2-ethylhexanoic acid, cerium salt

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Between 25 July 2012 and 19 October 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Well documented GLP study performed according to OECD Guideline 471, EU Method B.13/14 and EPA OPPTS 870.5100.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

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

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 trichloride

EC Number:

232-227-8

EC Name:

Cerium trichloride

Cas Number:

7790-86-5

Molecular formula:

CeCl3

IUPAC Name:

cerium(3+) trichloride

Test material form:

solid: crystalline

Method

Target gene:

Salmonella typhimurium
TA1537: his C 3076; rfa-; uvrB- (frame shift mutations)
TA98: his D 3052; rfa-; uvrB-; R-factor (frame shift mutations)
TA1535: his G 46; rfa-; uvrB- (base-pair mutations)
TA100: his G 46; rfa-; uvrB-; R-factor (base-pair mutations)

Escherichia coli
WP2 uvrA: trp-; uvrA- (base-pair substitution)

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:

S9 microsomal fraction from rats induced with phenobarbitone/beta-naphtoflavone

Test concentrations with justification for top dose:

Preliminary toxicity test: 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate (cerium trichloride as active ingredient)
Mutation test - experiment 1: 0, 50, 150, 500, 1500 and 5000 µg/plate (cerium trichloride as active ingredient)
Mutation test - experiment 2: 0, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate (cerium trichloride as active ingredient) - additional dose levels (5 and 15 µg/plate) and an expanded dose range were selected in order to achieve both four non-toxic dose levels and the toxic limit of the test item.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: sterile distilled water

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

2 µg/plate for WP2uvrA, 3 µg/plate for TA100, 5 µg/plate for TA1535; without S9-mix

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

80 µg/plate for TA1537; without S9 mix

Controls 3

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 4-Nitroquinoline-1-oxide

Remarks:

0.2 µg/plate for TA98; without S9 mix

Controls 4

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

1 µg/plate for TA100; 2 µg/plate for TA1535 and TA1537; 10 µg/plate for WP2uvrA; with S9-mix

Controls 5

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

5 µg/plate for TA98; with S9-mix

Details on test system and experimental conditions:

METHOD OF APPLICATION: plate incorporation (Mutation test - Experiment I) and preincubation method (Mutation test - Experiment II)

DURATION
- Preincubation period: 20 minutes (mutation test - experiment 2 - pre-incubation method)
- Exposure duration: 48 hours
- Selection time (if incubation with a selection agent): simultaneous with exposure

SELECTION AGENT (mutation assays): trace histidine or tryptophan supplemented

NUMBER OF REPLICATIONS: triplicate

DETERMINATION OF CYTOTOXICITY
- Method: reduction in number of revertant colonies and reduction of bacterial background lawn
- Preliminary toxicity test:
In order to select appropriate dose levels for use in the main test, a preliminary test was carried out to determine the toxicity of the test item. The concentrations tested were 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate (cerium trichloride as active ingredient). The test was performed by mixing 0.1 mL of bacterial culture (TA100 or WP2uvrA), 2 mL of molten, trace histidine or tryptophan supplemented, top agar, 0.1 mL of test item formulation and 0.5 mL of S9-mix or phosphate buffer and overlaying onto sterile plates of Vogel-Bonner Minimal agar (30 mL/plate). Ten concentrations of the test item formulation and a vehicle control (sterile distilled water) were tested. In addition, 0.1 mL of the maximum concentration of the test item and 2 mL of molten, trace histidine or tryptophan supplemented, top agar were overlaid onto a sterile nutrient agar plate in order to assess the sterility of the test item. After approximately 48 hours incubation at 37°C the plates were assessed for numbers of revertant colonies using an automated colony counter and examined for effects on the growth of the bacterial background lawn.

OTHER: S9 - mix was used at 10% in plates incubated with metabolic activation

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 as determined by UKEMS (Mahon et al (1989))
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.
A test item 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 item activity. Results of this type will be reported as equivocal.

Results and discussion

Test results

Test results 1

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:

cytotoxicity

Remarks:

Visible reduction in the growth of the bacterial background lawns of all of the tester strains (except TA98 dosed in the presence of S9-mix in Experiment 2) was observed in both the presence and absence of S9-mix at 5000 µg/plate in both experiments.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

.Visible reduction in the growth of the bacterial background lawns of WP2 uvr A was observed in both the presence and absence of S9-mix at 5000 µg/plate in both experiments.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

The test item caused a visible reduction in the growth of the bacterial background lawns of all of the tester strains (except TA98 dosed in the presence of S9-mix in Experiment 2) in both the presence and absence of S9-mix at 5000 µg/plate in both experiments. A slight reduction in TA98 revertant colony frequency was noted at 5000 µg/plate in both experiments. A slight reduction in TA98 revertant colony frequency was noted at 5000 µg/plate in Experiment 2 (presence of S9-mix) without a weakening of the bacterial background lawns. These results were not indicative of toxicity sufficiently severe enough to prevent the test item being tested up to the maximum recommended dose level of 5000 µg/plate. A test item precipitate (particulate in appearance) was noted at 5000 µg/plate in both the presence and absence of S9-mix in Experiments 1 and 2, this observation did not prevent the scoring of revertant colonies.

No significant increases in the frequency of revertant colonies were recorded for any of the strains of bacteria, at any dose level either with or without metabolic activation or exposure method.

All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies thus confirming the activity of the S9-mix and the sensitivity of the bacterial strains.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:

Interpretation of results:
negative both with and without metabolic activation

The test item, cerium trichloride, was considered to be non-mutagenic with and without metabolic activation, under the conditions of this test.