Cerium(3+) 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:

07 December 2016 to 10 January 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2008

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: Japanese Ministry of Economy, Trade and Industry, Japanese Ministry of Health, Labour and Welfare and Japanese Ministry of Agriculture, Forestry and Fisheries.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Cerium(3+) acetate

EC Number:

208-654-0

EC Name:

Cerium(3+) acetate

Cas Number:

537-00-8

Molecular formula:

C2H4O2.1/3Ce

IUPAC Name:

cerium(3+) triacetate

Test material form:

solid: granular

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Batch No.of test material: CEACK1/16
- Expiration date of the lot/batch: 17 October 2018

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature in the dark over silica gel

Method

Target gene:

S. typhimurium: Histidine locus
E. coli: Tryptophan locus

Species / strain

Species / strain 1

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: University of California, Berkeley, on culture discs, on 04 August 1995 and British Industrial Biological Research Association, on a nutrient agar plate, on 17 August 1987.
- Methods for maintenance in cell culture if applicable: All of the strains were stored at approximately -196 °C in a Statebourne liquid nitrogen freezer.

MEDIA USED
In this assay, overnight sub-cultures of the appropriate coded stock cultures were prepared in nutrient broth (Oxoid Limited; lot number 1865318 05/21) and incubated at 37 °C for approximately 10 hours. Each culture was monitored spectrophotometrically for turbidity with titres determined by viable count analysis on nutrient agar plates.

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: University of California, Berkeley, on culture discs, on 04 August 1995 and British Industrial Biological Research Association, on a nutrient agar plate, on 17 August 1987.
- Methods for maintenance in cell culture if applicable: All of the strains were stored at approximately -196 °C in a Statebourne liquid nitrogen freezer.

MEDIA USED
In this assay, overnight sub-cultures of the appropriate coded stock cultures were prepared in nutrient broth (Oxoid Limited; lot number 1865318 05/21) and incubated at 37 °C for approximately 10 hours. Each culture was monitored spectrophotometrically for turbidity with titres determined by viable count analysis on nutrient agar plates.

Metabolic activation:

with and without

Metabolic activation system:

S9-mix

Test concentrations with justification for top dose:

- Test for Mutagenicity: Experiment 1 - Plate Incorporation Method: 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate
- Test for Mutagenicity: Experiment 2 – Pre-Incubation Method: 15, 50, 150, 500, 1500 and 5000 µg/plate.
- The dose range used for Experiment 2 was determined by the results of Experiment 1. Six test material concentrations were selected in Experiment 2 in order to achieve both four non toxic dose levels and the toxic limit of the test material following the change in test methodology from plate incorporation to pre-incubation.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test material formed the best doseable suspension in dimethyl sulphoxide, therefore, this solvent was selected as the vehicle.

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

benzo(a)pyrene

other: 2-Aminoanthracene and 4-Nitroquinoline-1-oxide

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation) and pre-incubation.

EXPERIMENT 1: PLATE INCORPORATION METHOD
- Eight concentrations of the test material (1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate) were assayed in triplicate against each tester strain, using the direct plate incorporation method.
- 0.1 mL of the appropriate concentration of test material, solvent vehicle or appropriate positive control was added together with 0.1 mL of one of the bacterial strain cultures and 0.5 mL of phosphate buffer to 2 mL of molten, trace amino-acid supplemented media. These were then mixed and overlayed onto a Vogel Bonner agar plate. Negative (untreated) controls were also performed on the same day as the mutation test. Each concentration of the test material, appropriate positive, vehicle and negative controls, and each bacterial strain, was assayed in triplicate.
- For treatment with metabolic activation, the procedure was the same as described previously except that following the addition of the test material formulation and bacterial culture, 0.5 mL of S9 mix was added to the molten, trace amino-acid supplemented media instead of phosphate buffer.

EXPERIMENT 2: PRE-INCUBATION METHOD
The dose range used for Experiment 2 was determined by the results of Experiment 1 and was 15 to 5000 µg/plate.
- 0.1 mL of the appropriate bacterial strain culture, 0.5 mL of phosphate buffer and 0.1 mL of the test material formulation, solvent vehicle or 0.1 mL of appropriate positive control were incubated at 37 ± 3 °C for 20 minutes (with shaking) prior to addition of 2 mL of molten, trace amino-acid supplemented media and subsequent plating onto Vogel Bonner plates. Negative (untreated) controls were also performed on the same day as the mutation test employing the plate incorporation method. All testing for this experiment was performed in triplicate.
- For treatment with metabolic activation, the procedure was the same as described previously except that following the addition of the test material formulation and bacterial strain culture, 0.5 mL of S9 mix was added to the tube instead of phosphate buffer, prior to incubation at 37 ± 3 °C for 20 minutes (with shaking) and addition of molten, trace amino-acid supplemented media.

INCUBATION AND SCORING
For both experiments, all of the plates were incubated at 37 ± 3 °C for approximately 48 hours and scored for the presence of revertant colonies using an automated colony counting system. The plates were viewed microscopically for evidence of thinning (toxicity).

NUMBER OF REPLICATIONS: 3

ACCEPTABILITY CRITERIA
The reverse mutation assay may be considered valid if the following criteria are met:
- All bacterial strains must have demonstrated the required characteristics as determined by their respective strain checks.
- All tester strain cultures should exhibit a characteristic number of spontaneous revertants per plate in the vehicle and untreated controls (negative controls). Acceptable ranges are presented as follows; TA1535: 7 to 40, TA100: 60 to 200, TA1537: 2 to 30, TA98: 8 to 60 and WP2uvrA: 10 to 60.
- All tester strain cultures should be in the range of 0.9 to 9 x 10^9 bacteria per mL.
- Diagnostic mutagens (positive control chemicals) must be included to demonstrate both the intrinsic sensitivity of the tester strains to mutagen exposure and the integrity of the S9-mix. All of the positive control chemicals used in the study should induce marked increases in the frequency of revertant colonies, both with or without metabolic activation.
- There should be a minimum of four non-toxic test material dose levels.
- There should be no evidence of excessive contamination.

Evaluation criteria:

There are several criteria for determining a positive result. Any, one, or all of the following can be used to determine the overall result of the study:
1. A dose-related increase in mutant frequency over the dose range tested.
2. A reproducible increase at one or more concentrations.
3. Biological relevance against in-house historical control ranges.
4. Statistical analysis of data.
5. Fold increase greater than two times the concurrent solvent control for any tester strain (especially if accompanied by an out of historical range response (Cariello and Piegorsch, 1996)).
A test material will be considered non-mutagenic (negative) in the test system if the above criteria are not met.
Although most experiments will give clear positive or negative results, in some instances the data generated will prohibit making a definite judgment about test material activity. Results of this type will be reported as equivocal.

Statistics:

Statistical significance was confirmed by using Dunnetts Regression Analysis (* = p < 0.05) for those values that indicate statistically significant increases in the frequency of revertant colonies compared to the concurrent solvent control.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium, other: TA1535, TA100, TA1537 and TA98

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

Test results 2

Key result

Species / strain:

E. coli WP2 uvr A

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:

Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory). The amino acid supplemented top agar and the S9-mix used in both experiments was shown to be sterile. The test material formulation was also shown to be sterile.

The individual plate counts, the mean number of revertant colonies and the standard deviations, for the test material, positive and vehicle controls, both with and without metabolic activation, are presented in Table 1 for Experiment 1 and Table 2 for Experiment 2.

The maximum dose level of the test material in the first experiment was selected as the maximum recommended dose level of 5000 µg/plate. In the first mutation test (plate incorporation method), the test material induced a visible reduction in the growth of the bacterial background lawns of all of the tester strains initially from 1500 and 5000 µg/plate in the absence and presence S9-mix respectively. These results were not indicative of toxicity sufficiently severe enough to prevent the test material being tested up to the maximum recommended dose level of 5000 µg/plate in the second mutation test. The test material induced a similar toxic response in the second mutation test (pre-incubation method) with reduced bacterial background lawn growth noted at 5000 µg/plate in both the absence and presence of S9-mix. No toxicity was noted for TA1537 dosed in the absence of S9-mix and WP2uvrA dosed in the presence of S9-mix. A test material precipitate (particulate in appearance) was noted at 5000 µg/plate in the first mutation test (plate incorporation method) and from 1500 µg/plate in the second mutation test after incorporating the pre-incubation modification. The precipitate observation did not prevent the scoring of revertant colonies.

There were no significant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation (S9-mix) in Experiment 1 (plate incorporation method). Similarly, no significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation (S9-mix) in Experiment 2 (pre incubation method).

VALIDITY OF THE TEST
Results for the negative controls (spontaneous mutation rates) were considered to be acceptable. These data are for concurrent untreated control plates performed on the same day as the Mutation Test.
The vehicle (dimethyl sulphoxide) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

Applicant's summary and conclusion

Conclusions:

Under the conditions of this study, the test material was considered to be non-mutagenic.

Executive summary:

The potential of the test material to cause genetic toxicity to bacteria was determined in accordance with the standardised guidelines OECD 471, EU Method B13/14, USA EPA OCSPP870.5100 and The Japanese Ministry of Economy, Trade and Industry, Japanese Ministry of Health, Labour and Welfare and Japanese Ministry of Agriculture, Forestry and Fisheries. The genetic toxicity was examined using the reverse mutation assay ‘Ames Test’ under GLP conditions.

Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA were treated with suspensions of the test material using both the Ames plate incorporation and pre-incubation methods at up to eight dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system (10% liver S9 in standard co-factors). The dose range for Experiment 1 was pre-determined and was 1.5 to 5000 µg/plate. The experiment was repeated on a separate day (pre-incubation method) using fresh cultures of the bacterial strains and fresh test material formulations. The dose range was amended following the results of Experiment 1 and was 15 to 5000 µg/plate. Six test material concentrations were selected in Experiment 2 in order to achieve both four non toxic dose levels and the toxic limit of the test material following the change in test methodology.

The vehicle (dimethyl sulphoxide) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

The maximum dose level of the test material in the first experiment was selected as the maximum recommended dose level of 5000 µg/plate. In the first mutation test (plate incorporation method), the test material induced a visible reduction in the growth of the bacterial background lawns of all of the tester strains initially from 1500 and 5000 µg/plate in the absence and presence S9-mix respectively. These results were not indicative of toxicity sufficiently severe enough to prevent the test material being tested up to the maximum recommended dose level of 5000 µg/plate in the second mutation test. The test material induced a similar toxic response in the second mutation test (pre-incubation method) with reduced bacterial background lawn growth noted at 5000 µg/plate in both the absence and presence of S9-mix. No toxicity was noted for TA1537 dosed in the absence of S9-mix and WP2uvrA dosed in the presence of S9-mix. A test material precipitate (particulate in appearance) was noted at 5000 µg/plate in the first mutation test (plate incorporation method) and from 1500 µg/plate in the second mutation test after incorporating the pre-incubation modification. The precipitate observation did not prevent the scoring of revertant colonies.

There were no significant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation (S9-mix) in Experiment 1 (plate incorporation method). Similarly, no significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation (S9-mix) in Experiment 2 (pre‑incubation method). 

Under the conditions of this study, the test material was considered to be non-mutagenic.