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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Three key studies are available:

-In an Ames test according to OECD Guideline 471, EU Method B.13/14 and EPA OPPTS 870.5100 (Thompson, 2013), cerium ammonium nitrate proved to be negative for mutagenicity with and without metabolic activation.
- Chromosome aberration: performed with the read-across substance cerium trinitrate according to OECD 473 in human lymphocytes (Bowles A, 2013). According to the test results of the study, cerium trinitrate is considered to be non-clastogenic.

- In vitro mammalian cell gene mutation: performed with the read-across substance cerium trinitrate according to OECD 476 in Chinese hamster Ovary (CHO) cells (Morris, 2013). According to the test results of the study, cerium trinitrate is considered to be non-clastogenic.

A justification for the read across approach is included in section 13.

Link to relevant study records
Reference
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 7 August 2012 to 11 October 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
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 / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
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 ammonium nitrate as active ingredient)
Mutation test - experiment 1:
15, 50, 150, 500, 1500, 5000 µg/plate (cerium ammonium nitrate as active ingredient)
Mutation test - experiment 2:
5, 15, 50, 150, 500, 1500, 5000 µg/plate (cerium ammonium nitrate as active ingredient)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: sterile distilled water
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
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
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
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
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 ammonium nitrate 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). Then 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 judgement about test item activity. Results of this type will be reported as equivocal.
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:
A visible reduction in the growth of the bacterial background lawns of all the strains at 5000 µg/plate with and without S9-mix was observed in both experiments except TA1537 with S9-mix, where the reduction was also observed at 1500 µg/plate Exp. I only
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
A visible reduction in the growth of the bacterial background lawns of WP2 uvr A at 5000 µg/plate with and without S9-mix was observed in both experiments.
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Preliminary toxicity test:
Reductions in colony frequency were noted at 5000 µg/plate. The test item formulation and S9-mix used in this experiment were both shown to be sterile.

Mutation test:
The test item caused a visible reduction in the growth of the bacterial background lawns of all of the tester strains at 5000 µg/plate in both the absence and presence of S9-mix employing plate incorporation and pre-incubation methodologies (except TA1537, with S9-mix, where the bacterial background lawn was also toxic at 1500 µg/plate in Experiment 1 only). These results were not indicative of sufficiently severe toxicity to prevent the test item being tested up to the maximum recommended dose level of 5000 µg/plate. No test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix in both experiments.

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
Conclusions:
The test item, cerium ammonium nitrate, was considered to be non-mutagenic under the conditions of this test.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

According to REACH Annex IX section 8.4, column 2, no further in vivo testing is required as no positive results were obtained in any of the three in vitro studies performed according to REACH Annexes VII and VIII section 8.4.

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Genetic toxicity in vitro:

Bacterial reverse mutation assay:

Thompson (2013) performed an Ames (preliminary toxicity test, plate incorporation assay and preincubation method) test with S typhimuriums trains TA1535, TA1537, TA98 and TA100 and Escherichia colis train WP2uvrA in the presence and absence metabolic activation (10% S9 -mix).

 

Following test concentrations were applied in triplicate:

Preliminary toxicity test: 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate (cerium ammonium nitrate as active ingredient)

Mutation test - experiment 1: 15, 50, 150, 500, 1500, 5000 µg/plate (cerium ammonium nitrate as active ingredient)

Mutation test - experiment 2: 5, 15, 50, 150, 500, 1500, 5000 µg/plate (cerium ammonium nitrate as active ingredient)

 

The test item caused a visible reduction in the growth of the bacterial background lawns of all of the tester strains at 5000 µg/plate in both the absence and presence of S9-mix employing plate incorporation and pre-incubation methodologies (except TA1537, with S9-mix, where the bacterial background lawn was also toxic at 1500 µg/plate in Experiment 1 only). These results were not indicative of sufficiently severe toxicity to prevent the test item being tested up to the maximum recommended dose level of 5000 µg/plate. No test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix in both experiments. 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.

Solvent, negative and positive controls were valid. The test item, cerium ammonium nitrate, was considered to be non-mutagenic under the conditions of this test.

Chromosome Aberration:

Read-across to cerium trinitrate is suggested for this endpoint. The read-across justification is added in section 13 to the IUCLID file for extrapolation to cerium ammonium nitrate. Bowles A (2013) performed an in vitro Chromosome Aberration test in human lymphocytes (OECD Guideline 473 and EU Method B10). Two experiments were performed using different test concentrations with and without S9 activation (4h or 24h exposure; 20h expression period). In both experiments cerium trinitrate exhibited a modest dose-related inhibition of mitotic index in the dose levels tested. However, the substance did not induce any statistically significant increases in the frequency of cells with aberrations in either exposure group, which included a dose level that was generally within the optimal 50% mitotic inhibition. The test item did not induce a statistically significant increase in the numbers of polyploid cells at any dose level in any of the exposure groups. Cerium trinitrate was considered to be non-clastogenic with and without metabolic activation.

In vitro mammalian cell gene mutation:

Read-across to cerium trinitrate is suggested for this endpoint. The read-across justification is added in section 13 to the IUCLID file for extrapolation to cerium ammonium nitrate. Morris (2013) performed a CHO hprt forward mutation assay with the read-across substance cerium trinitrate, targeting the HPRT locus of Chinese hamster ovary (CHO) cells, according to OECD Guideline 476 and EU Method B.17. Chinese hamster Ovary (CHO) cells were treated with the test item at up to eight dose levels, in duplicate, together with vehicle (dimethylsulfoxide) and positive controls. Ethyl methane sulphonate (EMS) was used as the positive control for cultures in the absence of S9 and Dimethyl benzanthacene (DMBA) was used as the positive control in cultures with S9. Four treatment conditions were used for the test, i.e. in the

The technique used is a plate assay using tissue culture flasks and 6-thioguanine (6-TG) as the selective agent. The vehicle and positive control were considered to be valid and the test item did not demonstrate dose related increases in mutant frequency either with or without metabolic activation, either in the first or the second experiment. Therefore, the test item was considered to be non-mutagenic to CHO cells at the HPRT locus under the conditions of this test.



Justification for classification or non-classification

On the basis of the available information and according to the criteria of the DSD and CLP Regulation, cerium ammonium nitrate should not be classified as mutagenic.