Cesium formate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From September 29, 1994 to January 6, 1995

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1995

Report date:

1995

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: Department of Health. Report on Health and Social Subjects Number 35. Guidelines for the testing of chemicals for mutagenecity (1989)

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Test material

Test material information

Constituent 1

Reference substance name:

Cesium formate

EC Number:

222-492-8

EC Name:

Cesium formate

Cas Number:

3495-36-1

Molecular formula:

HCO2.Cs, Cs+HCOO-, CH2O2.Cs

IUPAC Name:

Caesium formate

Test material form:

solid

Details on test material:

- Name of test material (as cited in study report): Cesium Formate
- Physical state: White solid
- Storage condition of test material: Room temperature

Method

Target gene:

Not applicable

Species / strain

Species / strain / cell type:

lymphocytes: Human lymphocytes collected from healthy male donor blood

Details on mammalian cell type (if applicable):

No details

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9 fraction obtained from male Sprague Dawley rats treated withAroclor 1254 and mixed with co-factors

Test concentrations with justification for top dose:

Concentration of the test substance in first test-
9.8, 19.5, 39.1, 78.1, 156.3, 312.5, 625, 1250, 2500 and 5000 µg/mL both in the presence and absence of metabolic activation

Concentration of the test substance in second test-
After 18 h harvest
625, 1250, 2500 and 5000 µg/ml both in the absence and presence of metabolic activation

After 32 h harvest
In absence of S9 mix: 156.3, 312, 625, 1250, 2500 and 5000 µg/ml
In presence of S9 mix: 625, 1250, 2500 and 5000 µg/ml

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Water
- Justification for choice of solvent/vehicle: the test substance is fairly soluble in water (>500 mg/mL). No precipitate was formed at the highest concentration of 5000 µg/mL.
5000 µg/mL was chosen as the highest concentration due to possible artefactual effects on chromosomal aberrations due to high osmolality of higher concentrations.

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

water

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

dissolved in dimethyl sulfoxide, used at a concentration of 250, 500 and 750 µg/mL

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

water

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

dissolved in sterile distilled water, used at a concentration of 2.5, 5, 10, 15 µg/mL

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 48 h
- Exposure duration: 3 h
- Expression time (cells in growth medium):
First test: In presence of S9 mix: 15 h; In absence of S9 mix: 18 h
Second test: In presence of S9 mix: 15 or 29 h; In absence of S9 mix: 18 or 32 h

- Selection time (if incubation with a selection agent): Not applicable
- Fixation time (start of exposure up to fixation or harvest of cells):
First test: In presence of S9 mix: 17 h; In absence of S9 mix: 20 h
Second test: In presence of S9 mix: 17 or 31 h; In absence of S9 mix: 20 or 34 h

SELECTION AGENT (mutation assays): Not applicable
SPINDLE INHIBITOR (cytogenetic assays): Colchicine
STAIN (for cytogenetic assays): Giemsa with dilution factor 1 part Giemsa to 9 parts buffered distilled water

NUMBER OF REPLICATIONS: Duplicate cultures were used for each concentration for all tests

NUMBER OF CELLS EVALUATED: 1000 cells in each culture was examined for the proportion of mitotic cells (except for positive control treated cells)

DETERMINATION OF CYTOTOXICITY
- Method: Mitotic index

OTHER EXAMINATIONS:
- Determination of polyploidy: Not determined in this study
- Determination of endoreplication: Not determined in this study
- Other:
Metaphase analysis: Approximately 100 metaphase figures were examined from each culture for chromatid break, chromosome break, chromatid gap, chromatid exchange, chromosome exchange and chromosome gap.

The dose levels causing a decrease in mitotic index to 50 % of the control value were chosen as the highest dose for metaphase analysis. The intermediate and low doses were selected for the analysis.

Only cells with 44-46 chromosomes were analysed.

Evaluation criteria:

Chromosomal aberrations were scored according to the classification of ISCN1985 (International System for Human Cytogenetic Nomenclature)
Criteria for evaluating results:
Cells evaluated per dose group
1. Proportion of mitotic cells per 1000 cells in each culture recorded
2. Dose level causing approx. 50 % decrease in mitotic index (compared to the solvent control value) selected
3. 100 metaphase figures examined from each culture
4. Cells with 44-48 chromosomes analysed
5. Recording of polyploidy and endoreduplicated cells also done
6. Chromosomal aberration scoring done as per ISCN (1985)

Criteria for accepting validity of the test:
1. Negative and positive control values lie within current historical control range of laboratory

Criteria for judging response of the test:
Positive -
1. Statistically significant increases (P<0.01) in frequency of aberrant chromosomes in metaphase
2. Increases in frequency of aberrant chromosomes in metaphase exceeds 99 % of confidence limits for current historical control range
3. Reproducible increases between replicate cultures
4. Increases not associated with variations in pH, osmolality of the treatment or extreme toxicity
5. Evidence of dose-response relationship to support conclusion

Negative-
No statistically significant increases in frequency of aberrant chromosomes in metaphase observed above concurrent control frequencies

Statistics:

Fischer’s test was used to compare the number of aberrant metaphases figures in each treatment group with the solvent control (Fischer, 1973)

Results and discussion

Test results

Key result

Species / strain:

lymphocytes: Human

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: Not reported
- Effects of osmolality:
First test
An increase in the osmolality of the cultures at 5000 µg/mL was observed both in the absence and presence of S9 mix as compared to solvent control. However, this increase is not considered significant as these increases in osmolality were less than 50 mOsmol/kg

Second test (18 h harvest)
In the absence of S9 mix: No significant increase in the osmolality at 2500 µg/mL was observed.
In the presence of S9 mix: A significant increase of 52 mOsmol/kg above the mean solvent control value occurred at 5000 µg/mL.

Second test (32 h harvest)
In the absence of S9 mix: Significant increase in osmolality of 52 mOsmol/kg observed at 5000 µg/mL.
In the presence of S9 mix: No significant increase in osmolality was observed at any dose level.

- Evaporation from medium: Not reported
- Water solubility: the test substance is fairly soluble in water (>500 mg/mL).
- Precipitation: No precipitation occurred at the highest concentration of 5000 µg/mL
- Other confounding effects: None

RANGE-FINDING/SCREENING STUDIES: Screening studies are not performed in this study

COMPARISON WITH HISTORICAL CONTROL DATA: Not reported

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Mitotic index

First test
In the absence of S9 mix: the test substance reduced the mitotic index to 51 % of the solvent control value at 5000 µg/mL and to 88 % at 2500 µg/mL.
In the presence of S9 mix: Reduction in mitotic index to 71 % of the solvent control value at 5000 µg/mL.

Second test (18 h harvest)
In the absence of S9 mix: Reduction in the mitotic index to 40 % of the solvent control value at 5000 µg/mL and to 85 % at 2500 µg/mL
In the presence of S9 mix: Reduction in the mitotic index to 86 % of the solvent control value at 5000 µg/mL

Second test (32 h harvest)
In the absence of S9 mix: Reduction in the mitotic index to 50 % of the solvent control value at 5000 µg/mL and to 76 % at 1250 µg/mL
In the presence of S9 mix: No toxic responses were observed.

Any other information on results incl. tables

Metaphase analysis

First test:

Both in the presence as well as in the absence of S9 mix, no statistically significant increase in the proportion of abberant cells was observed.

Second test (18 h harvest):

In the absence of S9 mix: Significant increase (5.5 %, which falls just outside the historical control range) in the proportion of metaphase figures with chromosomal aberrations at 2500 µg/mL.

In the presence of S9 mix: No statistically significant increase in the proportion of metaphase figures were observed.

Second test (32 h harvest): No significant increase in the proportion of metaphase figures were observed both in the presence and absence of metabolic activation. Both the positive controls caused large significant increase in aberrant metaphase figures.

Applicant's summary and conclusion

Conclusions:

Under the study conditions evidence of cytotoxicity was observed from 1250 µg/mL in the absence of metabolic activation and only at 5000 µg/mL in the presence of metabolic activation. Based on the above results, it is concluded that the test substance shows no evidence of clastogenic activity in the in vitro human lymphocyte chromosomal aberration assay.

Executive summary:

A study was conducted to determine the clastogenic potential of the test substance according to OECD Guideline 473, in compliance with GLP. Duplicate cultures of human lymphocytes obtained from a healthy male donor were treated with the test substance (dissolved in water) both in the absence and presence of metabolic activation for 3 h. Concentrations of 9.8, 19.5, 39.1, 78.1, 156.3, 312.5, 625, 1250, 2500 and 5000 µg/mL were used in the first test where a harvest/fixation time of 17 or 20 h were kept. Concentrations of 625, 1250, 2500 and 5000 µg/mL were used for an 18 h harvest time in the second test both in the absence and presence of S9 mix. For a 32 h harvest test 156.3, 312, 625, 1250, 2500 and 5000 µg/mL was used in the absence of S9 mix and 625, 1250, 2500 and 5000 µg/mL in the presence of S9 mix. Cytotoxicity was determined by mitotic index. Metaphase analysis was performed for 100 cells per culture. Both the positive controls (cyclophosphamide and ethylmethane sulphonate) caused large, statistically significant increases in the proportion of abberant cells. The test substance caused no substantial increase in the proportion of metaphase figures containing chromosomal aberrations at any dose levels when compared with the solvent control. Under the study conditions evidence of cytotoxicity was observed from 1250 µg/mL in the absence of metabolic activation and only at 5000 µg/mL in the presence of metabolic activation. Based on the above results, it is concluded that the test substance shows no evidence of clastogenic activity in the in vitro human lymphocyte chromosomal aberration assay (Akhurst, 1995).