Cesium potassium fluoroaluminate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Since all the available in vitro tests showed negative results, cesium potassium fluoroaluminate is not to be considered as mutagenic. Further testing of in vivo genetic toxicity is not considered necessary.

Link to relevant study records

Reference

Reference 1

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

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

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.

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.

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

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.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 29 Oct 2003 to 28 Jan 2004

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

lymphocytes: human

Metabolic activation:

with and without

Metabolic activation system:

S9 mix from rat liver cells

Test concentrations with justification for top dose:

10, 33, 100, 333 and 1000 µg/ml

Vehicle / solvent:

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

Untreated negative controls:

yes

Remarks:

vehicle served as control

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

DOSE RANGE-FINDING TEST
ln order to select the appropriate dose levels for the chromosome aberration test cytotoxicity data were obtained in a dose range finding test. Cesium potassium fluoroaluminate was tested in the absence and in the presence of 1.8% (v/v) S9-fraction.
Lymphocyte cultures (0.4 ml blood of a healthy male donor was added to 5 ml or 4.8 ml culture medium, without and with metabolic activation respectively and 0.1 ml (9 mg/ml) Phytohaemagglutinin) were cultured for 48 h and thereafter exposed to selected doses of Cesium potassium fluoroaluminate for 3 h, 24 h and 48 h in the absence of S9-mix or for 3 h in the presence of S9-mix.
The highest tested concentration was determined by the solubility of Cesium potassium fluoroaluminate in the culture medium.
After 3 h exposure to Cesium potassium fluoroaluminate in the absence or presence of S9-mix, the cells were separated from the exposure medium by centrifugation (5 min, 150 g). The supernatant was removed and cells were rinsed with 5 ml HBSS. After a second centrifugation step, HBSS was removed and cells were resuspended in 5 ml culture medium and incubated for another 20-22 h (24 h fixation time). The cells which were exposed for 24 h and 48 h in the absence of S9-mix were not rinsed after exposure but were fixed immediately (24 h and 48 h fixation time).
Based on the results of the dose range finding test an appropriate range of dose levels was chosen for the cytogenetic assay considering the highest dose level was determined by the solubility.

CYTOGENETIC ASSAY
The cytogenetic assay was carried out with minor modifications as described by Evans, 1984 (2). Cesium potassium fluoroaluminate was tested in the absence and presence of 1.8% (v/v) S9-fraction in duplicate in two independent experiments.

First cytogenetic assay
Lymphocyte cultures were cultured for 48 h and thereafter exposed in duplicate to selected doses of Cesium potassium fluoroaluminate for 3 h in the absence and presence of S9-mix. After 3 h exposure, the cells were separated from the exposure medium by centrifugation (5 min, 150 g). The supernatant was removed and the cells were rinsed once with 5 ml HBSS. After a second centrifugation step, HBSS was removed and cells were resuspended in 5 ml culture medium and incubated for another 20-22 h (24 h fixation time).
Based on the mitotic index of the dose range finding test and the first cytogenetic assay and on the solubility of the test substance in the culture medium appropriate dose levels were selected for the second cytogenetic assay. The independent repeat was performed with the following modifications of experimental conditions.

Second cytogenetic assay
Lymphocyte cultures were cultured for 48 h and thereafter exposed in duplicate to selected doses of Cesium potassium fluoroaluminate for 24 h and 48 h in the absence of S9-mix or for 3 h in the presence of S9-mix.
After 3 h exposure, the cells exposed to Cesium potassium fluoroaluminate in the presence of S9-mix were rinsed once with 5 ml of HBSS and incubated in 5 ml culture medium for another 44-46 h (48 h fixation time).
The cells which were treated for 24 h and 48 h in the absence of S9-mix were not rinsed after exposure but were fixed immediately after 24 h and 48 h (24 h and 48 h fixation time).

CHROMOSOME PREPARATION
During the last 2.5 h of the culture period, cell division was arrested by addition of the spindle inhibitor colchicine (0.5 µg/ml medium). Thereafter the cell cultures were centrifuged for 5 min at 1300 rpm (150 g) and the supernatant was removed. Cells in the remaining cell pellet were swollen by a 5 min treatment with hypotonic 0.56% (w/v) potassium chloride solution at 37°C. After hypotonic treatment, cells were fixed with 3 changes of methanol: acetic acid fixative (3:1 v/v).

PREPARATION OF SLIDES
Fixed cells were dropped onto cleaned slides which were immersed for 24 hours in a 1:1 mixture of 96% (v/v) ethanol/ether and cleaned with a tissue. The slides were marked with the NOTOX study identification number and group number. Two slides were prepared per culture.
Slides were allowed to dry and thereafter stained for 10-30 min with 5% (v/v) Giemsa solution in tap water. Thereafter slides were rinsed in tap-water and allowed to dry. The dry slides were cleared by dipping them in xylene before they were embedded in MicroMount and mounted with a coverslip.

Mitotic index/dose selection for scoring of the cytogenetic assay
The mitotic index of each culture was determined by counting the number of metaphases per 1000 cells. At least three analysable concentrations were used for scoring of the cytogenetic assay. ln case of cytotoxicity, chromosomes of metaphase spreads were analysed of those cultures with an inhibition of the mitotic index of about 50% or greater whereas the mitotic index of the lowest dose level was approximately the same as the mitotic index of the solvent control. Also cultures treated with an intermediate dose were examined for chromosome aberrations. ln case a test substance is not cytotoxic, the highest concentration scored was the concentration in which the test substance precipitated in the culture medium.

Analysis of slides for chromosome aberrations
To prevent bias, all slides were randomly coded before examination of chromosome aberrations and scored. An adhesive label with NOTOX study identification number and code was stuck over the marked slide. 100 metaphase chromosome spreads per culture were examined by light microscopy for chromosome aberrations. ln case the number of aberrant cells, gaps excluded, was ≥ 25 in 50 metaphases no more metaphases were examined. Only metaphases containing 46 ± 2 centromeres (chromosomes) were analysed. The number of cells with aberrations and the number of aberrations were calculated.

Evaluation criteria:

A chromosome aberration test was considered acceptable if it met the following criteria:
a) The number of chromosome aberrations found in the solvent control cultures should reasonably be within the laboratory historical control data range.
b) The positive control substances should produce a statistically significant (Chi-square test, P < 0.05) increase in the number of cells with chromosome aberrations.
c) A homogeneous response between the replicate cultures is observed.

A test substance was considered positive (clastogenic) in the chromosome aberration test if:
a) it induced a dose-related statistically significant (Chi-square test, P < 0.05) increase in the number of cells with chromosome aberrations.
b) a statistically significant and biologically relevant increase in the frequencies of the number of cells with chromosome aberrations was observed in the absence of a clear dose-response relationship.

A test substance was considered negative (not clastogenic) in the chromosome aberration test if none of the tested concentrations induced a statistically significant (Chi-square test, P < 0.05) increase in the number of cells with chromosome aberrations.
The preceding criteria are not absolute and other modifying factors might enter into the final evaluation decision.

Statistics:

Chi-square test

Species / strain:

lymphocytes: human

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 1000 µg/ml

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

DOSE RANGE-FINDING TEST
At a concentration of 1000 µg/ml Cesium potassium fluoroaluminate precipitated in the culture medium. Therefore, a concentration of 1000 µg/ml was used as the highest concentration of Cesium potassium fluoroaluminate.
ln the dose range finding test blood cultures were treated with 10, 33, 100, 333 and 1000 µg Cesium potassium fluoroaluminate/ml culture medium with and without S9-mix. Table 1 shows the mitotic index of cultures (from blood of a healthy male donor) treated with various Cesium potassium fluoroaluminate concentrations or with the negative control substance.

FIRST CYTOGENETIC ASSAY
Based on the results of the dose range finding test the following dose levels were selected for the cytogenetic assay:
With and without S9-mix: 10, 33, 100, 333 and 1000 µg Cesium potassium fluoroaluminate/ml culture medium (3 h exposure time, 24 h fixation time). Table 2 shows the mitotic index of cultures (from blood of a healthy male donor) treated with various Cesium potassium fluoroaluminate (Cesium 5.64% w/w) concentrations or with the positive or negative control substances.
The following dose levels were selected for scoring of chromosome aberrations:
With and without S9-mix: 100, 333 and 1000 µg Cesium potassium fluoroaluminate/ml culture medium (3 h exposure time, 24 h fixation time).
Both in the absence and presence of S9-mix Cesium potassium fluoroaluminate did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations (Tables 3, 4).

SECOND CYTOGENETIC ASSAY
To obtain more information about the possible clastogenicity of Cesium potassium fluoroaluminate, a second cytogenetic assay was performed in which human lymphocytes were continuously exposed to Cesium potassium fluoroaluminate in the absence of S9 mix for 24 or 48 hours. ln the presence of S9-mix, cells were fixed after 48 hours following a 3 hour exposure to Cesium potassium fluoroaluminate. The following dose levels were selected for the second cytogenetic assay:
Without S9-mix: 100, 333 and 1000 µg Cesium potassium fluoroaluminate/ml culture medium (24 h and 48 h exposure time, 24 h and 48 h fixation time)
With S9-mix: 100, 333 and 1000 µg Cesium potassium fluoroaluminate/ml culture medium (3 h exposure time, 48 h fixation time)

Table 5 shows the mitotic index of cultures (from blood of a healthy male donor) treated with various Cesium potassium fluoroaluminate (Cesium 5.64% w/w) concentrations or with the positive or negative contrai substances. Based on these observations all doses were selected for scoring of chromosome aberrations.

ln the absence of S9-mix, at the 24 h continuous exposure time, Cesium potassium fluoroaluminate did not induce a statistically significant increase in the number of cells with chromosome aberrations (Table 6).
ln the absence of S9-mix, at the 48 h continuous exposure time, Cesium potassium fluoroaluminate induced a statistically significant increase in the number of cells with chromosome aberrations at the highest, precipitating concentration only, both when gaps were included and excluded (Table 7). ln the presence of S9-mix, Cesium potassium fluoroaluminate did not induce a statistically significant increase in the number of cells with chromosome aberrations (Table 8).

EVALUATION OF RESULTS
The ability of Cesium potassium fluoroaluminate to induce chromosome aberrations in human peripheral lymphocytes was investigated in two independent experiments. The mitotic indices of cultures treated with various Cesium potassium fluoroaluminate concentrations or with the negative control substances are presented in Tables 1, 2 and 5. The scores for the number of aberrant cells (gaps included and excluded) and the number of the various types of chromosome aberrations at the various concentrations of Cesium potassium fluoroaluminate are presented in Tables 3, 4 and 6-8.

The number of cells with chromosome aberrations found in the solvent control cultures was within the laboratory historical control data range. The positive control chemicals (MMC-C and CP) both produced statistically significant increases in the frequency of aberrant cells. lt was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.
Generally, slides of cultures exposed to the precipitating concentration of 1000 µg/ml Cesium potassium fluoroaluminate were of moderate to poor quality. This effect was substance and dose-related, and should be taken into account for the interpretation of the results.

First cytogenetic assay
Cesium potassium fluoroaluminate did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in the absence and in the presence of S9-mix.

Second cytogenetic assay
ln the absence of S9-mix, at the 24 h continuous exposure time, Cesium potassium fluoroaluminate did not induce a statistically significant increase in the number of cells with chromosome aberrations.

ln the absence of S9-mix, at the 48 h continuous exposure time, Cesium potassium fluoroaluminate induced a statistically significant increase in the number of cells with chromosome aberrations at the highest tested, precipitating concentration only, both when gaps were included and excluded.

Since the increase in the number of cells with chromosome aberrations was not dose related but observed only at a precipitating concentration at the continuous exposure time of 48 hours, the type of aberrations observed were only breaks and gaps and the metaphases on the slides of the cultures exposed to the highest concentration were of poor quality due to the precipitate, it is possible that the observed increase in the number of chromosome aberrations is not caused by a DNA-compound interaction, but is the result of an aspecific, indirect cell damaging action finally resulting in chromosome aberrations. However, in this type of chromosome aberration assays, discrimination between clastogenic and toxic compounds cannot be irrefutably attained.
ln the presence of S9-mix, Cesium potassium fluoroaluminate did not induce a statistically significant increase in the number of cells with chromosome aberrations.

See all tables results in attachment.

Conclusions:

Cesium potassium fluoroaluminate is not considered as clastogenic in human lymphocytes.

Executive summary:

This study describes the effect of cesium potassium fluoroaluminate on the number of chromosome aberrations in cultured peripheral human lymphocytes in the presence and absence of a metabolic activation system (Aroclor-1254 induced rat liver S9-mix). The possible clastogenicity of Cesium potassium fluoroaluminate was tested in two independent experiments, according to the OECD 473 guideline and under GLP conditions.

 

ln the first cytogenetic assay, cesium potassium fluoroaluminate was tested up to 1000 µg/ml for a 3 h exposure time with a 24 h fixation time in the absence and presence of S9-mix. The test substance precipitated in the culture medium at this dose level. ln the second cytogenetic assay, cesium potassium fluoroaluminate was tested up to 1000 µg/ml for a 24 h and 48 h continuous exposure time with a 24 h and 48 h fixation time in the absence of S9-mix. ln the presence of 1.8% (v/v) S9-fraction the test substance was tested up to 1000 µg/ml for a 3 h exposure time with a 48 h fixation time. Cesium potassium fluoroaluminate precipitated in the culture medium at this dose level. 

Positive control chemicals (mitomycin C and cyclophosphamide) both produced a statistically significant increase in the incidence of cells with chromosome aberrations, indicating that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly. Generally, slides of cultures exposed to the precipitating concentration of 1000 µg/ml Cesium potassium fluoroaluminate were of moderate to poor quality. This effect was substance and dose-related, and should be taken into account for the interpretation of the results.

 

First cytoqenetic assay

Cesium potassium fluoroaluminate did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in the absence and in the presence of S9-mix.

 

Second cytoqenetic assay

ln the presence of S9-mix, Cesium potassium fluoroaluminate did not induce a statistically significant increase in the number of cells with chromosome aberrations.

ln the absence of S9-mix, at the 24 h continuous exposure time, Cesium potassium fluoroaluminate did not induce a statistically significant increase in the number of cells with chromosome aberrations. ln the absence of S9-mix, at the 48 h continuous exposure time, Cesium potassium fluoroaluminate induced a statistically significant increase in the number of cells with chromosome aberrations at the highest tested, precipitating concentration only, both when gaps were included and excluded. Since the increase in the number of cells with chromosome aberrations was not dose related but observed only at a precipitating concentration at the continuous exposure time of 48 hours, the type of aberrations observed were only breaks and gaps and the metaphases on the slides of the cultures exposed to the highest concentration were of poor quality due to the precipitate, it is possible that the observed increase in the number of chromosome aberrations is not caused by a DNA-compound interaction, but is the result of an aspecific, indirect cell damaging action finally resulting in chromosome aberrations. However, in this type of chromosome aberration assays, discrimination between clastogenic and toxic compounds cannot be irrefutably attained.

Conclusion:

It is concluded that the test is valid and that Cesium potassium fluoroaluminate is not considered as clastogenic in human lymphocytes under the experimental conditions described in this study.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Reason / purpose for cross-reference:

read-across source

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Conclusions:

Based on read-across from multiconstituent aluminium potassium fluoride, the substance cesium potassium fluoroaluminate is considered as not mutagenic at the TK-locus of mouse lymphoma L5178Y cells.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

In vitro gene mutation study in bacteria

Cesium potassium fluoroaluminate was examined for mutagenic activity in the Ames test (OECD guideline 471 and GLP compliant) using Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and the tryptophan-requiring Escherichia coli strain WP2 uvrA, in the absence and presence of metabolic activation. One bacterial reverse mutation test was performed. All strains were treated with five concentrations of the test substance, ranging from 3 to 5000 µg/plate. Negative controls and positive controls were run simultaneously with the test substance.The mean number of his+ and trp+ revertant colonies of the negative controls were within the acceptable range and the positive controls gave the expected increase in the mean number of revertant colonies. The test substance was not toxic to any strain, in both the absence and presence of S9-mix, as neither a decrease in the mean number of revertants nor a clearing of the background lawn of bacterial growth compared to the negative controls was observed. In both the absence and presence of S9-mix in all strains, cesium potassium fluoroaluminate did not induce a minimal 2-fold and/or dose related increase in the mean number of revertant colonies compared to the background spontaneous reversion rate observed with the negative control. 

In vitro gene mutation study in mammalian cells

Since no in vitro gene mutation study in mammalian cells is available for cesium potassium fluoroaluminate, the results from the structural analogue multiconstituent aluminium potassium fluoride are used instead (for details see Read-across justification as attached in section 13).

In a study according to OECD guideline 476 and in compliance with GLP, multiconstituent aluminium potassium fluoride was examined for its potential to induce gene mutations at the TK-locus of cultured mouse lymphoma L5178Y cells (TNO, 2010a). One assay was conducted in which 7 duplicate cultures were treated for 24 hours and 4 hours in the absence and presence of S9-mix, respectively. The test substance was suspended in dimethyl sulfoxide (DMSO) prior to testing. Treatment with the positive controls yielded the expected significant increases in mutant frequency compared to the negative control.

The highest concentration of aluminium potassium fluoride tested and evaluated for mutagenicity was 10 mmol/L in both the absence and presence of S9-mix. Aluminium potassium fluoride was cytotoxic in both the absence and presence of S9-mix. In the absence of S9-mix cytotoxicity resulted in a reduction in initial cell yield and suspension growth at and above 1.2 mmol/L. The relative total growth (RTG) value at the highest concentration tested and evaluated for mutagenicity (10 mmol/L) was 29% (mean of duplicate cultures). In the presence of S9-mix, cytotoxicity was observed at and above 2.5 mmol/L; the RTG at the highest concentration tested and evaluated for mutagenicity (10 mmol/L) was 61% (mean of duplicate cultures).In both the absence and presence of S9 -mix no increase in mutant frequency was observed at any test substance concentration evaluated.

In vitro cytogenicity / chromosome aberration study in mammalian cells

The ability of cesium potassium fluoroaluminate to induce chromosome aberrations in cultured peripheral human lymphocytes in the presence and absence of a metabolic activation system

(Aroclor-1254 induced rat liver S9-mix) was investigated in a study performed according to OECD Guideline 473 and under GLP. The possible clastogenicity of the test substance was investigated in two independant experiments. ln the first cytogenetic assay, cesium potassium fluoroaluminate was tested up to 1000 µg/ml for a 3 h exposure time with a 24 h fixation time in the absence and presence of S9-mix. The test substance precipitated in the culture medium at this dose level. ln the second cytogenetic assay, cesium potassium fluoroaluminate was tested up to 1000 µg/ml for a 24 h and 48 h continuous exposure time with a 24 h and 48 h fixation time in the absence of S9-mix. ln the presence of 1.8% (v/v) S9-fraction test substance was tested up to 1000 µg/ml for a 3 h exposure time with a 48 h fixation time. Cesium potassium fluoroaluminate precipitated in the culture medium at this dose level.

Positive controls produced a statistically significant increase in the incidence of cells with chromosome aberrations, indicating that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly. Generally, slides of cultures exposed to the precipitating concentration of 1000 µg/ml cesium potassium fluoroaluminate were of moderate to poor quality. This effect was substance and dose-related, and should be taken into account for the interpretation of the results.

 

First cytogenetic assay

Both in the absence and presence of S9-mix cesium potassium fluoroaluminate did not induce a statistically or biologically significant increase in the number of cells with chromosome aberrations.

Second cytogenetic assay

ln the presence of S9-mix, cesium potassium fluoroaluminate did not induce a statistically significant increase in the number of cells with chromosome aberrations. ln the absence of S9-mix, at the 24 h continuous exposure time, cesium potassium fluoroaluminate did not induce a statistically significant increase in the number of cells with chromosome aberrations. ln the absence of S9-mix, at the 48 h continuous exposure time, cesium potassium fluoroaluminate induced a statistically significant increase in the number of cells with chromosome aberrations at the highest tested, precipitating concentration only, both when gaps were included and excluded. Since the increase in the number of cells with chromosome aberrations was not dose related but observed only at a precipitating concentration at the continuous exposure time of 48 hours, the type of aberrations observed were only breaks and gaps and the metaphases on the slides of the cultures exposed to the highest concentration were of poor quality due to the precipitate, it is possible that the observed increase in the number of chromosome aberrations is not caused by a DNA-compound interaction, but is the result of an aspecific, indirect cell damaging action finally resulting in chromosome aberrations. However, in this type of chromosome aberration assays, discrimination between clastogenic and toxic compounds cannot be irrefutably attained.

It is concluded that the test is valid and that cesium potassium fluoroaluminate is not clastogenic in human lymphocytes under the experimental conditions described in this study.

Justification for classification or non-classification

Based on the available in vitro genotoxicity studies, cesium potassium fluoroaluminate does not need to be classified for genotoxicity in accordance with EU Classification, Labeling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.