

WITHOUT S9 mix:

Dose (µg/plate)	TA 1535	TA 1537	TA 98	TA 100	WP2 uvrA
Positive control	803 ± 23	515 ± 44	594 ± 16	766 ± 118	692 ± 23
Solvent control	16 ± 1	13 ± 3	28 ± 3	121 ± 17	7 ± 2
3				126 ± 14	6 ± 1
10				115 ± 2	6 ± 2
33				118 ± 12	9 ± 3
100	16 ± 2	16 ± 2	31 ± 4	104 ± 12	10 ± 1
333	14 ± 2	14 ± 1	35 ± 9	98 ± 9	7 ± 3
1000	20 ± 5	16 ± 1	28 ± 2	107 ± 12	9 ± 2
3330	16 ± 1	16 ± 2	24 ± 1	77 ± 5	6 ± 1
5000	15 ± 2	10 ± 2	27 ± 4	81 ± 10	7 ± 1

WITH S9 mix:

Dose (µg/plate)	TA 1535	TA 1537	TA 98	TA 100	WP2 uvrA
Positive control	352 ± 19	333 ± 11	795 ± 21	771 ± 20	220 ± 18
Solvent control	19 ± 2	16 ± 3	40 ± 3	122 ± 16	7 ± 3
3				113 ± 13	6 ± 2
10				114 ± 9	8 ± 3
33				115 ± 15	7 ± 3
100	21 ± 2	11 ± 2	34 ± 2	101 ± 12	5 ± 2
333	20 ± 2	9 ± 1	36 ± 2	96 ± 9	8 ± 3
1000	17 ± 3	11 ± 2	39 ± 2	102 ± 3	8 ± 1
3330	18 ± 2	15 ± 2	30 ± 1	79 ± 4	7 ± 2
5000	16 ± 1	12 ± 2	34 ± 6	84 ± 6	8 ± 1

 

Experiment 2: Mutagenic response of Cesium potassium fluoroaluminate in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay

WITHOUT S9 mix:

Dose (µg/plate)	TA 1535	TA 1537	TA 98	TA 100	TA 100 WP2 uvrA
Positive control	627 ± 30	568 ± 56	666 ± 44	1008 ± 88	957 ± 48
Solvent control	15 ± 3	5 ± 2	17 ± 3	106 ± 2	11 ± 4
100	10 ± 4	7 ± 4	18 ± 3	104 ± 5	12 ± 5
333	11 ± 2	5 ± 3	15 ± 3	130 ± 8	12 ± 3
1000	11 ± 3	7 ± 4	15 ± 3	116 ± 2	11 ± 1
3330	11 ± 1	6 ± 3	14 ± 3	92 ± 9	8 ± 2
5000	9 ± 3	4 ± 1	12 ± 2	78 ± 12	11 ± 5

WITH S9 mix:

Dose (µg/plate)	TA 1535	TA 1537	TA 98	TA 100	WP2 uvrA
Positive control	117± 17	91 ± 26	383 ± 15	811 ± 102	128 ± 12
Solvent control	14 ± 2	4 ± 1	26 ± 3	90 ± 8	18 ± 2
100	8 ± 3	4 ± 2	24 ± 3	104 ± 6	17 ± 3
333	10 ± 3	5 ± 2	28 ± 6	122 ± 2	14 ± 4
1000	6 ± 0	5 ± 3	24 ± 3	105 ± 5	15 ± 4
3330	7 ± 1	4 ± 2	21 ± 5	103 ± 10	17 ± 2
5000	9 ± 3	3 ± 1	19 ± 4	105 ± 5	10 ± 2

Solvent control: 0.1 ml ethanol

The S9-mix contained 9.5% (v/v) S9 fraction

Applicant's summary and conclusion

Conclusions:

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

Executive summary:

Cesium potassium fluoroaluminate was tested in the Salmonella typhimurium reverse mutation assay with four histidine-requiring strains of Salmonella typhimurium (TA1535, TA 1537, TA 100 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 (Aroclor-1254 induced rat liver S9-mix), according to the OECD 471 guideline and under GLP conditions. The test substance was suspended in ethanol.

 

ln the dose range finding test, Cesium potassium fluoroaluminate was tested up to concentrations of 5000 µg/plate in the absence and presence of S9-mix in the strains TA100 and WP2uvrA.

ln the first and in the second mutation assay, Cesium potassium fluoroaluminate was tested up to concentrations of 5000 µg/plate in the absence and presence of S9-mix. In dose range-finding, first and second experiment, the bacterial background lawn was not reduced at any of the concentrations tested and no decrease in the number of revertants was observed.

The presence of 5 and 9.5% (v/v) liver microsomal activation did not influence these findings. Cesium potassium fluoroaluminate did not induce a dose-related, two-fold increase in the number of revertant (His+) colonies in each of the four tester strains (TA 1535, 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. Based on the results of this study it is concluded that Cesium potassium is not mutagenic in the Salmonella typhimurium and in the Escherichia coli reverse mutation assay.