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Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames

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

Chromosome Aberration Assay ( Read-across performed with structurally similar substance)

Cerium trinitrate was considered not to induce any statistically significant increases in the frequency of cells with aberrations and, therefore was considered to be non-clastogenic with and without metabolic activation.

CHO HPRT

The test material did not induce any toxicologically significant or dose-related increases in mutant frequency per survivor in either the presence or absence of metabolic activation in either of the two experiments. The test material was therefore considered to be non-mutagenic to CHO cells at the HPRT locus under the conditions of this test.

Link to relevant study records

Referenceopen allclose all

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
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1997
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
2008
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
no
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
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

Target gene:
S. typhimurium: Histidine locus
E. coli: Tryptophan locus
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 / 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.
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.
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
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.
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.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
30 August 2012 to 22 March 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
Read-across performed with structurally similar substance.
Reason / purpose for cross-reference:
other: read-across target
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: UK Department of Health Guidelines for Testing of Chemicals for Mutagenicity
Deviations:
not specified
GLP compliance:
yes (incl. QA statement)
Type of assay:
in vitro mammalian chromosome aberration test
Species / strain / cell type:
lymphocytes: Human
Details on mammalian cell type (if applicable):
- Type and identity of media: Cells were grown in Eagle's minimal essential medium with HEPES buffer (MEM), supplemented "in-house" with L-glutamine, penicillin/streptomycin, amphotericin B and 10% foetal bovine serum (FBS), at approximately 37°C with 5% CO2 in humidified air. The lymhocytes of fresh heparinised whole blood were stimulated to divide by the addition of phytohaemagglutinin (PHA).
- Properly maintained: yes
Metabolic activation:
with and without
Metabolic activation system:
rat liver homogenate metabolising system (S9) induced by a mixture of phenobarbitone and beta-naphthoflavone
Test concentrations with justification for top dose:
Four hours exposure with 20-hour expression period without S9: 101.9, 203.75, 407.5, 815, 1222.5 and 1630 µg/mL
Four hours exposure with 20-hour expression period with S9 (2%): 203.75, 407.5, 815, 1630, 2445, 3260 µg/mL
24-hour exposure without S9: 101.9, 203.75, 407.5, 815, 1222.5 and 1630 µg/mL
Four hours exposure with 20-hour expression period with S9 (1%): 203.75, 407.5, 815, 1630, 2445, 3260 µg/mL

Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Dimethyl sulphoxide (DMSO)
- Justification for choice of solvent/vehicle: The test material was insoluble in aqueous media at 32.6 mg/mL but was soluble in dimethyl sulphoxide at 326 mg/mL in solubility checks performed in-house. Dimethyl sulphoxide at 326 mg/ml was, therefore, selected as the vehicle.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
Remarks:
0.4 and 0.2 µg/mL mitomycin C was used in experiment 1 and 2 respectively in the absence of S9. It was dissolved in Minimal Essential Medium. In the presence of S9, cyclophosphamide was used at 5 µg/mL in both experiments. It was dissolved in DMSO.
Details on test system and experimental conditions:
- Exposure duration: Four and 24 hours
- Expression time: 20 hours

SPINDLE INHIBITOR: Colcemid
STAIN: Giemsa

NUMBER OF CELLS EVALUATED: 2000 lymphocyte cell nuclei were counted and the number of cells in metaphase recorded and expressed as the mitotic index and as a percentage of the vehicle control value.

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

OTHER EXAMINATIONS:
- Determination of polyploidy: Cells with 69 chromosomes or more were scored as polyploid.
Evaluation criteria:
Where possible the first 100 consecutive well-spread metaphases from each culture were counted, where there were approximately 30 to 50 % of cells with aberrations, slide evaluation was terminated at 50 cells. If the cell had 44 - 48 chromosomes, any gaps, breaks or rearrangements were noted according to the simplified system of Savage (1976). Cells with chromosome aberrations were reviewed as necessary by a senior cytogeneticist prior to decoding the slides. Cells with chromosome aberrations were reviewed as necessary by a senior cytogeneticist prior to decoding the slides. A positive response was recorded for a particular treatment if the % cells with aberrations, excluding gaps, markedly exceeded that seen in the concurrent control, either with or without a clear dose-relationship. For modest increases in aberration frequency a dose response relationship is generally required and appropriate statistical tests may be applied in order to record a positive response.
Statistics:
The frequency of cells with aberrations excluding gaps and the frequency of polyploidy cells was compared, where necessary, with the concurrent vehicle control value using Fisher's Exact test.
Key result
Species / strain:
lymphocytes: Human lymphocytes
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Qualitative observations in that inhibition of mitotic index was observed and 52% and 82% mitotic inhibition was achieved at 815 and 1222.5 µg/mL, respectively, in the absence of S9. In the presence of S9, 26% mitotic inhibition was achieved at 2445 µg/mL
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
Above 2445 µg/mL metaphases could not be accurately assessed due to the presence of excessive precipitate on the slides.

The maximum dose level selected for metaphase analysis was therefore 815 µg/mL and 2445 µg/mL in the absence and presence of S9, respectively. The toxicity observed at 1222.5 µg/mL, in the absence of S9, was considered to be excessive and precluded the dose from chromosome analysis.

Cerium trinitrate 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 material did not induce a statistically significant increase in the numbers of polyploid cells at any dose level in any of the exposure groups.

All vehicle controls had frequencies of cells with aberrations within the range expected for normal human lymphocytes, determined by the in-house historical range. All the positive controls induced statistically significant increases in the frequency of cells with aberrations indicating that the sensitivity of the assay and the efficacy of the S9-mix were validated.

Conclusions:
Cerium trinitrate was considered not to induce any statistically significant increases in the frequency of cells with aberrations and, therefore was considered to be non-clastogenic with and without metabolic activation.
Executive summary:

The genetic toxicity of the test material was investigated in accordance with the standardised guidelines OECD 473 and EU Method B.10, under GLP conditions.

Human lymphocytes were treated with the test material in the chromosome aberration test, with and without metabolic activation in the form of S9 mix.

In both experiments cerium trinitrate exhibited a modest dose-related inhibition of mitotic index in the dose levels tested. The test material did not induce any statistically significant increases in the frequency of cells with aberrations in the exposure groups dosed in the presence or absence of S9, which included either at least one dose level where precipitate on the slide was observed or 50 % mitotic inhibition.

Cerium trinitrate was considered not to induce any statistically significant increases in the frequency of cells with aberrations and, therefore was considered to be non-clastogenic with and without metabolic activation.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study conducted on read-across material
Justification for type of information:
Read-across performed with structurally similar substance.
Reason / purpose for cross-reference:
read-across source
Key result
Species / strain:
lymphocytes: human
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Qualitative observations in that inhibition of mitotic index was observed, & 52% and 82% mitotic inhibition was achieved at 815 and 1222.5 µg/mL, respectively, in the absence of S9. In the presence of S9, 26% mitotic inhibition was achieved at 2445 µg/mL
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
02 August 2012 to 03 January 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
Read-across performed with structurally similar substance.
Reason / purpose for cross-reference:
other: read-across target
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
mammalian cell gene mutation assay
Target gene:
HPRT locus of Chinese Hamster Ovary (CHO) cells
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
- Type and identity of media: Cells were routinely cultured in Hams F12 medium, supplemented with 5% foetal bovine serum and antibiotics (Penicillin/Streptomycin at 100 units/100 µg per mL) at 37°C with 5% CO2 in air.
- Periodically "cleansed" against high spontaneous background: yes: Cell stocks spontaneously mutate at a low but significant rate. Before stocks of cells were frozen down they were cleansed of HPRT-mutants by culturing in HAT medium for 4 days. This is Hams F12 growth medium supplemented with hypoxanthine, aminopterin and thymidine. After 4 days in medium containing HAT, the cells were passaged into HAT-free medium and grown for 4 to 7 days. Bulk frozen stocks of HAT cleansed cells were frozen down, with fresh cultures being recovered from frozen before each experiment.
Metabolic activation:
with and without
Metabolic activation system:
S9
Test concentrations with justification for top dose:
The dose ranges selected for Experiment 1 and Experiment 2 were based on the results of the preliminary cytotoxicity test and were as follows:
4-hour without S9: 76.41, 152.81, 305.63, 611.25, 916.88, 1222.5 µg/mL
4-hour with S9 (2%): 76.41, 152.81, 305.63, 611.25, 1222.5, 2445 µg/mL
24-hour without S9: 19.1, 38.2, 76.41, 152.81, 305.63, 458.44, 611.25, 1222.5 µg/mL
4-hour with S9 (1%): 76.41, 152.81, 305.36, 611.25, 1222.5, 2445 µg/mL

The concentrations of the test material were calculated based on the anhydrous form.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
Hams F12 medium
True negative controls:
no
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
ethylmethanesulphonate
Remarks:
ethyl methane sulphonate used as positive control at 500 and 750 µg/mL without S9; dimethyl benzanthracene used as positive control at 0.5 and 1.0 µg/mL in cultures with S9
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: not applicable
- Exposure duration: Experiment 1: 4 hours exposure in the presence of S9 and 4 hours in the absence of S9; in experiment 2: 4 hours exposure in the presence of S9 and 24 hours of exposure in the absence of S9
- Expression time (cells in growth medium): 7 days
- Fixation time (start of exposure up to fixation or harvest of cells): Cytotoxicity flasks were incubated for 6 to 7 days then fixed with methanol and stained with Giemsa. Colonies were manually counted and recorded to estimate cytotoxicity.

SELECTION AGENT (mutation assays): 6-thioguanine to determine mutant frequency

NUMBER OF REPLICATIONS: triplicate for determination of cloning efficiency; 5 replicates per group for determination of the mutant frequency

NUMBER OF CELLS EVALUATED: 200 cells/25 m² (cloning efficiency); 2 x 1E05 cells/75 cm² (mutant frequency)

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency

Evaluation criteria:
For a test material dose level to be considered as positive the mutant frequency value must exceed the vehicle control value by 20 x 1E-06 per survivor in order to compensate for random fluctuation in the 0 to 25 x 1E-06 background mutant frequencies that is typical for this assay. A single dose level that meets the minimum criterion for a positive response within a range of assayed concentrations is not sufficient to evaluate the test item as a mutagen. Test results to be obtained for either with or without-metabolic activation conditions before this conclusion can be made, are detailed in the field 'Any other information on materials and methods incl. tables'.
Statistics:
If a test material gives a marked and dose-related increase in the mutant frequency over the vehicle controls it will be designated as mutagenic and statistical analysis will not be required. However, if weaker responses are observed then statistical analysis be performed using the SPSS program or a suitable alternative. All weak responses wil be assessed by the Study Director for biological relevance and justified in the report.

CALCULATIONS
The cloning efficiency (CE), mean plate counts, % control, mutant plate counts, mutant frequency/1E06 and mutant frequency/1E06 survival rate were calculated using the following formulae:

CE% = (mean CE counts/200) x 100
% control = (CE% of dose IDx/CE%) of dose ID0) x 100
MF1E-06 for each dose = total mutant plate counts
MFSV for each dose = (MF 1E-06/CE) x 100
Dose ID0 = vehicle control values
Dose IDx = dose level values

The calculations were performed using an Excel spreadsheet which may result in minor variations in the calculated values when compared to manual calculations due to rounding up differences.
Key result
Species / strain:
Chinese hamster Ovary (CHO)
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
- Precipitation: Experiment 1: a precipitate of the test material was seen at the end of exposure at and above 76.41 µg/mL in the absence of S9 and at and above 611.25 µg/mL in the presence of S9. In the absence of S9 the precipitate became greasy/oily at and above 152.81 µg/mL; Experiment 2: a precipitate of the test material was seen at the end of the exposure period at and above 152.81 µg/mL in both exposure groups.
- Other confounding effects: The osmolality did not increase by more than 50 mOsm when the test material was dosed into media at the dose levels investigated. However, there was a decrease in the pH value of greater than one pH unit when the test material was dosed into media. To help reduce this difference HEPES was added (supplement with buffering capacity) to the culture media at a final concentration of 20mM and this brought the pH readings into an acceptable range (6.21- 6.98).

RANGE-FINDING/SCREENING STUDIES:
A dose range of 9.55, 19.1, 38.2, 76.41, 152.81, 305.63, 611.25, 1222.5 and 2445 µg/mL was used in the preliminary cytotoxicity test. The maximum recommended dose level was 3260 µg/mL, but due to the water content of the test material not being corrected for, the maximum dose tested was 2445 µg/mL. However, since the main tests were limited by toxicity and precipitate this error was considered to be of little consequence and it is considered that the test material was adequately tested.
A precipitate of the test material was observed at the end of exposure at and above 38.2 µg/mL in the 4-hour exposure group in the absence of S9 and at and above 76.41 µg/mL in the 4-hour exposure group in the presence of S9 and in the 24-hour exposure group.

The results of the individual flask counts and their analysis: it can be seen that there was marked toxicity in the 4-hour exposure group in the absence of S9 and in the 24-hour exposure group where dose related reductions in the cloning efficiency can be seen. The 4-hour exposure group in the presence of S9 demonstrated no marked toxicity throughout the test material dose range.

The maximum dose level selected for the main experiments was based on the toxicity seen in the preliminary toxicity test and was 1225 µg/mL for the 4-hour exposure group in the absence of S9 and for the 24 hour exposure group of experiment 2. For the 4-hour exposure groups in the presence of S9 there was no marked toxicity observed throughout the dose range and therefore the maximum dose tested (2445 µg/mL) was selected for the main experiments.

COMPARISON WITH HISTORICAL CONTROL DATA: The vehicle (solvent) controls gave mutant frequencies within the range expected of CHO cells at the HPRT locus.
The positive control treatments, both in the presence and absence of metabolic activation, gave significant increases in the mutant frequency indicating the satisfactory performance of the test and of the metabolising system.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
The test material was markedly toxic in the 4-hour exposures in the absence of S9 and in the 24-hour exposure in the absence of S9 but demonstrated only modest toxicity in the 4-hour exposure in the presence of S9.
In Experiment 1, dose related toxicity was achieved at day 0 in the absence of S9 and no marked toxicity was seen in the presence of S9 at day 0.
The 24-hour exposure group demonstrated marked toxicity at day 0 in Experiment 2 and the 4-hour exposure group in the presence of S9 again demonstrated only modest toxicity at day 0.

OTHER INFORMATION: The concentrations of the test material were originally calculated based on the anhydrous form of the test material and no correction for the water content of the molecule was made (25 % water content). Although the test material has no longer been tested to the maximum recommended 10mM dose level, the presence of toxicity in the absence of S9 means that the test material has been adequately tested in these exposure groups. In the presence of S9 there was no marked toxicity at the dose levels tested. However it is considered that higher dose levels would have been affected by lowered pH greater than the acceptable 1 pH unit and would therefore have been excluded for this reason. Therefore, it is considered that the test item has been adequately tested.

Acceptance criteria:

An assay will normally be considered acceptable for the evaluation of the test results only if all the following criteria are satisfied. The with and without metabolic activation portions of mutation assays are usually performed concurrently, but each portion is, in fact, an independent assay with its own positive and negative controls. Activation or non-activation assays will be repeated independently, as needed, to satisfy the acceptance criteria.

1) The average absolute cloning efficiency of negative controls should be between 70 and 115 % with allowances being made for errors in cell counts and dilutions during cloning and assay variables. Assays in the 50 to 70 % range may be accepted but this will be dependent on the scientific judgement of the study director. All assays below 50 % cloning efficiency will be unacceptable.

2) The background (spontaneous) mutant frequency of the vehicle controls is generally in the range of 0 to 25 x 1E-06. The background values for the with and without-activation segments of a test may vary even though the same stock populations of cells may be used for concurrent assays. Assays with backgrounds greater than 35 x 1E-06 will not be used for the evaluation of a test material.

3) Assays will only be acceptable without positive control data (los due to contamination or technical error) if the test material clearly shows mutagenic activity. Negative or equivocal mutagenic responses by the test material must have a positive control mutant frequency that is markedly elevated over the concurrent negative control.

4) Test materials with little or no mutagenic activity, should include an acceptable assay where concentrations of the test material have reduced the clonal survival to approximately 10 to 15 % of the average of the negative controls, reached the maximum recommended dose (10 mM or 5 mg/mL) or twice the solubility limit of the test material in culture medium. Where a test material is excessively toxic, with a steep response curve, a concentration that is at least 75 % of the toxic dose level should be used. There is no maximum toxicity requirement for test materials that are clearly mutagenic.

5) Mutant frequencies are normally derived from sets of five dishes for mutant colony count and three dishes for variable colony counts. To allow for contamination losses it is acceptable to score a minimum of four mutant selection dishes and two viability dishes.

6) Five dose levels of test material, in duplicate, in each assay will normally be assessed for mutant frequency. A minimum of four analysed duplicate dose levels is considered necessary in order to accept a single assay for evaluation of the test material.

Conclusions:
The test material did not induce any toxicologically significant or dose-related increases in mutant frequency per survivor in either the presence or absence of metabolic activation in either of the two experiments. The test material was therefore considered to be non-mutagenic to CHO cells at the HPRT locus under the conditions of this test.
Executive summary:

The genetic toxicity of the test material was investigated in accordance with the standardised guidelines OECD 476, EU Method B.17 and EPA OPPTS 870.5300 under GLP conditions.

The effects of the test material were determines using the HPRT assay with Chinese Hamster Ovary (CHO) cells. The dose ranges selected for Experiment 1 and Experiment 2 were based on the results of the preliminary cytotoxicity test and were as follows: 4-hour without S9: 76.41, 152.81, 305.63, 611.25, 916.88, 1222.5 µg/mL, 4-hour with S9 (2%): 76.41, 152.81, 305.63, 611.25, 1222.5, 2445 µg/mL, 24-hour without S9: 19.1, 38.2, 76.41, 152.81, 305.63, 458.44, 611.25, 1222.5 µg/mL and 4-hour with S9 (1%): 76.41, 152.81, 305.36, 611.25, 1222.5, 2445 µg/mL.

The test material was markedly toxic in the 4-hour exposures in the absence of S9 and in the 24-hour exposure in the absence of S9 but demonstrated only modest toxicity in the 4-hour exposure in the presence of S9. In Experiment 1, dose related toxicity was achieved at day 0 in the absence of S9 and no marked toxicity was seen in the presence of S9 at day 0. The 24-hour exposure group demonstrated marked toxicity at day 0 in Experiment 2 and the 4-hour exposure group in the presence of S9 again demonstrated only modest toxicity at day 0.

The test material did not induce any toxicologically significant or dose-related increases in mutant frequency per survivor in either the presence or absence of metabolic activation in either of the two experiments. The test material was therefore considered to be non-mutagenic to CHO cells at the HPRT locus under the conditions of this test.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study conducted on read-across material
Justification for type of information:
Read-across performed with structurally similar substance.
Reason / purpose for cross-reference:
read-across source
Key result
Species / strain:
Chinese hamster Ovary (CHO)
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
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Ames Test

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. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

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.

Chromosome Aberration Assay ( Read-across performed with structurally similar substance)

The genetic toxicity of the test material was investigated in accordance with the standardised guidelines OECD 473 and EU Method B.10, under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

Human lymphocytes were treated with the test material in the chromosome aberration test, with and without metabolic activation in the form of S9 mix.

In both experiments cerium trinitrate exhibited a modest dose-related inhibition of mitotic index in the dose levels tested. The test material did not induce any statistically significant increases in the frequency of cells with aberrations in the exposure groups dosed in the presence or absence of S9, which included either at least one dose level where precipitate on the slide was observed or 50 % mitotic inhibition.

Cerium trinitrate was considered not to induce any statistically significant increases in the frequency of cells with aberrations and, therefore was considered to be non-clastogenic with and without metabolic activation.

CHO HPRT

The genetic toxicity of the test material was investigated in accordance with the standardised guidelines OECD 476, EU Method B.17 and EPA OPPTS 870.5300 under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

The effects of the test material were determines using the HPRT assay with Chinese Hamster Ovary (CHO) cells. The dose ranges selected for Experiment 1 and Experiment 2 were based on the results of the preliminary cytotoxicity test and were as follows: 4-hour without S9: 76.41, 152.81, 305.63, 611.25, 916.88, 1222.5 µg/mL, 4-hour with S9 (2%): 76.41, 152.81, 305.63, 611.25, 1222.5, 2445 µg/mL, 24-hour without S9: 19.1, 38.2, 76.41, 152.81, 305.63, 458.44, 611.25, 1222.5 µg/mL and 4-hour with S9 (1%): 76.41, 152.81, 305.36, 611.25, 1222.5, 2445 µg/mL.

The test material was markedly toxic in the 4-hour exposures in the absence of S9 and in the 24-hour exposure in the absence of S9 but demonstrated only modest toxicity in the 4-hour exposure in the presence of S9. In Experiment 1, dose related toxicity was achieved at day 0 in the absence of S9 and no marked toxicity was seen in the presence of S9 at day 0. The 24-hour exposure group demonstrated marked toxicity at day 0 in Experiment 2 and the 4-hour exposure group in the presence of S9 again demonstrated only modest toxicity at day 0.

The test material did not induce any toxicologically significant or dose-related increases in mutant frequency per survivor in either the presence or absence of metabolic activation in either of the two experiments. The test material was therefore considered to be non-mutagenic to CHO cells at the HPRT locus under the conditions of this test.

Justification for classification or non-classification

In accordance with the criteria for classification as defined in Annex I, Regulation (EC) No. 1272/2008, the substance does not require classification with respect to genetic toxicity.