Cesium acetate

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 10 Jan, 1997 to 11 April, 1997

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1997

Report date:

1997

Materials and methods

Test guideline

Qualifier:

equivalent or similar 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

Reference substance name:

Cesium acetate

EC Number:

222-248-0

EC Name:

Cesium acetate

Cas Number:

3396-11-0

Molecular formula:

C2H4O2.Cs

IUPAC Name:

cesium acetate

Test material form:

liquid

Remarks:

Clear

Details on test material:

- Name of test material (as cited in study report): Cesium acetate
- Physical state: Clear liquid
- Analytical purity: 96%
- Storage condition of test material: Room temperature; protected from exposure to light

Method

Species / strain

Species / strain 1

Species / strain / cell type:

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

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced rat liver S9

Test concentrations with justification for top dose:

Preliminary test: 6.7, 10, 33, 67, 100, 333, 667, 1000, 3333 and 5000 μg/plate

Experiment 1 (initial mutagenicity assay): 100, 333, 1000, 3333 and 5000 μg/plate
Experiment 1 (independent repeat assay): 100, 333, 1000, 3333 and 5000 μg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Sterile distilled water
- Justification for choice of solvent/vehicle: A solubility test was conducted to select the vehicle. The test was conducted using one or more of the following solvents in the order of preference as listed: purified water, dimethylsulfoxide, ethanol and acetone. The test substance was tested to determine the vehicle, selected in order of preference, that permitted preparation of the highest soluble or workable stock concentration, up to 500 mg/mL.

Controls

Controls 1

Untreated negative controls:

yes

Remarks:

Concurrent untreated controls

Negative solvent / vehicle controls:

yes

Remarks:

Sterile distilled water

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: 2-nitrofluorene at 1 μg/plate for TA98

Remarks:

Without metabolic activation

Controls 2

Untreated negative controls:

yes

Remarks:

Concurrent untreated controls

Negative solvent / vehicle controls:

yes

Remarks:

Sterile distilled water

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene at 1 μg/plate for TA98

Remarks:

With metabolic activation

Controls 3

Untreated negative controls:

yes

Remarks:

Concurrent untreated controls

Negative solvent / vehicle controls:

yes

Remarks:

Sterile distilled water

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: Sodium azide at 1 μg/plate for TA100

Remarks:

Without metabolic activation

Controls 4

Untreated negative controls:

yes

Remarks:

Concurrent untreated controls

Negative solvent / vehicle controls:

yes

Remarks:

Sterile distilled water

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene at 1 μg/plate for TA100

Remarks:

With metabolic activation

Controls 5

Untreated negative controls:

yes

Remarks:

Concurrent untreated controls

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

other:

Positive control substance:

other: Sodium azide at 1 μg/plate for TA1535

Remarks:

Without metabolic activation

Controls 6

Untreated negative controls:

yes

Remarks:

Concurrent untreated controls

Negative solvent / vehicle controls:

yes

Remarks:

Sterile distilled water

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene at 1 μg/plate for TA1535

Remarks:

With metabolic activation

Controls 7

Untreated negative controls:

yes

Remarks:

Concurrent untreated controls

Negative solvent / vehicle controls:

yes

Remarks:

Sterile distilled water

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: 9-aminoacridine at 75 μg/plate for TA1537

Remarks:

Without metabolic activation

Controls 8

Untreated negative controls:

yes

Remarks:

Concurrent untreated controls

Negative solvent / vehicle controls:

yes

Remarks:

Sterile distilled water

True negative controls:

not specified

Positive controls:

not specified

Positive control substance:

other: 2-aminoanthracene at at 1 μg/plate for TA1537

Remarks:

With metabolic activation

Controls 9

Untreated negative controls:

yes

Remarks:

Concurrent untreated controls

Negative solvent / vehicle controls:

yes

Remarks:

Sterile distilled water

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: Methyl methanesulfonate at at 1000 μg/plate for WP2 uvrA

Remarks:

Without metabolic activation

Controls 10

Untreated negative controls:

yes

Remarks:

Concurrent untreated controls

Negative solvent / vehicle controls:

yes

Remarks:

Sterile distilled water

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene at 10 μg/plate for WP2 uvrA

Remarks:

With metabolic activation

Details on test system and experimental conditions:

Test System: The tester strains used were the Salmonella typhimurium histidine auxotrophs TA98, TA100, TA1535 and TA1537 as described by Ames et al. (1975) and Escherichia coli tester strain WP2 uvrA. Tester strains TA98 and TA1537 are reverted from histidine dependence (auxotrophy) to histidine independence (prototrophy) by frameshift mutagens. Tester strain TA1535 is reverted by mutagens that cause basepair substitutions. Tester strain TA100 is reverted by mutagens that cause both frameshift and basepair substitution mutations. Specificity of the reversion mechanism in E. coli is sensitive to base-pair substitution mutations, rather than frameshift mutations (Green and Muriel, 1976). Overnight cultures were prepared by inoculating from the appropriate master plate or from the appropriate frozen permanent stock into a vessel containing 50 mL of culture medium. To assure that cultures were harvested in late log phase, the length of incubation was controlled and monitored. Following inoculation, each flask was placed in a resting shaker/incubator at room temperature. The shaker/incubator was programmed to begin shaking at approx 125 rpm at 37±2°C approx 12 h before the anticipated time of harvest. Each culture was monitored spectrophotometrically for turbidity and was harvested at a percent transmittance yielding a titer of approx 109 cells/mL. The actual titers were determined by viable count assays on nutrient agar plates.

Metabolic Activation System: Aroclor 1254-induced rat liver S9 was used as the metabolic activation system.

Solubility Test: A solubility test was conducted to select the vehicle. The test was conducted using one or more of the following solvents in the order of preference as listed: purified water, dimethylsulfoxide, ethanol and acetone. The test substance was tested to determine the vehicle, selected in order of preference that permitted preparation of the highest soluble or workable stock concentration, up to 500 mg/mL.

Preliminary Toxicity Assay
The preliminary toxicity assay was used to establish the dose-range over which the test substance would be assayed. Ten dose levels of the test substance were plated, one plate per dose, with overnight cultures of TA98, TA100, TA1535, TA1537 and WP2 uvrA on selective minimal agar in both the presence and absence of rat liver S9 activation.

Mutagenicity Assay
The mutagenicity assay (initial and independent repeat assays) was used to evaluate the mutagenic potential of the test substance. A minimum of five dose levels of test substance along with appropriate vehicle and positive controls were plated with tester strains TA98, TA100, TA1535, TA1537 and WP2 uvrA in the presence and absence of rat liver S9 activation. All dose levels of test substance, vehicle controls and positive controls were plated in triplicate.

Plating and Scoring Procedures
The test system was exposed to the test substance via the plate incorporation methodology originally described by Ames et al. (1975) and updated b Maron and Ames (1983). On the day of its use, minimal top agar, containing 0.8 % agar (w/v) and 0.5 % NaCl (w/v), was melted and supplemented with L-histidine, D-biotin and L-tryptophan solution to a final concentration of 50 µM each. Top agar not used with S9 or Sham mix was supplemented with 25 mL of water for each 100 mL of minimal top agar. For the preparation of media and reagents, all references to water imply sterile, deionized water produced by the Milli-Q reagent water system. Bottom agar was Vogel-Bonner minimal medium E (Vogel and Bonner, 1956) containing 1.5 % (w/v) agar. Nutrient bottom agar was Vogel-Bonner minimal medium E containing 1.5 % (w/v) agar and supplemented with 2.5 % (W/V) Oxoid Nutrient Broth No.2 (dry powder). Nutrient Broth was Vogel-Bonner salt solution supplemented with 2.5 % (w/v) Oxoid Nutrient Broth No.2 (dry powder). Each plate was labeled with a code system that identified the test substance, test phase, dose level, tester strain, and activation, as described in detail in Microbiological Associates, Inc.'s Standard Operating Procedures. Test substance dilutions were prepared immediately before use. One-half (0.5) mL of S9 or Sham mix, 100 µL of tester strain and 50 µL of vehicle or test substance were added to 2 mL of molten selective top agar at 45+2°C. After vortexing, the mixture was overlaid onto the surface of 25 mL of minimal bottom agar. When plating the positive controls, the test substance aliquot was replaced by a 50 µL aliquot of appropriate positive control. After the overlay had solidified, the plates were inverted and incubated for approx 48 to 72 hours at 37±2°C. Plates that were not counted immediately following the incubation period were stored at 4±2°C until colony counting could be conducted. The condition of the bacterial background lawn was evaluated for evidence of test substance toxicity by using a dissecting microscope. Precipitate was evaluated by visual examination without magnification. Toxicity and degree of precipitation were scored relative to the vehicle control plate. Revertant colonies for a given tester strain and activation condition were counted either entirely by automated colony counter or entirely by hand unless the assay was the preliminary toxicity assay or the plate exhibited toxicity. Plates with sufficient test substance precipitate to interfere with automated colony counting were counted manually.

Evaluation criteria:

For each replicate plating, the mean and standard deviation of the number of revertants per plate were calculated and are reported. For the test substance to be evaluated positive, it must cause a dose-related increase in the mean revertants per plate of at least one tester strain with a minimum of two increasing concentrations of test substance. Data sets for strains TA1535 and TA1537 were judged positive if the increase in mean revertants at the peak of the dose response is equal to or greater than three times the mean vehicle control value. Data sets for strains TA98, TA100 and WP2 uvrA were judged positive if the increase in mean revertants at the peak of the dose response is equal to or greater than two times the mean vehicle control value.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Solubility Test: Water was selected as the solvent of choice based on solubility of the test substance and compatibility with the target cells. The test substance was soluble in water at approximately 500 mg/mL, the maximum concentration tested.

Preliminary Toxicity Assay: In the preliminary toxicity assay, the maximum dose tested was 5000 µg/plate; this dose was achieved using a concentration of 100 mg/mL and 50 µg/plate plating aliquot. Neither precipitate nor appreciable toxicity was observed. Based on the findings of the toxicity assay, the maximum dose plated in the mutagenicity assay was 5000 µg/plate.

Applicant's summary and conclusion

Conclusions:

Under the study conditions, the test substance was considered to be non-mutagenic

Executive summary:

A study was performed to evaluate the mutagenic potential of the test substance according to a protocol similar to OECD Guideline 471, in compliance with GLP. Tester strains TA98, TA100, TA1535 and TA1537 of Salmonella typhimurium and strain and WP2 uvrA of Escherichia coli in the presence and absence of S9 were exposed to the test substance. The assay was performed in two phases using the plate incorporation method. The first phase, the preliminary toxicity assay, was used to establish the dose range for the mutagenicity assay. The second phase, the mutagenicity assay (initial and independent repeat assays), was used to evaluate the mutagenic potential of the test substance. Water was selected as the solvent of choice based on solubility of the test substance and compatibility with the target cells. In the preliminary toxicity assay, the maximum dose tested was 5000 µg/plate; this dose was achieved using a concentration of 100 mg/mL and 5 µg/plate plating aliquot. Neither precipitate nor appreciable toxicity was observed. Based on the findings of the toxicity assay, the maximum dose plated in the mutagenicity assay was 5000 µg/plate. No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test substance either with or without metabolic activation in the mutagenicity assays. Under the study conditions, the test substance was considered to be non-mutagenic (Wagner, 1997).