Registration Dossier
Registration Dossier
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EC number: 909-709-8 | CAS number: -
Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
In vitro gene mutation in bacteria
No indication of mutagenicity was observed in an Ames test performed with a representative nanoform of the reaction mass of cerium dioxide and zirconium dioxide. The results from Ames studies performed with bulk or nano cerium dioxide, bulk zirconium dioxide, and zirconium dioxide with a small w/w % of yttrium in it, were supportive of this finding and therefore it can be concluded that the reaction mass of cerium dioxide and zirconium dioxide is not mutagenic in bacteria.
In vitro gene mutation in mammalian cells
In the absence of experimental information on the reaction mass itself, this endpoint was covered by read across from the results of in vitro gene mutation assays in mammalian cells performed with (bulk) cerium dioxide and (bulk) zirconium dioxide. In these studies no mutagenic activity was observed with and without metabolic activation under the conditions of the tests. Therefore, no mutagenic activity is to be expected from the reaction mass of cerium dioxide and zirconium dioxide either.
In vitro (and in vivo) cytogenicity
In the absence of experimental information on the reaction mass itself, this endpoint was covered by read across from an in vitro chromosome aberration study performed with (bulk) zirconium dioxide, which was negative with and without metabolic activation under the conditions of the test, and an in vivo mouse micronucleus test performed with (bulk) cerium dioxide, which was negative as well. Therefore, no clastogenicity is to be expected from the reaction mass of cerium dioxide and zirconium dioxide either.
Link to relevant study records
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- from 21-MAY-2007 to 23-NOV-2007
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- bacterial reverse mutation assay
- Species / strain / cell type:
- S. typhimurium, other: TA1535, TA1537, TA98, TA100 and TA102
- Metabolic activation:
- with and without
- Metabolic activation system:
- Liver S9 fraction of rats induced with Aroclor 1254
- Test concentrations with justification for top dose:
- 312.5, 625, 1250, 2500 and 5000 µg/plate for the three experiments, with or without S9 mix
- Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: homogeneous suspension to the naked eye
- Volume of vehicle/solvent in the medium: 0.05 mL per 2.60 mL medium - Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: sodium azide (TA1535 and TA100 without S9 mix, 1 µg/plate); 9-aminoacridine (TA1537 without S9 mix, 50 µg/plate); 2-nitrofluorene (TA98 without S9 mix, 0.5 µg/plate); mitomycin C (TA102 without S9 mix, 0.5 µg/plate)
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: 2-anthramine (TA1535, TA1537 and TA98 with S9 mix, 2 µg/plate; TA102 with S9 mix, 10 µg/plate); benzo(a)pyrene (TA100 with S9 mix, 5 µg/plate)
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: All experiments were performed according to the direct plate incorporation method except for the second test with S9 mix, which was performed according to the pre-incubation method
DURATION
- Pre-incubation period: 60 minutes, 37°C
- Exposure duration: 48 to 72 hours
NUMBER OF REPLICATES: three plates/dose-level
OTHER: SCORING METHOD: automated - Evaluation criteria:
- A reproducible 2-fold increase (for the TA98, TA100 and TA102 strains) or 3-fold increase (for the TA1535 and TA1537 strains) in the number of revertants compared with the vehicle controls, in any strain at any dose-level and/or evidence of a dose-response was considered as a positive result. Reference to historical data, or other considerations of biological relevance were taken into account in the evaluation of the data obtained.
- Statistics:
- not concerned
- Key result
- Species / strain:
- S. typhimurium, other: TA1535, TA1537, TA98, TA100 and TA102
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity, but tested up to precipitating concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: A moderate to strong precipitate was observed in the Petri plates when scoring the revertants at dose-levels >= 312.5 µg/plate (the precipitate did not interfere with the scoring).
RANGE-FINDING/SCREENING STUDIES
To assess the toxicity of the test item to the bacteria, six dose-levels (one plate/dose-level) were tested in the TA98, TA100 and TA102 strains, with and without S9 mix. The evaluation of the toxicity was performed on the basis of the observation of the decrease in the number of revertant colonies and/or a thinning of the bacterial lawn.
A moderate to strong precipitate was observed in the Petri plates when scoring the revertants at dose-levels >= 100 µg/plate. No noteworthy toxicity was noted towards the three strains used, either with or without S9 mix.
COMPARISON WITH HISTORICAL CONTROL DATA: The control data were in the range of the historical control data observed in the laboratory. - Remarks on result:
- other: all strains/cell types tested
- Conclusions:
- Under the experimental conditions of the test, the reaction mass of cerium dioxide and zirconium dioxide did not show any mutagenic activity in the bacterial mutation test with Salmonella typhimurium.
- Executive summary:
The objective of this study was to evaluate the potential of the reaction mass of cerium dioxide and zirconium dioxide to induce reverse gene mutations in Salmonella typhimurium.
The study was performed according to international guidelines (OECD 471, Commission Directive No. B13/14) and in compliance with the Principles of Good Laboratory Practice Regulations.
The test item was tested in two independent experiments, with and without a metabolic activation system, i.e. S9 mix, prepared from a liver post mitochondrial fraction (S9 fraction) of rats induced with Aroclor1254. A third experiment was performed with S9 mix.
Salmonella typhimurium TA1535, TA1537, TA98, TA100 and TA102 were used. Each strain was exposed to at least five dose-levels of the test item (three plates/dose-level). After 48 to 72 hours of incubation at 37°C, the revertant colonies were scored.
Solvent control (DMSO) and positive controls were used.
The number of revertants for the vehicle and positive controls was as specified in the acceptance criteria. The study was therefore considered valid.
The selected treatment-levels ranged from 312.5 to 5000 µg/plate, either with or without S9 mix.
A moderate to strong precipitate was observed in the Petri plates when scoring the revertants at dose-levels >= 312.5 µg/plate. No noteworthy toxicity was induced in any of the five tester strains.
The test item did not induce any noteworthy increase in the number of revertants which could be considered as relevant, either with or without S9 mix, in any of the five tester strains.
Under the experimental conditions of the test, the reaction mass of cerium dioxide and zirconium dioxide did not show any mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium.
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- experimental study
- Adequacy of study:
- supporting study
- Study period:
- Not specified
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- comparable to guideline study
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Deviations:
- not applicable
- GLP compliance:
- not specified
- Type of assay:
- bacterial forward mutation assay
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Details on mammalian cell type (if applicable):
- Obtained from B.N. Ames, USA
- Species / strain / cell type:
- S. typhimurium TA 1538
- Details on mammalian cell type (if applicable):
- Obtained from B.N. Ames, USA
- Species / strain / cell type:
- E. coli WP2 uvr A
- Details on mammalian cell type (if applicable):
- Obtained from M. Ishizawa, Japan
- Metabolic activation:
- with and without
- Metabolic activation system:
- Polychlorinated biphenyl-pretreated male Sprague-Dawley rat liver S9 fraction
- Test concentrations with justification for top dose:
- 0 (vehicles), 1, 5, 10, 50, 100, 500, 1000 or 5000 µg/plate
- Vehicle / solvent:
- Vehicle(s)/solvent(s) used: water (diluent), DMSO and acetone (negative controls)
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- Remarks:
- water, DMSO and acetone
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 4-nitroquinoline-N-oxide
- 9-aminoacridine
- N-ethyl-N-nitro-N-nitrosoguanidine
- other: 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide (AF-2)
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- Remarks:
- water, DMSO and acetone
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- benzo(a)pyrene
- other: 2-aminoanthracene
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: pre-incubation
DURATION
- Pre-incubation period: 20 minutes at 37°C
- Incubation period: 48 hours at 37°C
SELECTION AGENT (mutation assays): histidine and biotin (S. typhimurium); tryptophan (E. coli)
NUMBER OF REPLICATIONS: duplicates
OTHER: Concentrations inducing growth inhibition noted - Evaluation criteria:
- Number of revertant colonies scored with an automated colony counter
- Statistics:
- No data
- 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:
- no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
- Remarks:
- Growth inhibition noted at 5000 µg/plate for TA1535 and TA1537 with or without S9
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Species / strain:
- S. typhimurium TA 1538
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
- 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:
- no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Conclusions:
- No mutagenic activity in this Ames test up to the limit concentration of 5000 µg/plate with or without metabolic activation
- Executive summary:
The mutagenic potential of Cerium Oxide was tested in a bacterial reverse mutation (Ames) test. The test substance was applied on five strains of Salmonella typhimurium (TA100, TA98, TA1535, TA1537 and TA1538) and one strain of Escherichia coli (WP2uvrA), using the preincubation method, at concentrations of 0 (water, DMSO and acetone), 1, 5, 10, 50, 100, 500, 1000 or 5000 µg/plate, with or without metabolic activation. The appropriate positive controls were included and responded adequately.
Whatever the test concentration and the presence or absence or metabolic activation, no significant increase in the number of revertant colonies per plate over controls occurred.
Therefore Cerium Oxide showed no mutagenic activity in this bacterial reverse mutation (Ames) test using the preincubation method up to the limit concentration of 5000 µg/plate with or without metabolic activation.
This study is classified as acceptable. It is compatible with the OECD 471 guideline requirements on bacterial reverse mutation test.
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Study period:
- 08 November 2005 - 03 January 2006
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- in vitro mammalian cell gene mutation test using the Hprt and xprt genes
- Target gene:
- HPRT locus of V79 Chinese Hamster cells
- Species / strain / cell type:
- Chinese hamster lung fibroblasts (V79)
- Details on mammalian cell type (if applicable):
- MEDIA USED
- Type and identity of media: Minimal Essential Medium supplemented with 10% fetal calf serum
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes - Metabolic activation:
- with and without
- Metabolic activation system:
- Phenobarbital/beta-Naphtoflavone-induced rat liver S9 fraction
- Test concentrations with justification for top dose:
- Experiment I:
- 4 hours without S9: 28.1, 56.3, 112.5, 225, 450 and 1800 µg/mL
- 4 hours wIth S9: 28.1, 56.3, 112.5, 225, 450 and 1800 µg/mL
Experiment II:
- 24 hours without S9: 28.1, 56.3, 112.5, 225, 450 and 1800 µg/mL
- 4 hours with S9: 14.1, 28.1, 56.3, 112.5, 225 and 1800 µg/mL - Vehicle / solvent:
- - Solvent used: deionized water
- Untreated negative controls:
- yes
- Remarks:
- culture medium
- Negative solvent / vehicle controls:
- yes
- Remarks:
- deionized water
- True negative controls:
- yes
- Remarks:
- untreated cells
- Positive controls:
- yes
- Positive control substance:
- ethylmethanesulphonate
- Untreated negative controls:
- yes
- Remarks:
- culture medium
- Negative solvent / vehicle controls:
- yes
- Remarks:
- deionized water
- True negative controls:
- yes
- Remarks:
- untreated cells
- Positive controls:
- yes
- Positive control substance:
- 7,12-dimethylbenzanthracene
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium
DURATION
- Preincubation period: not applicable
- Exposure duration: 4 or 24 hours
- Expression time (cells in growth medium): approx. 7 days
- Selection time (if incubation with a selection agent): not specified
- Fixation time (start of exposure up to fixation or harvest of cells): not specified
SELECTION AGENT (mutation assays): 6-thioguanine
STAIN (for cytogenetic assays): 10% methylene blue in 0.01% KOH solution
NUMBER OF REPLICATIONS: duplicate cultures in 2 independent experiments
NUMBER OF CELLS EVALUATED: 5x10E2 (cytotoxicity, cloning efficiency I) - 1.5x10E6 (mutant frequency)
DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency I (cytotoxicity); cloning efficiency II (cell viability)
OTHER EXAMINATIONS:
- Mutant colonies per 10E6 cells = mean number of mutant colonies per flask found after plating in 6-thioguanine containing medium x 10E6 divided by the number of cells survived
- Induction factor = mutant colonies per 10E6 cells / mutant colonies per 10E6 cells of the corresponding solvent control - Evaluation criteria:
- The assay is considered acceptable if:
- the numbers of mutant colonies per 10E6 cells in negative and/or solvent controls fall with the test facility historical data range
- the positive control substances produce a significant increase in mutant colony frequencies
- the cloning efficiency II value of the negative and/or solvent controls exceed 0.5
A test substance is considered as positive if it induces either a concentration-related increase in the mutation frequency or a reproducible and positive response at one of the test points (at least three times above the spontaneous mutation frequency). - Statistics:
- Since the distribution of mutant cells does not follow known statistical models, no adequate statistical method was available
- Species / strain:
- Chinese hamster lung fibroblasts (V79)
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity, but tested up to precipitating concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
Precipitation was observed by the naked eye in all parts of pre-experiment at 112.5 µg/mL and above. In the first main experiment (4-hour exposure), precipitation was seen at 225 µg/mL and above with or without S9. In the second main experiment, precipitation was observed at 225 µg/mL and above in the absence of S9 (24-hour exposure) and at 112.5 µg/mL and above in the presence of S9 (4-hour exposure).
RANGE-FINDING/SCREENING STUDIES:
A pre-test was performed in order to determine the concentration range for the mutagenicity experiments, using same experimental conditions as described for the main experiments. In the pre-test, the colony forming ability of approximately 500 single cells after exposure to the test substance was observed and compared to the controls.
The highest concentration used in the pre-test was chosen with regard to the purity (99.8 %) and the molecular weight of the test item (172.12 g/mol). Test item concentrations between 14.1 and 1800 µg/mL (approximately 10 mM) were used to evaluate toxicity in the presence (4 h treatment) and absence (4 h and 24 h treatment) of metabolic activation. No relevant toxic effect (relative cloning efficiency at or below 50 %) occurred up to the high-est concentration of both treatment periods with and without metabolic activation. Precipitation was noted at 112.5 µg/mL and above in the absence and presence of metabolic activation at both treatment intervals (4 and 24 hours). There was no relevant shift of osmolarity and pH values of the medium even at the maximum concentration of the test item.
COMPARISON WITH HISTORICAL CONTROL DATA:
In both experiments (with or without S9), the range of the negative and solvent controls was from 2.1 to 12.3 mutants per 10E6 cells. The range of the cells exposed to the test substance was from 0.9 to 26.3 mutants per 10E6 cells. However all experimental points remained within the historical control data range. - Conclusions:
- No evidence of gene mutations at the HPRT locus in V79 cells up to the concentration of 1800 µg/mL with or without S9
- Executive summary:
The potential of Cerium Oxide to induce gene mutations at the HPRT locus in Chinese Hamster V79 cells was tested. The assay was performed in two independent experiments, each using duplicate cultures. In the first experiment, the cells were exposed to the test substance suspended in deionized water at concentrations ranging from 28.1 to 1800 µg/mL (equivalent to10 mM) for 4 hours with or without metabolic activation (S9). In the second experiment, cell exposure was 24 hours at concentrations ranging from 28.1 to 1800 µg/mL in the absence of S9 and 4 hours at concentrations ranging from 14.1 to 1800 µg/mL in the presence of S9. The cells were evaluated for mutant frequency at selected test concentrations. Positive controls consisted of 1.2 or 2.4 mM ethylmethane sulfonate and 7.7 µM 7,12-dimethylbenz(a)anthracene without and with S9, respectively.
Precipitation was observed from 225 µg/mL up to the maximum concentration with and without S9 (4 h treatment) in the first main experiment and without S9 mix in the second experiment (24 h treatment). It was also observed from 112.5 µg/mL up to the maximum concentration in the second experiment with S9 (4 h treatment). No relevant cytotoxic effects indicated by relative cloning efficiency lower than 50% were observed up to 1800 µg/mL in both main assays with or without S9. No significant and reproducible dose-dependent increases in the mutation frequency were observed in both main experiments.
Appropriate reference mutagens used as positive controls showed a distinct increase in induced mutant colonies, indicating that the tests were sensitive and valid.
Therefore, Cerium Oxide did not induce gene mutations at the HPRT locus in V79 cells up to the concentration of 1800 µg/mL with or without S9.
This study is classified as acceptable. It satisfies the OECD 476 guideline requirements on In vitro Mammalian Cell Gene Mutation Test.
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- experimental study
- Adequacy of study:
- supporting study
- Study period:
- 2008-03-04 to 2008-04-02
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Remarks:
- Certificate provided by Rheinlandpfalz
- Type of assay:
- bacterial reverse mutation assay
- Species / strain / cell type:
- S. typhimurium, other: TA97a, TA98, TA100, TA102, TA1535
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9
- Test concentrations with justification for top dose:
- 4998, 1499, 500, and 50 µg/plate - Experiment one
4998, 2499, and 1250 µg/plate - Experiment two
As the test item was not soluble in any suitable solvent, a stock suspension containing 50 g/L was prepared and diluted as necessary. - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: DMSO; water
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: 4-Nitro-1,2-phenylene diamine in DMSO (without at 80 µg for strains TA 97a, TA98 and TA102); Sodium azide in deionised water (without at 6 µg for strains TA100 and TA1535)
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: Benzo-a-pyrene; 2-Amino-anthracene in DMSO (with at 40 µg for stain TA98); 2-Aminoanthracene in DMSO (with at 3 µg for strains TA97a, TA100, TA102 and TA1535)
- Details on test system and experimental conditions:
- METHOD OF APPLICATION:
- In agar (plate incorporation) - Experiment one
Per strain and dose, four plates with and four plates without S9 mix were used. 10 mL of the test solution of the appropriate concentration were membrane filtrated (size of pores was 0.2 µm) into sterile vessels. Top agar basis was melted in a microwave oven, after melting, 10 mL of histidine-biotin-solution 0.5 mmol per 100 mL basis was added and the bottle was placed in the water bath at 45 degrees C.
0.1 mL of the appropriate solution of the test item was given into a sterile tube. After mixing with 0.1 mL overnight culture of the respective strain and 0.5 mL phosphate buffer (only for treatments without S9) or 0.5 mL S9 mix, 2 mL Top-Agar were added. The mixture was gently vortexed, then poured on a minimal glucose plate and distributed evenly, using a Drigalski spatula. The plates were closed, covered with brown paper and left to harden for a few minutes, then inverted and placed in the dark incubator at 37 degrees C.
- Pre-incubation - Experiment two
Per strain and dose, four plates with and four plates without S9 mix were used. 10 mL of the test solution of the appropriate concentration were membrane filtrated into sterile vessels. Top agar basis was melted in a microwave oven, after melting, 10 mL of histidine-biotin-solution 0.5 mmol per 100 mL basis was added and the bottle was placed in the water bath at 45 degrees C.
0.1 mL of the appropriate solution of the test item was given into a sterile tube. After mixing with 0.1 mL overnight culture of the respective strain, 0.5 mL phosphate buffer (only for treatments without S9) or 0.5 mL S9 mix were added. The mixture was incubated in an incubation chamber at 37 degrees C for 20 minutes. During this time the vessels were aerated through careful shaking. Then 2 mL top agar was added. The mixture was vortexed gently, then poured on a minimal glucose plate and distributed evenly, using a Drigalski spatula. The plates were closed, covered with brown paper and left to harden for a few minutes, then inverted and placed in the dark incubator at 37 degrees C.
DURATION
- Pre-incubation period: 20 minutes at 37 degrees C
- Exposure duration: 48 hours at 37 degrees C - Both experiments
NUMBER OF REPLICATIONS: 4
NUMBER OF CELLS EVALUATED: at least 10^9 cells/mL correlating to 100 colonies / plate - Evaluation criteria:
- A test substance is considered to have mutagenic potential, if a significant, reproducible increase of revertant colonies per plate (increase factor >/= 2) in at least one strain can be observed. A concentration-related increase over the range tested can also be taken as a sign of mutagenic activity.
- Statistics:
- The colonies were counted visually, the numbers were recorded. A spreadsheet software (Microsoft Excel) was used to calculate mean values and standard deviations as well as the increase factor of revertant induction.
- Species / strain:
- S. typhimurium, other: TA97a, TA98, TA100, TA102, TA1535
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Additional information on results:
- - The test item did not show mutagenic effects in both experiments. The number of revertant colonies was not increased in comparison with the spontaneous revertants (solvent only).
- Cytotoxicity of the test item was not detected. The background lawn was visible and the number of revertants was not significantly decreased.
- No toxicity was observed - Remarks on result:
- other: all strains/cell types tested
- Conclusions:
- Interpretation of results: negative
CC10 zirconium oxide is considered as "not mutagenic under the conditions of the test." - Endpoint:
- in vitro cytogenicity / chromosome aberration study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- From 2010-04-19 to 2010-05-18
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
- Deviations:
- yes
- Remarks:
- In the dose range finding study/first cytogenetic assay during incubation period, temperature was outside the range of 37.0±1.0°C as specified in the protocol with a minimum of 31.3°C for approx 1.5 hour. This deviation had no effects on the results
- GLP compliance:
- yes
- Type of assay:
- in vitro mammalian chromosome aberration test
- Target gene:
- Not applicable
- Species / strain / cell type:
- lymphocytes: cultured peripheral human lymphocytes
- Details on mammalian cell type (if applicable):
- See section 'Any other information on materials and methods incl. tables'
- Additional strain / cell type characteristics:
- not specified
- Metabolic activation:
- with and without
- Metabolic activation system:
- Rat liver microsomal enzymes were routinely prepared from adult male Wistar rats (6), which were obtained from Charles River (Sulzfeld, Germany) (S9 fraction)
- Test concentrations with justification for top dose:
- Dose range finding test/first cytogenetic assay: at 3 h exposure time: 10, 33 and 100 µg zirconium dioxide/mL culture medium with and without S9-mix; at 24 and 48 h continuous exposure time blood cultures were treated with 1, 3, 10, 33, 100, 333 and 1000 µg zirconium dioxide/mL culture medium without S9-mix
Second cytogenicity test: without S9-mix: 10, 33 and 100 µg/mL culture medium (24 and 48 h exposure time, 24 h and 48 h fixation time); with S9-mix: 10, 33 and 100 µg/mL culture medium (3 h exposure time, 48 h fixation time) - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: no data - Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- mitomycin C
- Remarks:
- Without metabolic activation (-S9-mix); solvent for positive controls: Hanks' Balanced Salt Solution (HBSS) without calcium and magnesium
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- Remarks:
- With metabolic activation (+S9-mix); solvent for positive controls: Hanks' Balanced Salt Solution (HBSS) without calcium and magnesium
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium
DURATION
- Preincubation period: not applicable
- Exposure duration: 24 and 48 h in the absence of S9-mix or for 3 h in the presence of S9 mix (second cytogenetic assay)
- Expression time (cells in growth medium): after 3 h exposure, the cells exposed to zirconium dioxide in the presence of S9-mix were separated from the exposure medium by centrifugation (5 min, 365 g). The supernatant was removed and the cells were rinsed once with 5 mL of HBSS and incubated in 5 mL culture medium for another 44-46 h; the cells that were treated for 24 h and 48 h in the absence of S9-mix were not rinsed after exposure but were fixed immediately after 24 h and 48 h (24 h and 48 h fixation time)
- Selection time (if incubation with a selection agent): not applicable
- Fixation time (start of exposure up to fixation or harvest of cells): see above
SELECTION AGENT (mutation assays): not applicable
SPINDLE INHIBITOR (cytogenetic assays): colchicine (0.5 µg/mL medium) (Acros Organics, Belgium) - during the last 2.5-3 h of the culture period
STAIN (for cytogenetic assays): Cell cultures were centrifuged for 5 min at 1300 rpm (365 g) and the supernatant was removed. Cells in the remaining cell pellet were swollen by a 5 min treatment with hypotonic 0.56% (w/v) potassium chloride (Merck) solution at 37°C. After hypotonic treatment, cells were fixed with 3 changes of methanol (Merck): acetic acid (Merck) fixative (3:1 v/v). Fixed cells were dropped onto cleaned slides, which were immersed in a 1:1 mixture of 96% (v/v) ethanol (Merck)/ether (Merck) and cleaned with a tissue. The slides were marked with the NOTOX study identification number and group number. At least two slides were prepared per culture. Slides were allowed to dry and thereafter stained for 10-30 min with 5% (v/v) Giemsa (Merck) solution in tap water. Thereafter slides were rinsed in tap-water and allowed to dry. The dry slides were cleared by dipping them in xylene (Klinipath, Duiven, The Netherlands) before they were embedded in Pertex (Klinipath) and mounted with a coverslip.
NUMBER OF REPLICATIONS: duplicate cultures
NUMBER OF CELLS EVALUATED: To prevent bias, all slides were randomly coded before examination of chromosome aberrations and scored. An adhesive label with NOTOX study identification number and code was placed over the marked slide. One hundred metaphase chromosome spreads per culture were examined by light microscopy for chromosome aberrations. in case the number of aberrant cells, gaps excluded, was > or = 25 in 50 metaphases, no more metaphases were examined. Only metaphases containing 46 ± 2 centromeres (chromosomes) were analysed. The number of cells with aberrations and the number of aberrations were calculated.
DETERMINATION OF CYTOTOXICITY
- Method: mitotic index: The mitotic index of each culture was determined by counting the number of metaphases per 1000 cells. At least three analysable concentrations were used for scoring of the cytogenetic assay. The highest concentration analysed was based on the solubility of zirconium dioxide in the culture medium. However, the extent of precipitation may not interfere with the scoring of chromosome aberrations.
OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: yes
- Other: no
OTHER: Test substance preparation: Zirconium dioxide was suspended in dimethyl sulfoxide of spectroscopic quality (SeccoSolv, Merck, Darmstadt, Germany) at concentrations of 0.3 mg/mL and above. the stock solution was treated with ultrasonic waves to obtain a homogeneous suspension. Zirconium dioxide was dissolved in dimethyl sulfoxide at concentrations of 0.1 mg/mL and below. Zirconium dioxide concentrations were used within 2.5 hours after preparation. The final concentration of the solvent in the culture medium was 1.0% (v/v) - Evaluation criteria:
- A test substance was considered positive (clastogenic) in the chromosome aberration test if:
a) It induced a dose-related statistically significant (Chi-square test, one-side, p < 0.05) increase in the number of cells with chromosome aberrations.
b) A statistically significant and biologically relevant increase in the frequencies of the number of cells with chromosome aberrations was observed in the absence of a clear dose-response relationship.
A test substance was considered negative (not clastogenic) in the chromosome aberration test if none of the tested concentrations induced a statistically significant (Chi-square test, one-sided, p < 0.05) increase in the number of cells with chromosome aberrations. The preceding criteria are not absolute and other modifying factors might enter into the final evaluation decision. - Statistics:
- The incidence of aberrant cells (cells with one or more chromosome aberrations, gaps included or excluded) for each exposure group outside the laboratory historical control data range was compared to that of the solvent control using Chi-square statistics:
X²=[(N-1) (ad-bc)²]/[(a+b) (c+d) (a+c) (b+d)]
where b = the total number of aberrant cells in the control cultures, d = the total number of non aberrant cells in the control cultures, n0 = the total number of cells scored in the control cultures, a = the total number of aberrant cells in treated cultures to be compared with the control, c = the total number of non aberrant cells in treated cultures to be compared with the control, n1 = the total number of cells scored in the treated cultures, N = sum of n0 and n1
If P [X² > [(N-1) (ad-bc)²]/[(a+b) (c+d) (a+c) (b+d)]] (one-tailed) is small (p< 0.05) the hypothesis that the incidence of cells with chromosome aberrations is the same for both the treated and the solvent control group is rejected and the number of aberrant cells in the test group is considered to be significantly different from the control group at the 95% confidence interval. - Key result
- Species / strain:
- lymphocytes: cultured peripheral human lymphocytes
- Remarks:
- all strains/cell types tested
- Metabolic activation:
- without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Remarks:
- The mitotic index of the test substance didn't reach 50% of the control value for all tested concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Key result
- Species / strain:
- lymphocytes: cultured peripheral human lymphocytes
- Remarks:
- all strains/cell types tested
- Metabolic activation:
- with
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Remarks:
- The mitotic index of the test substance didn't reach 50% of the control value for all tested concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no data
- Effects of osmolality: no data
- Evaporation from medium: no data
- Water solubility: no data
- Precipitation: yes
RANGE-FINDING/SCREENING STUDIES: In order to select the appropriate dose levels for the chromosome aberration test cytotoxicity data were obtained in a dose range finding test. Zirconium dioxide was tested in the absence and presence of 1.8% (v/v) S9-fraction. Lymphocytes (0.4 mL blood of a healthy male donor + 5 mL or 4.8 mL culture medium + (+ or - S9) + 0.1 mL (9 mg/mL) Phytohaemagglutinin) were cultured for 48 h and thereafter exposed to selected doses of zirconium dioxide for 3h, 24h, and 48h in the absence of S9-mix or for 3 h in the presence of S9-mix. The highest tested concentration was determined by the solubility of zirconium dioxide in the culture medium at the 3h exposure time. At a concentration of 100 µg/mL zirconium dioxide precipitated in the culture medium. The lymphocytes were cultured in duplicate at the 3 h exposure time and appropriate vehicle and positive controls were included. At the 24h and 48h exposure time, zirconium dioxide was tested beyond the limit of solubility to obtain adequate toxicity data. After 3 h exposure to zirconium dioxide in the absence or presence fo S9-mix, the cells were separated from the exposure medium by centrifugation (5 min, 365 g). The supernatant was removed and cells were rinsed with 5 mL HBSS. After a second centrifugation step, HBSS was removed and cells were resuspended in 5 mL culture medium and incubated for another 20 - 22 h (24 h fixation time). The cells that were exposed for 24 h and 48 h in the absence of S9-mix were not rinsed after exposure but were fixed immediately (24 and 48h fixation time). Cytotoxicity of zirconium dioxide in the lymphocyte cultures cultures was determined using the mitotic index. No cytotoxicity was observed in the duplicate cultures of the 3 h exposure time and the slides were scored for chromosome aberrations. The first cytogenetic assay was ommited. Based on the results of the dose range finding test an appropriate range of dose levels was chosen for the second cytogenetic assay considering the highest dose level was determined by the solubility.
COMPARISON WITH HISTORICAL CONTROL DATA: The number of cells with chromosome aberrations found in the solvent control cultures was within the laboratory historical control data range. The number of polyploid cells and cells with endoreduplicated chromosomes in the solvent control cultures was within the laboratory historical control data range. The positive control chemicals (MMC-C and CP) both produced statistically significant increases in the mutation frequency of aberrant cells. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly. - Conclusions:
- Interpretation of results: negative with and without metabolic activation
Finally, it is concluded that this test is valid and that zirconium dioxide is not clastogenic in human lymphocytes under the experimental conditions of this test. - Endpoint:
- in vitro gene mutation study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
- Deviations:
- yes
- Remarks:
- Deviations of temperature and humidity caused by adjustment after opening of the incubator door. However the study integrity was not adversely affected by the deviations.
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
- Deviations:
- yes
- Remarks:
- Deviations of temperature and humidity caused by adjustment after opening of the incubator door. However the study integrity was not adversely affected by the deviations.
- GLP compliance:
- yes (incl. QA statement)
- Remarks:
- Food and Consumer Product Safety Authority (VWA), Prinses Beatrixlaan 2, 2595 AL Den Haag, Postbus 19508, 2500,CM Den Haag, The Netherlands
- Type of assay:
- in vitro mammalian cell gene mutation tests using the thymidine kinase gene
- Target gene:
- thymidine-kinase (TK) locus L5178Y
- Species / strain / cell type:
- mouse lymphoma L5178Y cells
- Details on mammalian cell type (if applicable):
- - Properly maintained: yes
- Periodically checked for Mycoplasma contamination: no data
- Periodically checked for karyotype stability: no data
- Periodically "cleansed" against high spontaneous background: no data - Additional strain / cell type characteristics:
- not specified
- Metabolic activation:
- with and without
- Metabolic activation system:
- rat liver S9-mix induced by a combination of phenobarbital and beta-naphtoflavone
- Test concentrations with justification for top dose:
- 0.03, 0.1, 1, 3, 10, 33 and 100 µg/mL
- Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: no data - Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- methylmethanesulfonate
- Remarks:
- Without metabolic activation; MMS was dissolved in dimethyl sulfoxide. The stock solutions of MMS were prepared immediately before use.
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- Remarks:
- With metabolic activation; CP was dissolved in Hanks' balanced salt solution (HBSS) without calcium and magnesium. The stock solutions of CP were stored in aliquots at < or = -15°C in the dark and one sample was thawed immediately before use.
- Details on test system and experimental conditions:
- In a first experiment, cell cultures were exposed for 3 hours to zirconium dioxide in exposure medium in the absence and presence of S9-mix. In a second experiment, cell cultures were exposed to zirconium dioxide in exposure medium for 24 hours in the absence of S9-mix and for 3 hours in the presence of S9-mix.
METHOD OF APPLICATION: in medium
DURATION
- Preincubation period: not applicable
- Exposure duration: 3 hours or 24 hours
- Expression time (cells in growth medium): 48 hours
- Selection time (if incubation with a selection agent): 11 or 12 days (TFT selection)
- Fixation time (start of exposure up to fixation or harvest of cells): 2 hours (MTT staining)
SELECTION AGENT (mutation assays): TFT
SPINDLE INHIBITOR (cytogenetic assays): not applicable
STAIN (for cytogenetic assays): not applicable
NUMBER OF REPLICATIONS: 2 independent experiments
NUMBER OF CELLS EVALUATED: for the determination of mutation frequency a total number of 9.6 x 1E05 cells/concentration were plated in five 96-well microtiter plates, each well containing 2000 cells in selective medium, with the exception of the positive control groups (MMS and CP) where a total number of 9.6 x 1E05 cells/concentration were plated in ten 96-well microtiter plates, each well containing 1000 cells in selective medium (trifluorothymidine-selection).
DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency
OTHER:
- Determination of polyploidy: not applicable
- Determination of endoreplication: not applicable
- Type and identity of media: horse serum was inactivated by incubiation at 56°C for at least 30 minutes. Basic medium: RPMI 1640 Hepes buffered medium (Dutch modificiation) containing penicillin/streptomycin (50 U/mL and 50 µg/mL, respectively), 1 mM sodium pyruvate and 2 mM L-glutamin. Growth medium: basic medium, supplemented with 10% (v/v) heat-inactivated horse serum (=R10 medium). Exposure medium: for 3 hour exposure: cells were exposed to the test substance in basic medium supplemented with 5% (v/v) heat-inactivated horse serum (R5-medium). For 24 hour exposure: cells were exposed to the test substance in basic medium supplemented with 10% (v/v) heat-inactivated horse serum (R10-medium). Selective medium consisted of basic medium supplemented with 20% (v/v) heat-inactivated horse serum (total amount of serum = 20%, R20) and 5 µg/mL trifluorothymidine (TFT) (Sigma). Non-selective medium consisted of basic medium supplemented with 20% (v/v) heat-inactivated horse serum (total amount of serum = 20%, R20).
- State of the suspension/solution according to the concentration: at a concentration of 0.12 mg/mL and higher zirconium dioxide was suspended in dimethyl sulfoxide (DMSO, SeccoSolv, Merck Darmdstadt, Germany). At a concentration of 0.04 mg/mL and lower the test substance was dissolved in dimethyl sulfoxide. The stock solution was treated with ultrasonic waves to obtain a homogeneous suspension. Zirconium dixoide concentrations were used within 1 hour after preparation. The final concentration of the solvent in the exposure medium was 0.8% (v/v). - Evaluation criteria:
- The global evaluation factor (GEF) has been defined as the mean of the negative/solvent mutation frequency distribution plus one standard deviation. For the micro well version of the assay the GEF is 126. A test substance is considered positive (mutagenic) in the mutation assay if it induces a mutation frequency of more then mutation frequency (controls) + 126 in a dose-dependent manner. An observed increase should be biologically relevant and will be compared with the historical control data range. A test substance is considered equivocal (questionable) in the mutation assay if no clear conclusion for positive or negative result can be made after an additional confirmation study. A test substance is considered negative (not mutagenic) in the mutation assay if: a) non of the tested concentrations reaches a mutation frequency of mutation frequency (controls) + 126; b) the results are confirmed in an independent repeated test.
- Species / strain:
- mouse lymphoma L5178Y cells
- Remarks:
- all strains/cell types tested
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Remarks:
- first and second experiment
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH:no data
- Effects of osmolality: no data
- Evaporation from medium: no data
- Water solubility: no data
- Precipitation: Zirconium dioxide precipitated in the exposure medium at concentration of 100 µg/mL and above. Zirconium dioxide was tested beyond the limit of solubility to obtain adequate cytotoxicity data, the concentration used as the highest test substance concentration for the dose range finding test was 333 µg/mL
- Other confounding effects: no data
RANGE-FINDING/SCREENING STUDIES: L5178Y mouse lymphoma cells were treated with a test substance concentration range of 3 to 333 µg/mL in the absence of S9-mix with a 3 and 24 hour treatment period and in the presence of S9-mix with a 3 hour treatment period. After 3 hours of treatment: both in the absence and presence of S9-mix, no toxicity in the relative suspension growth was observed up to and including the highest test substance concentration of 333 µg/mL compared to the suspension growth of the solvent control. After 24 hours of treatment with various concentrations of Zirconium dioxide, no toxicity in the relative suspension growth was observed up to and including the highest test substance concentration of 333 µg/mL compared to the suspension growth of the solvent control.
COMPARISON WITH HISTORICAL CONTROL DATA: The spontaneous mutation frequencies in the solvent-treated control cultures were between the minimum and maximum value of the historical control data range.
ADDITIONAL INFORMATION ON CYTOTOXICITY: No toxicity was observed and all dose levels were evaluated in the absence and presence of S9-mix. - Conclusions:
- Interpretation of results: negative with and without metabolic activation
In conclusion, zirconium dioxide is not mutagenic in the TK mutation test system under the specified experimental conditions. - Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- experimental study
- Adequacy of study:
- supporting study
- Study period:
- Not specified
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- study well documented, meets generally accepted scientific principles, acceptable for assessment
- Reason / purpose for cross-reference:
- reference to same study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Deviations:
- not specified
- GLP compliance:
- not specified
- Type of assay:
- bacterial reverse mutation assay
- Target gene:
- No data
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Species / strain / cell type:
- S. typhimurium TA 102
- Test concentrations with justification for top dose:
- 50 - 5000 µg/plate (in triplicate)
- Positive controls:
- yes
- Positive control substance:
- 4-nitroquinoline-N-oxide
- 9-aminoacridine
- N-ethyl-N-nitro-N-nitrosoguanidine
- mitomycin C
- Positive controls:
- yes
- Positive control substance:
- benzo(a)pyrene
- other: 2-aminoanthracene (2AA) at 1 µg/plate for TA100, 2 µg/plate for TA1535 and 1537 with S9; 1,8-Dihydroxyanthraquinone (DAN) at 10 µg/plate for TA102 with S9
- 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:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Species / strain:
- S. typhimurium TA 102
- 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:
- - Non-nano Cerium oxide precipitation was not observed on the plates at any dose-level tested in either the presence or absence of metabolic activation.
- No toxicity of the cerium oxide as no visible reduction in the growth of the bacteria background lawn at any dose level was observed both with and without metabolic activation.
- All of the positive control chemicals used in the study induced marked increase in the frequency of revertant colonies thus confirming the activity of the S9-mix and the sensitivity of the bacterial strains. - Conclusions:
- Cerium oxide (non-nanoparticular) was non-mutagenic in this Ames test at concentrations between 50 and 5000 µg/plate
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- weight of evidence
- Justification for type of information:
- Read across based on the results of two in vitro gene mutation studies in mammalian cells performed with zirconium dioxide and cerium dioxide. The read across justification document is attached to IUCLID Section 13.
- Reason / purpose for cross-reference:
- read-across source
- Reason / purpose for cross-reference:
- read-across source
- Key result
- Species / strain:
- other: read across conclusion
- Remarks:
- The in vitro gene mutation studies in mammalian cells performed with zirconium dioxide and cerium dioxide were used in a weight-of-evidence approach to conclude on the reaction mass. The reaction mass was concluded not to be mutagenic.
- Remarks on result:
- other: The in vitro gene mutation studies in mammalian cells performed with zirconium dioxide and cerium dioxide were used in a weight-of-evidence approach to conclude on the reaction mass. The reaction mass was concluded not to be mutagenic.
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- experimental study
- Adequacy of study:
- supporting study
- Study period:
- Not specified
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- study well documented, meets generally accepted scientific principles, acceptable for assessment
- Reason / purpose for cross-reference:
- reference to same study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Deviations:
- not specified
- GLP compliance:
- not specified
- Type of assay:
- bacterial reverse mutation assay
- Target gene:
- No data
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Species / strain / cell type:
- S. typhimurium TA 102
- Test concentrations with justification for top dose:
- 50 - 5000 µg/plate (in triplicate)
- Positive controls:
- yes
- Positive control substance:
- 4-nitroquinoline-N-oxide
- 9-aminoacridine
- N-ethyl-N-nitro-N-nitrosoguanidine
- mitomycin C
- Positive controls:
- yes
- Positive control substance:
- benzo(a)pyrene
- other: 2-aminoanthracene: at 1 µg/plate for TA100, 2 µg/plate for TA1535 and TA1537 (with S9) / 1,8-dihydroxyanthraquinone: at 10 µg/plate for TA102 (with S9)
- 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:
- no cytotoxicity, but tested up to precipitating concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Species / strain:
- S. typhimurium TA 102
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity, but tested up to precipitating concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- - A cream-coloured film was observed at 1500 μg/plate and above with an associated precipitate at 5000 μg/plate. This observation did not, however, prevent the scoring of revertant colonies and confirmed that the samples were tested up to maximal dose level.
- No toxicity of the cerium oxide as no visible reduction in the growth of the bacteria background lawn at any dose level was observed both with and without metabolic activation.
- All of the positive control chemicals used in the study induced marked increase in the frequency of revertant colonies thus confirming the activity of the S9-mix and the sensitivity of the bacterial strains. - Conclusions:
- Cerium oxide (nanoparticular) was non-mutagenic in this Ames test at concentrations between 50 and 5000 µg/plate
- 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:
- key study
- Justification for type of information:
- Read across based on a study performed with (bulk) zirconium dioxide. The read across justification document is attached to IUCLID Section 13.
- Reason / purpose for cross-reference:
- read-across source
- Key result
- Species / strain:
- other: read across conclusion
- Remarks:
- The reaction mass was concluded not to be clastogenic, based on an in vitro chromosome aberration study in mammalian cells performed with zirconium dioxide, in combination with an in vivo mouse micronucleus test performed with cerium dioxide.
- Remarks on result:
- other: The reaction mass was concluded not to be clastogenic, based on an in vitro chromosome aberration study in mammalian cells performed with zirconium dioxide, in combination with an in vivo mouse micronucleus test performed with cerium dioxide.
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- experimental study
- Adequacy of study:
- supporting study
- Study period:
- 1997-10-20 to 1997-12-19
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- 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
- Target gene:
- histidine locus (Salmonella typhimurium)
tryptophan locus (Escherichia coli) - 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 induced rat liver
- Test concentrations with justification for top dose:
- Dose-setting test: 5, 10, 50, 500, 1000, and 5000 µg/plate
Final test: 0.156, 0.313, 0.625, 1.25, 2.5 and 5 µg/plate - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: distilled water
- Justification for choice of solvent/vehicle: no data - Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- distilled water
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: 2-(2-furyl)-3-(5-nitro-2-furyl) acrylamide (AF-2)
- Remarks:
- For strains: TA 98, TA 100 and WP2 uvrA without S9 mix
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- distilled water
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- sodium azide
- Remarks:
- For strains: TA 1535 without S9 mix
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- distilled water
- True negative controls:
- not specified
- Positive controls:
- yes
- Positive control substance:
- other: 2-methoxy-6-Chloro-9-[3-(2-chloroethyl)-aminopropylamino] acridine ¿ 2HCl(ICR-191)
- Remarks:
- For strains: TA 1537 without S9 mix
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- distilled water
- True negative controls:
- not specified
- Positive controls:
- yes
- Positive control substance:
- other: 2-aminoanthracene (2AA)
- Remarks:
- For strains: TA 98, TA 100, TA 1535, TA 1537 and WP2 uvrA with S9 mix
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: pre-incubation
DURATION
- Pre-incubation period: no data
- Exposure duration: at least 48 hours
- Expression time (cells in growth medium): not applicable
- Selection time (if incubation with a selection agent): at least 48 hours
- Fixation time (start of exposure up to fixation or harvest of cells): not applicable
SELECTION AGENT (mutation assays): histidine and tryptophan
SPINDLE INHIBITOR (cytogenetic assays): not applicable
STAIN (for cytogenetic assays): not applicable
NUMBER OF REPLICATIONS: 3
NUMBER OF CELLS EVALUATED: no data
DETERMINATION OF CYTOTOXICITY
- Method: reduction of bacterial background lawn
OTHER EXAMINATIONS:
- Determination of polyploidy: not applicable
- Determination of endoreplication: not applicable
- Other: not applicable
OTHER: NaN3 was dissolved in distilled water; AF-2 ICR-191 and 2AA were dissolved in DMSO. - Evaluation criteria:
- It is determined positive if the number of revertant colonies of the test agent treatment group depends on the dosage and increased twice or more as the negative control and reproducibility is acknowledged. Other cases are determined negative.
Unpredictable situations that may affect the credibility of the test, and not following the test plan.
For not following the test plan, the minimum glucose agar plating medium Lot No. AN550JM (manufactured on Oct. 3, 1997) was used in addition, while only AN510IM was to be used according to the plan. It was determined, however, that it would have no bad effect to the test.
There was no other unpredictable situation that may affect the credibility of the test.
No other situation that may adversely affect the credibility of the test, or no other incident of not following the test plan was acknowledged. - Statistics:
- Statistical analyses were not done.
- 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:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- 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:
- not examined
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no data
- Effects of osmolality: no data
- Evaporation from medium: no data
- Water solubility: insoluble in water or oil
- Precipitation: Precipitation thought to be the test agent of this test was seen in 5 µg/plate regardless of presence/absence of S9 mix
- Other confounding effects: no data
RANGE-FINDING/SCREENING STUDIES:
5000 µg/plate as the maximum, the seven doses; 1000, 500, 100, 50, 10 and 5 µg/plate, were set. As a result, depositions which seem to be the test agent were seen in 5 µg/plate, regardless of the presence/absence of S9 mix.
COMPARISON WITH HISTORICAL CONTROL DATA:
It was confirmed that the positive control agent induces mutation in the test strains, and that the numbers of revertant colonies as well as the negative control value were within the range of the historical data of this institute, thus it was confirmed that the test was conducted properly.
ADDITIONAL INFORMATION ON CYTOTOXICITY: no data - Remarks on result:
- other: all strains/cell types tested
- Conclusions:
- It is determined that yttrium zirconium oxide does not have reverse mutation inducing capacity under the conditions of this test.
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- supporting study
- Justification for type of information:
- Supporting information on in vitro mutagenicity in bacteria (Ames studies) for bulk and nano cerium dioxide, bulk zirconium dioxide, and zirconium dioxide with a small w/w % of yttrium in it. Read across justification document is attached to IUCLID Section 13.
- Reason / purpose for cross-reference:
- read-across source
- Reason / purpose for cross-reference:
- read-across source
- Reason / purpose for cross-reference:
- read-across source
- Reason / purpose for cross-reference:
- read-across source
- Reason / purpose for cross-reference:
- read-across source
- Reason / purpose for cross-reference:
- read-across source
- Species / strain:
- other: read across conclusion
- Remarks:
- Studies performed with bulk and nano cerium dioxide, bulk zirconium dioxide, and yttrium zirconium oxide, support the findings in the key study performed with a representative nanoform of the reaction mass of cerium dioxide and zirconium dioxide.
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
Referenceopen allclose all
Table 1: First experiment (direct plate incorporation) - Mean revertant colony counts
|
TA 1535 |
TA 1537 |
TA 98 |
||||||
Conc. |
- MA |
+ MA |
Cytotoxic |
- MA |
+ MA |
Cytotoxic |
- MA |
+ MA |
Cytotoxic |
0* |
30 |
21 |
No |
9 |
13 |
No |
26 |
23 |
No |
312.5 |
27 |
28 |
No (Mp) |
5 |
11 |
No (Mp) |
36 |
22 |
No (Mp) |
625 |
32 |
20 |
No (Mp) |
9 |
11 |
No (Mp) |
38 |
39 |
No (Mp) |
1250 |
32 |
26 |
No (Mp) |
9 |
7 |
No (Mp) |
31 |
27 |
No (Mp) |
2500 |
29 |
19 |
No (Sp) |
10 |
8 |
No (Sp) |
49 |
27 |
No (Sp) |
5000 |
21 |
23 |
No (Sp) |
8 |
6 |
No (Sp) |
50 |
52 |
No (Sp) |
Positive control |
541 |
195 |
No |
386 |
103 |
No |
183 |
1641 |
No |
|
TA 100 |
TA 102 |
||||
Conc. |
- MA |
+ MA |
Cytotoxic |
- MA |
+ MA |
Cytotoxic |
0* |
143 |
157 |
No |
415 |
440 |
No |
312.5 |
133 |
127 |
No (Mp) |
368 |
619 |
No (Mp) |
625 |
153 |
111 |
No (Mp) |
569 |
710 |
No (Mp) |
1250 |
195 |
133 |
No (Mp) |
485 |
586 |
No (Mp) |
2500 |
138 |
90 |
No (Sp) |
513 |
563 |
No (Sp) |
5000 |
141 |
129 |
No (Sp) |
570 |
587 |
No (Sp) |
Positive control |
515 |
364 |
No |
2249 |
2795 |
No |
*solvent control with DMSO
Mp : Moderate precipitate
Sp : Strong precipitate
MA : Metabolic activation
Table 2: Second experiment (direct plate incorporation without S9 mix and preincubation with S9 mix) - Mean revertant colony count
|
TA 1535 |
TA 1537 |
TA 98 |
||||||
Conc. |
- MA |
+ MA |
Cytotoxic |
- MA |
+ MA |
Cytotoxic |
- MA |
+ MA |
Cytotoxic |
0* |
18 |
15 |
No |
8 |
6 |
No |
28 |
34 |
No |
312.5 |
20 |
15 |
No (Mp) |
5 |
9 |
No (Mp) |
53 |
43 |
No (Mp) |
625 |
17 |
11 |
No (Mp) |
5 |
6 |
No (Mp) |
32 |
36 |
No (Mp) |
1250 |
18 |
14 |
No (Mp) |
6 |
10 |
No (Mp) |
49 |
44 |
No (Mp) |
2500 |
19 |
14 |
No (Sp) |
4 |
5 |
No (Sp) |
26 |
25 |
No (Sp) |
5000 |
9 |
10 |
No (Sp) |
6 |
8 |
No (Sp) |
37 |
42 |
No (Sp) |
Positive control |
551 |
154 |
No |
847 |
131 |
No |
218 |
1122 |
No |
|
TA 100 |
TA 102 |
||||
Conc. |
- MA |
+ MA |
Cytotoxic |
- MA |
+ MA |
Cytotoxic |
0* |
130 |
107 |
No |
440 |
558 |
No |
312.5 |
153 |
131 |
No (Mp) |
437 |
588 |
No (Mp) |
625 |
149 |
135 |
No (Mp) |
455 |
442 |
No (Mp) |
1250 |
131 |
136 |
No (Mp) |
512 |
436 |
No (Mp) |
2500 |
127 |
102 |
No (Sp) |
469 |
531 |
No (Sp) |
5000 |
149 |
118 |
No (Sp) |
477 |
452 |
No (Sp) |
Positive control |
639 |
771 |
No |
1992 |
3017 |
No |
*solvent control with DMSO
Mp : Moderate precipitate
Sp : Strong precipitate
MA : Metabolic activation
Table 3: Third experiment (direct plate incorporation with S9 mix) - Mean revertant colony count
|
TA 98 |
||
Conc. |
- MA |
+ MA |
Cytotoxic |
0* |
- |
32 |
No |
312.5 |
- |
37 |
No (Mp) |
625 |
- |
40 |
No (Mp) |
1250 |
- |
37 |
No (Mp) |
2500 |
- |
34 |
No (Sp) |
5000 |
- |
40 |
No (Sp) |
Positive control |
- |
1650 |
No |
*solvent control with DMSO
Mp : Moderate precipitate
Sp : Strong precipitate
MA : Metabolic activation
Summary table of mutagenicity assay results:
|
Dose (µg/plate) |
Number of revertant colonies per plate (Mean ± SD) |
|
|
|
|
|
|
|
|
|
|
|
|
|
TA100 |
|
TA1535 |
|
WP2 uvrA |
|
TA98 |
|
TA1537 |
|
TA1538 |
|
|
|
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
Negative control Water DMSO Acetone |
0 0 0 |
149 ± 17.1 150 ± 16.8 179 ± 15.0 |
161 ± 16.2 154 ± 17.5 136 ± 7.0 |
28 ± 6.9 30 ± 5.6 23 ± 1.5 |
15 ± 3.6 15 ± 7.1 8 ± 1.5 |
32 ± 7.3 30 ± 9.7 30 ± 7.5 |
33 ± 10.3 34 ± 10.9 26 ± 4.0 |
29 ± 6.2 32 ± 7.7 24 ± 3.0 |
39 ± 8.6 42 ± 10.1 32 ± 1.0 |
16 ± 6.4 18 ± 8.6 10 ± 3.0 |
21 ± 8.1 22 ± 6.3 18 ± 2.5 |
21 ± 5.5 22 ± 7.3 21 ± 0.5 |
28 ± 7.0 28 ± 5.7 26 ± 1.5 |
Positive control AF-2
ENNG 9AC 4NQO B(a)P 2AA |
0.01 0.05 5 80 0.25 5 5 |
501 ± 84.7 nt nt nt nt nt nt |
nt nt nt nt nt 1084 ± 236.3 nt |
nt nt 1101 ± 683.1 nt nt nt nt |
nt nt nt nt nt nt 440 ± 198.6 |
nt 1082 ± 293.7 nt nt nt nt nt |
nt nt nt nt nt nt 359 ± 127.0 |
nt 278 ± 64.8 nt nt nt nt nt |
nt nt nt nt nt 809 ± 108.4 nt |
nt nt nt 889 ± 275.7 nt nt nt |
nt nt nt nt nt 313 ± 41.6 nt |
nt nt nt nt 270 ± 66.2 nt nt |
nt nt nt nt nt 354 ± 89.4 nt |
Test substance - Cerium(IV) oxide |
1 5 10 50 100 500 1000 5000 |
150 160 187 169 139 156 168 163 |
177 192 190 186 212 206 199 188 |
30 30 36 34 30 35 37 30* |
11 17 14 19 19 14 14 10* |
25 24 32 25 25 26 25 21 |
30 31 30 25 25 26 25 22 |
25 27 23 21 22 19 28 28 |
34 36 37 29 40 35 33 42 |
6 5 6 10 8 10 12 9* |
11 15 14 12 11 12 17 12* |
25 34 29 23 35 23 35 22 |
31 40 43 50 43 41 38 32 |
AF-2: 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide
ENNG: N-ethyl-N'-nitro-N-nitrosoguanidine
9AC: 9-aminoacridine
4NQO: 4-nitroquinoline-1-oxide
B(a)P: benzo(a)pyrene
2AA: 2-aminoanthracene
* Growth inhibition observed
nt: not tested
Summary result table:
|
Concentration (µg/mL) |
S9 mix |
Relative cloning efficiency I (%) |
Relative cloning efficiency II (%) |
Mutant colonies per 10E6 cells |
Induction factor |
Relative cloning efficiency I (%) |
Relative cloning efficiency II (%) |
Mutant colonies per 10E6 cells |
Induction fact |
Experiment I / 4-h treatment |
|
|
Culture I |
|
|
|
Culture II |
|
|
|
Negative control |
0 |
- |
100.0 |
100.0 |
5.7 |
- |
100.0 |
100.0 |
7.2 |
- |
Solvent control (water) |
0 |
- |
100.0 |
100.0 |
12.3 |
1.0 |
100.0 |
100.0 |
7.7 |
1.0 |
Positive control (EMS) |
300.0 |
- |
30.2 |
80.2 |
137.5 |
24.0 |
30.1 |
86.9 |
67.3 |
9.4 |
Test item |
28.1 56.3 112.5 225.0 (p) 450.0 (p) 1800.0 (p) |
- - - - - - |
103.6 105.2 89.3 101.4 94.2 103.0 |
culture discontinued* 97.7 89.3 82.1 102.0 84.9 |
culture discontinued* 3.7 5.4 7.2 5.6 5.4 |
culture discontinued* 0.3 0.4 0.6 0.5 0.4 |
95.8 102.7 91.4 97.5 83.6 100.6 |
culture discontinued* 98.1 95.8 92.3 98.5 98.8 |
culture discontinued* 6.7 6.9 10.6 7.9 7.1 |
culture discontinued* 0.9 0.9 1.4 1.0 0.9 |
Experiment II / 24-h treatment |
|
|
Culture I |
|
|
|
Culture II |
|
|
|
Negative control |
0 |
+ |
100.0 |
100.0 |
5.7 |
- |
100.0 |
100.0 |
8.4 |
- |
Solvent control (water) |
0 |
+ |
100.0 |
100.0 |
3.0 |
1.0 |
100.0 |
100.0 |
5.2 |
1.0 |
Positive control (DMBA) |
2.0 |
+ |
24.1 |
80.2 |
510.3 |
172.2 |
77.8 |
55.8 |
1034.3 |
200.6 |
Test item |
14.1 28.1 56.3 112.5 (p) 225.0 (p) 1800.0 (p) |
+ + + + + + |
96.4 93.4 91.5 102.4 112.2 107.5 |
64.3 73.2 73.6 74.1 32.9 culture discontinued* |
8.1 4.5 3.1 6.8 15.7 culture discontinued* |
2.7 1.5 1.1 2.3 5.3 culture discontinued* |
89.0 88.9 82.2 85.2 88.6 76.0 |
66.1 72.8 81.6 74.2 95.4 culture discontinued* |
3.4 8.5 5.5 2.3 4.1 culture discontinued* |
0.7 1.7 1.1 0.4 0.8 culture discontinued* |
(p) Precipitation visible to unaided eye
* Since a minimum of 4 analyzable concentrations is required
Mean Revertants First Experiment:
| Strain | 97a | 98 | 100 | 102 | 1535 | ||||||
| Induction | -S9 | +S9 | -S9 | +S9 | -S9 | +S9 | -S9 | +S9 | -S9 | +S9 | |
| H2O | Mean | 139 | 109 | 13 | 11 | 152 | 185 | 212 | 192 | 15 | 16 |
| sd | 58.2 | 11.6 | 1.7 | 3.8 | 25.1 | 31.1 | 22.0 | 58.2 | 3.6 | 5.7 | |
| DMSO | Mean | 136 | 155 | 8 | 10 | 206 | 178 | 204 | 221 | 18 | 15 |
| sd | 16.8 | 49.5 | 4.7 | 3.4 | 30.2 | 34.9 | 12.4 | 73.3 | 2.4 | 5.4 | |
| Pos Contr | Mean | 1001 | 1001 | 1001 | 1001 | 1001 | 1001 | 1001 | 1001 | 1001 | 1001 |
| sd | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | |
| f(I) | 7.36 | 6.46 | 125.1 | 100.1 | 6.59 | 5.62 | 4.91 | 4.53 | 66.73 | 66.73 | |
| 4998 µg/pl. | Mean | 171 | 100 | 10 | 9 | 129 | 183 | 198 | 209 | 19 | 14 |
| sd | 28 | 8 | 3 | 1 | 11 | 17 | 18 | 68 | 6 | 2 | |
| f(I) | 1.23 | 0.92 | 0.77 | 0.82 | 0.85 | 0.99 | 0.93 | 1.09 | 1.27 | 0.88 | |
| 1499 µg/pl. | Mean | 156 | 146 | 12 | 9 | 170 | 160 | 198 | 178 | 15 | 18 |
| sd | 17 | 29 | 3 | 3 | 19 | 30 | 32 | 54 | 4 | 3 | |
| f(I) | 1.12 | 1.36 | 0.92 | 0.82 | 1.12 | 0.86 | 0.93 | 0.93 | 1.00 | 1.13 | |
| 500 µg/pl. | Mean | 139 | 133 | 16 | 8 | 160 | 152 | 157 | 176 | 13 | 15 |
| sd | 43 | 10 | 4 | 2 | 25 | 59 | 31 | 51 | 2 | 4 | |
| f(I) | 1.00 | 1.22 | 1.23 | 0.73 | 1.05 | 0.82 | 0.74 | 0.92 | 0.87 | 0.94 | |
| 150 µg/pl. | Mean | 134 | 113 | 10 | 9 | 152 | 178 | 209 | 168 | 15 | 12 |
| sd | 41 | 7 | 4 | 2 | 17 | 37 | 50 | 45 | 4 | 4 | |
| f(I) | 0.96 | 1.04 | 0.77 | 0.82 | 1.00 | 0.96 | 0.99 | 0.88 | 1.00 | 0.75 | |
| 50 µg/pl. | Mean | 135 | 145 | 10 | 6 | 145 | 127 | 218 | 200 | 17 | 20 |
| sd | 42 | 34 | 4 | 2 | 11 | 26 | 18 | 54 | 2 | 5 | |
| f(I) | 0.97 | 1.33 | 0.77 | 0.55 | 0.95 | 0.69 | 1.03 | 1.04 | 1.13 | 1.25 |
In this table ">1000" is represented by "1001"
Mean Revertants Second Experiment:
| Strain | 97a | 98 | 100 | 102 | 1535 | ||||||
| Induction | -S9 | +S9 | -S9 | +S9 | -S9 | +S9 | -S9 | +S9 | -S9 | +S9 | |
| H2O | Mean | 99 | 118 | 5 | 12 | 160 | 151 | 156 | 137 | 15 | 13 |
| sd | 54.9 | 13.6 | 1.7 | 3.9 | 23.5 | 20.1 | 14.0 | 25.0 | 1.8 | 4.9 | |
| DMSO | Mean | 152 | 115 | 7 | 12 | 142 | 133 | 167 | 164 | 8 | 11 |
| sd | 9.6 | 6.1 | 0.8 | 3.4 | 17.0 | 1.9 | 8.6 | 31.0 | 2.1 | 2.2 | |
| Pos.Contr. | Mean | 1001 | 1001 | 1001 | 1001 | 1001 | 1001 | 1001 | 1001 | 1001 | 1001 |
| sd | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | |
| f(I) | 6.59 | 8.70 | 143.0 | 83.42 | 6.26 | 7.53 | 5.99 | 6.10 | 66.73 | 91.00 | |
| 4998 µg/pl. | Mean | 126 | 115 | 8 | 11 | 112 | 187 | 141 | 156 | 10 | 15 |
| sd | 35 | 45 | 4 | 5 | 10 | 46 | 15 | 27 | 3 | 5 | |
| f(I) | 1.27 | 0.97 | 1.60 | 0.92 | 0.70 | 1.24 | 0.90 | 1.14 | 0.67 | 1.15 | |
| 2499 µg/pl. | Mean | 123 | 142 | 8 | 7 | 150 | 130 | 185 | 143 | 12 | 9 |
| sd | 39 | 24 | 4 | 6 | 14 | 47 | 13 | 46 | 6 | 3 | |
| f(I) | 1.24 | 1.20 | 1.60 | 0.58 | 0.94 | 0.86 | 1.19 | 1.04 | 0.80 | 0.69 | |
| 1250 µg/pl. | Mean | 145 | 149 | 10 | 9 | 164 | 157 | 204 | 182 | 15 | 12 |
| sd | 10 | 10 | 5 | 1 | 5 | 16 | 9 | 33 | 5 | 2 | |
| f(I) | 1.46 | 1.26 | 2.00 | 0.75 | 1.03 | 1.04 | 1.31 | 1.33 | 1.00 | 0.92 |
In this table "> 1000" is represented by "1001"
Results:
Both in the absence and presence of S9-mix zirconium dioxide did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in two independent experiments.
No effects of zirconium dioxide on the number of polyploid cells and cells with endoreduplicated chromosomes were observed both in the absence and presence of S9-mix. Therefore it can be concluded that zirconium dioxide does not disturb mitotic processes and cell cycle progression and does not induce numerical chromosome aberrations under the experimental conditions of this test.
Table 1: Mitotic index of human lymphocyte cultures treated with zirconium dioxide at the 24 h and 48 h continuous exposure time in the dose range finding test.
| Zirconium dioxide concentration (µg/mL) | Number of metaphases per 1000 cells | |
| Absolute | Percentage of control | |
| Without metabolic activation (-S9 -mix) | ||
| 24 h exposure time, 24 h fixation time | ||
| Control a) | 36 | 100 |
| 1 | 33 | 92 |
| 3 | 32 | 89 |
| 10 | 31 | 86 |
| 33 | 36 | 100 |
| 100 b) | 34 | 94 |
| 333 c) | 38 | 106 |
| 1000 c) | 38 | 106 |
| 48 h exposure time, 48 h fixation time | ||
| Control a) | 42 | 100 |
| 1 | 44 | 105 |
| 3 | 44 | 105 |
| 10 | 42 | 100 |
| 33 | 39 | 93 |
| 100 b) | 42 | 100 |
| 333 c) | 44 | 105 |
| 1000 c) | 44 | 105 |
a) Dimethyl sulfoxide
b) Zirconium dioxide precipitated in the culture medium
c) Zirconium dioxide precipitated heavily in the culture medium which would interfere with the scoring of chromosome aberrations
Table 2: Mitotic index of human lymphocyte cultures treated with zirconium dioxide at the 3 h exposure time in the dose range finding test (first cytogenetic assay)
| Zirconium dioxide concentration (µg/mL) | Number of metaphases per 1000 cells | |
| Without metabolic activation (-S9 -mix) | Absolute | Percentage of control |
| 3 h exposure time, 24 h fixation time | ||
| Control b) | 46 - 51 | 100 |
| 10 | 48 - 50 | 101 |
| 33 | 47 - 49 | 99 |
| 100 | 51 - 53 | 107 |
| MMC-C; 0.5 µg/mL | 38 - 33 | 73 |
| With metabolic activation (+ S9 -mix) | ||
| Control b) | 54 -54 | 100 |
| 10 | 50 - 49 | 92 |
| 33 | 55 - 54 | 101 |
| 100 c) | 50 - 53 | 95 |
| CP; 10 µg/mL | 21 - 28 | 45 |
a) Duplicate cultures
b) Dimethyl sulfoxide
c) Zirconium dioxide precipitated in the culture medium
Table 3: Mitotic index of human lymphocyte cultures treated with zirconium dioxide in the second cytogenetic assay
| Zirconium dioxide concentration (µg/mL) | Number of metaphases per 1000 cells | |
| Absolute | Percentage of control | |
| Without metabolic activation (-S9 -mix) | ||
| 24 h exposure time, 24 h fixation time | ||
| Control b) | 65 -68 | 100 |
| 10 | 63 - 69 | 99 |
| 33 | 60 65 | 94 |
| 100 c) | 58 -61 | 89 |
| MMC-C; 0.2 µg/mL | 31 - 35 | 50 |
| 48 h exposure time, 48 h fixation time | ||
| Control b) | 71 - 68 | 100 |
| 10 | 65 - 69 | 96 |
| 33 | 68 - 66 | 96 |
| 100 c) | 62 - 60 | 88 |
| MMC-C; 0.1 µg/mL | 53 - 55 | 78 |
| With metabolic activation (+S9 -mix) | ||
| 3 h exposure time, 48 h fixation time | ||
| Control b) | 75 - 77 | 100 |
| 10 | 72 - 76 | 97 |
| 33 | 79 - 79 | 104 |
| 100 | 78 - 75 | 101 |
| CP; 10 µg/mL | 28 - 25 | d) |
a) Duplicate cultures
b) Dimethyl sulfoxide
d) Zirconium dioxide precipitated in the culture medium
e) CP was fixed after 24 hours. Therefore, the mitotic index could not be calculated as percentage of control.
The growth rate over the two-day expression period for cultured treated with DMSO was between 20 and 28 (3 hours treatment) and 40 and 50 (24 hours treatment).
Mutation frequencies in cultures treated with positive control chemicals were increased by 26- and 14-fold for MMS in the absence of S9-mix, and by 19-fold for CP in the presence of S9-mix, in the first and second experiment respectively. It was therefore concluded that the test conditions, both in the absence and presence of S9-mix, were appropriate for the detection of a mutagenic response and that the metabolic activation system (S9-mix) functioned properly. In addition the observed mutation frequencies of the positive control substances were within the acceptability criteria of this assay.
Experiment 1: Cytotoxic and mutagenic response of zirconium dioxide in the mouse lymphoma L5178Y test system (3 hours treatment)
Without metabolic activation
| dose (µg/mL) | RSG (%) | CE day2 (%) | RS day2 (%) | RTG (%) | Mutation frequency x 1E-06 | |||
| total | (small | large) | ||||||
| SC1 | 100 | 118 | 100 | 100 | 53 | 31 | 20 | |
| SC2 | 100 | 113 | 100 | 100 | 51 | 31 | 19 | |
| 0.03 | 112 | 101 | 87 | 98 | 50 | 23 | 25 | |
| 0.1 | 105 | 110 | 95 | 100 | 54 | 29 | 23 | |
| 0.3 | 110 | 94 | 81 | 90 | 54 | 26 | 26 | |
| 1 | 117 | 111 | 96 | 113 | 50 | 21 | 28 | |
| 3 | 112 | 101 | 87 | 97 | 49 | 25 | 22 | |
| 10 | 106 | 98 | 85 | 90 | 58 | 34 | 23 | |
| 33 | 102 | 97 | 84 | 85 | 58 | 30 | 27 | |
| 100 (1) | 103 | 105 | 91 | 94 | 52 | 29 | 22 | |
| MMS | 66 | 57 | 49 | 32 | 1334 | 804 | 318 | |
With 8% (v/v) metabolic activation
| dose (µg/mL) | RSG (%) | CE day2 (%) | RS day2 (%) | RTG (%) | Mutation frequency x 1E-06 | ||
| total | (small | large) | |||||
| SC1 | 100 | 88 | 100 | 100 | 54 | 32 | 21 |
| SC2 | 100 | 89 | 100 | 100 | 53 | 29 | 23 |
| 0.03 | 100 | 102 | 116 | 116 | 53 | 34 | 18 |
| 0.1 | 99 | 83 | 94 | 93 | 54 | 38 | 15 |
| 0.3 | 99 | 79 | 90 | 89 | 59 | 32 | 26 |
| 1 | 100 | 81 | 92 | 93 | 67 | 33 | 33 |
| 3 | 92 | 74 | 83 | 77 | 78 | 47 | 29 |
| 10 | 99 | 86 | 98 | 97 | 60 | 31 | 27 |
| 33 | 92 | 90 | 102 | 94 | 56 | 33 | 21 |
| 100 (1) | 100 | 77 | 87 | 87 | 61 | 32 | 28 |
| CP | 53 | 72 | 82 | 44 | 1000 | 674 | 191 |
Note: all calculations were made without rounding off
RSG = Relative Suspension Growth; CE = Cloning efficiency; RS = Relative Survival; RTG = Relative Total Growth; SC = Solvent Control = DMSO; MMS = Methylmethanesulfonate; CP = cyclophosphamide
(1) zirconium dioxide precipitated in the exposure medium
Experiment 2: Cytotoxic and mutagenic response of zirconium dioxide in the mouse lymphoma L5178Y test system (24 hours)
Without metabolic activation
| dose (µg/mL) | RSG (%) | CE day2 (%) | RS day2 (%) | RTG (%) | Mutation frequency x 1E-06 | ||
| total | (small | large) | |||||
| SC1 | 100 | 118 | 100 | 100 | 57 | 32 | 23 |
| SC2 | 100 | 104 | 100 | 100 | 63 | 36 | 25 |
| 0.03 | 120 | 88 | 79 | 95 | 72 | 43 | 27 |
| 0.1 | 127 | 107 | 96 | 122 | 66 | 34 | 29 |
| 0.3 | 137 | 120 | 108 | 148 | 50 | 29 | 20 |
| 1 | 127 | 111 | 100 | 128 | 54 | 34 | 18 |
| 3 | 139 | 110 | 99 | 138 | 55 | 37 | 17 |
| 10 | 140 | 91 | 82 | 115 | 80 | 48 | 29 |
| 33 | 138 | 115 | 103 | 143 | 69 | 41 | 25 |
| 100 (1) | 153 | 97 | 87 | 133 | 54 | 38 | 15 |
| MMS | 119 | 77 | 69 | 83 | 815 | 564 | 157 |
With 12% (v/v) metabolic activation:
| dose (µg/mL) | RSG (%) | CE day2 (%) | RS day2 (%) | RTG (%) | Mutation frequency x 1E-06 | ||
| total | (small | large) | |||||
| SC1 | 100 | 111 | 100 | 100 | 67 | 40 | 25 |
| SC2 | 100 | 80 | 100 | 100 | 85 | 44 | 37 |
| 0.03 | 107 | 77 | 80 | 86 | 85 | 57 | 26 |
| 0.1 | 97 | 86 | 90 | 87 | 86 | 45 | 37 |
| 0.3 | 99 | 102 | 107 | 105 | 64 | 34 | 28 |
| 1 | 97 | 107 | 111 | 108 | 69 | 43 | 24 |
| 3 | 99 | 97 | 101 | 100 | 75 | 53 | 20 |
| 10 | 90 | 99 | 104 | 93 | 77 | 54 | 20 |
| 33 | 89 | 107 | 111 | 99 | 94 | 49 | 40 |
| 100 (1) | 91 | 102 | 107 | 97 | 71 | 45 | 24 |
| CP | 42 | 54 | 56 | 24 | 1422 | 832 | 355 |
(1) = Zirconium dioxide precipitated in the exposure medium
Note: all calculations were made without rounding off
RSG = Relative Suspension Growth; CE = Cloning efficiency; RS = Relative Survival; RTG = Relative Total Growth; SC = Solvent control = DMSO; MMS = Methylmethanesulfonate; CP = Cyclophosphamid (1) = Zirconium dioxide precipitated in the exposure medium
There were no significant increases in the frequency of revertant colonies recorded for any of the strains of Salmonella, at any dose level, either with or without metabolic activation, as detailed in the table below.
Summary table of mean revertant colonies (non-nano CeO2):
|
Dose (µg/plate) |
TA100 |
|
TA1535 |
|
TA102 |
|
TA98 |
|
TA1537 |
|
|
|
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
Experiment 1 |
|
|
|
|
|
|
|
|
|
|
|
DMSO |
100 µL |
83±7 |
64 ± 5 |
40 ± 2 |
12 ± 2 |
309 ± 24 |
333 ± 29 |
21 ± 5 |
26 ± 4 |
10 ± 2 |
17 ± 3 |
Cerium oxide (non-nano) |
50 150 500 1500 5000 |
85 ± 12 96 ± 7 80 ± 12 69 ± 10 84 ± 7 |
73 ± 11 70 ± 9 76 ± 10 75 ± 11 73 ± 9 |
37 ± 5 34 ± 3 35 ± 7 34 ± 2 36 ± 3 |
13 ± 1 11 ± 2 11 ± 2 13 ± 6 10 ± 2 |
333 ± 28 341 ± 21 345 ± 9 338 ± 10 313 ± 21 |
361 ± 29 359 ± 7 367 ± 11 350 ± 26 354 ± 14 |
19 ± 4 19 ± 3 22 ± 7 16 ± 2 16 ± 4 |
29 ± 2 29 ± 3 29 ± 6 25 ± 3 31 ± 4 |
12 ± 2 13 ± 3 13 ± 3 12 ± 5 11 ± 1 |
19 ± 6 18 ± 3 20 ± 4 21 ± 5 22 ± 1 |
Experiment 2 |
|
|
|
|
|
|
|
|
|
|
|
DMSO |
100 µL |
93 ± 16 |
105 ± 13 |
27 ± 7 |
9 ± 1 |
345 ± 17 |
374 ± 13 |
20 ± 3 |
27 ± 7 |
8 ± 3 |
13 ± 4 |
Cerium oxide (non-nano) |
50 150 500 1500 5000 |
87±10 95 ± 4 94 ± 13 82 ± 2 107 ± 2 |
115 ± 10 91 ± 8 89 ± 4 99 ± 2 97 ± 13 |
32 ± 2 28 ± 7 37 ± 5 33 ± 12 26 ± 4 |
11 ± 7 14 ± 2 9 ± 1 9 ± 3 10 ± 2 |
349 ± 42 343 ± 20 349 ± 27 351 ± 22 350 ± 49 |
352 ± 31 397 ± 21 364 ± 23 380 ± 10 382 ± 21 |
16 ± 7 20 ± 3 18 ± 2 17 ± 2 18 ± 4 |
23 ± 2 23 ± 2 29 ± 3 28 ± 4 23 ± 6 |
15 ± 4 8 ± 4 9 ± 1 8 ± 4 15 ± 5 |
19 ± 4 19 ± 5 17 ± 3 21 ± 6 19 ± 3 |
There were no significant increases in the frequency of revertant colonies recorded for any of the strains of Salmonella, at any dose level, either with or without metabolic activation, as detailed in the table below.
Table: Summary of mean revertant colonies (nano Ce02)
Substance |
Dose Level (μg/plate) |
TA100 |
TA1535 |
TA102 |
TA98 |
TA1537 |
|||||
(-S9) |
(+S9) |
(-S9) |
(+S9) |
(-S9) |
(+S9) |
(-S9) |
(+S9) |
(-S9) |
(+S9) |
||
Experiment 1 |
|||||||||||
DMSO |
100μL |
118 ± 7 |
97 ± 13 |
39 ± 3 |
32 ± 6 |
347 ± 29 |
351 ± 4 |
28 ± 3 |
36 ± 7 |
13 ± 5 |
22 ± 2 |
Cerium Oxide Nano |
50 |
100 ± 3 |
109 ± 17 |
37 ± 2 |
29 ± 13 |
356 ± 10 |
363 ± 25 |
23 ± 5 |
36 ± 2 |
11 ± 5 |
18 ± 4 |
150 |
88 ± 13 |
115 ± 9 |
40 ± 3 |
36 ± 1 |
360 ± 13 |
381 ± 20 |
25 ± 6 |
35 ± 6 |
12 ± 5 |
15 ± 5 |
|
500 |
84 ± 16 |
114 ± 20 |
33 ± 3 |
36 ± 3 |
377 ± 12 |
350 ± 17 |
26 ± 6 |
35 ± 7 |
14 ± 5 |
17 ± 4 |
|
1500 |
101 ± 13 |
115 ± 8 |
28 ± 4 |
32 ± 9 |
343 ± 38 |
330 ± 17 |
22 ± 3 |
35 ± 1 |
14 ± 4 |
24 ± 2 |
|
5000 |
101 ± 4 |
113 ± 7 |
39 ± 3 |
38 ± 2 |
343 ± 16 |
332 ± 14 |
23 ± 4 |
37 ± 4 |
12 ± 2 |
20 ± 5 |
|
Experiment 2 |
|||||||||||
DMSO |
100μL |
97 ± 14 |
132 ± 8 |
28 ± 6 |
30 ± 11 |
394 ± 9 |
380 ± 25 |
21 ± 5 |
38 ± 7 |
12 ± 10 |
15 ± 6 |
Cerium Oxide Nano |
50 |
99 ± 19 |
123 ± 17 |
32 ± 2 |
29 ± 5 |
359 ± 28 |
397 ± 39 |
21 ± 4 |
33 ± 10 |
12 ± 1 |
13 ± 6 |
150 |
98 ± 20 |
127 ± 10 |
35 ± 6 |
40 ± 6 |
361 ± 32 |
391 ± 32 |
22 ± 4 |
32 ± 2 |
14 ± 7 |
12 ± 4 |
|
500 |
84 ± 6 |
126 ± 11 |
35 ± 11 |
32 ± 4 |
382 ± 29 |
350 ± 7 |
21 ± 4 |
32 ± 5 |
14 ± 4 |
16 ± 4 |
|
1500 |
92 ± 8 |
133 ± 7 |
36 ± 8 |
36 ± 7 |
361 ± 43 |
313 ± 29 |
19 ± 5 |
37 ± 4 |
9 ± 3 |
12 ± 3 |
|
5000 |
95 ± 3 |
121 ± 22 |
28 ± 5 |
29 ± 3 |
371 ± 24 |
353 ± 36 |
19 ± 3 |
26 ± 3 |
9 ± 1 |
17 ± 2 |
|
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Genetic toxicity in vivo
Description of key information
For the endpoint on in vitro cytogenicity in mammalian cells, a study is only available for zirconium dioxide, and not for cerium dioxide. However, for cerium dioxide, an in vivo mouse micronucleus test is available. Therefore, in the absence of experimental information on the reaction mass itself, the endpoint was covered by read across from the in vivo mouse micronucleus test performed with (bulk) cerium dioxide, which was negative, in combination with an in vitro chromosome aberration study performed with (bulk) zirconium dioxide, which was negative with and without metabolic activation under the conditions of the test. Therefore, no clastogenicity is to be expected from the reaction mass of cerium dioxide and zirconium dioxide either.
Link to relevant study records
- Endpoint:
- in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- Not specified
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
- Deviations:
- no
- GLP compliance:
- yes
- Type of assay:
- mammalian erythrocyte micronucleus test
- Species:
- mouse
- Strain:
- Swiss
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Iffa Credo, 69210 L'Arbresle France
- Age at study initiation: 5 weeks old
- Weight on day of dosing: 30 - 34 g (males) / 23 - 29 g (females)
- Housing: by groups of 5 of the same sex in polycarbonate cages with stainless steel lid
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 5 days
ENVIRONMENTAL CONDITIONS
- Temperature: 21 ± 2°C
- Humidity: 50 ± 20%
- Air changes (per hr): filtered and non-recycled fresh air
- Photoperiod: 12 hrs dark / 12 hrs light
IN-LIFE DATES: Not specified - Route of administration:
- oral: gavage
- Vehicle:
- - Vehicle used: 1% carboxymethylcellulose solution
- Justification for choice of solvent/vehicle: appropriate to oral suspensions
- Concentration of test material in vehicle: 100 mg/mL
- Amount of vehicle: 20 mL/kg bw
- Lot/batch no. (if required): Sigma 58C-0156 - Details on exposure:
- PREPARATION OF DOSING SOLUTIONS: Immediately before use, by putting the test substance in suspension
- Duration of treatment / exposure:
- Single administration
- Frequency of treatment:
- Single administration
- Post exposure period:
- 24 hours (test substance, negative control, positive control) and 48 hours (test substance, negative control)
- Dose / conc.:
- 2 000 mg/kg bw (total dose)
- Remarks:
- Basis: nominal
- No. of animals per sex per dose:
- 5
- Control animals:
- yes, concurrent vehicle
- Positive control(s):
- - Nature: cyclophosphamide
- Justification for choice of positive control: not provided
- Route of administration: Oral (gavage)
- Dose: 50 mg/kg bw - Tissues and cell types examined:
- Femur bone marrow erythrocytes
- Details of tissue and slide preparation:
- - Femur bone marrow eluted out with fetal calf serum and cell suspension centrifuged
- Supernatant removed and cells in sediment resuspended by shaking
- One drop of cell suspension spread on a coded slide
- Slides air-dried and stained by May-Grünwald Giemsa
- Two slides prepared per animal but only one used for scoring - Evaluation criteria:
- - 2000 polychromatic erythrocytes scored for micronuclei per animal
- Ratio between polychromatic and normochromatic erythrocytes (PE/NE ratio) calculated by scoring 1000 erythrocytes per animal
- Substance considered clastogenic if statistical increase in mean number of micronucleated polychromatic erythrocytes for at least one of the sampling time compared to negative controls and this increase doubles the number of micronucleated polychromatic erythrocytes in the test facility's historical data (1.4 +/- 0.6 per thousand) - Statistics:
- - Intergroup comparison of mean numbers of micronucleated polychromatic erythrocytes using Kastenbaum and Bowman's test (5% significance level)
- Intergroup comparison of PE/NE ratios using Student's t test - Key result
- Sex:
- male/female
- Genotoxicity:
- negative
- Remarks:
- at 2000 mg/kg bw (nominal)
- Toxicity:
- no effects
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- RESULTS OF RANGE-FINDING STUDY
No clinical signs in 3 males and 3 females given a single oral dose of 2000 mg/kg bw.
RESULTS OF DEFINITIVE STUDY
See summary table below. - Conclusions:
- No evidence of clastogenicity in this mouse bone marrow micronucleus assay at the limit dose of 2000 mg/kg.
- Executive summary:
The clastogenic potential of Cerium Oxide was tested in an oral mouse bone marrow micronucleus assay. Swiss OF1 5-week old mice (5/sex per group) were given a single oral administration of 2000 mg/kg Cerium Oxide by gavage. Two test substance-treated groups, two vehicle (1% carboxymethylcellulose)-treated control groups and one positive control (cyclophosphamide) group were used. Euthanasia was performed 24 and 48 hours after dosing for substance-treated and negative control groups. Positive controls were euthanasied 24 hours after dosing. For each animal, 2000 polychromatic erythrocytes from femur bone marrow were microscopically examined for micronuclei. The polychromatic to normochromatic erythrocytes (PE/NE) ratio was determined from 1000 erythrocytes per mouse.
At both sampling times, there were no statistical differences from negative control values in the number of micronucleated polychromatic erythrocytes and the PE/NE ratio from substance-treated animals.
An appropriate reference mutagen used as positive control showed a significant increase in micronucleated polychromatic erythrocytes together with a significant decrease in the PE/NE ratio, indicating that the test was sensitive and valid.
Therefore, Cerium Oxide did not induce cytogenetic damage in this micronucleus test in mice treated by the oral route at the limit dose of 2000 mg/kg.
This study is classified as acceptable. It satisfies the OECD 474 guideline requirements on Mammalian Erythrocyte Micronucleus Test.
- Endpoint:
- in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- key study
- Justification for type of information:
- Read across based on an in vivo mouse micronucleus study performed with (bulk) cerium dioxide. The read across justification document is attached to IUCLID Section 13.
- Reason / purpose for cross-reference:
- read-across source
- Key result
- Remarks on result:
- other: read across conclusion
- Remarks:
- The reaction mass was concluded not to be clastogenic, based on an in vivo mouse micronucleus test performed with cerium dioxide, in combination with an in vitro chromosome aberration study in mammalian cells performed with zirconium dioxide.
Referenceopen allclose all
Mean number of micronucleated polychromatic erythrocytes and PE/NE ratio:
Time of sacrifice (hours post-dosing) |
Group |
Number of polychromatic erythrocytes / 1000 polychromatic erythrocytes (mean ± standard deviation) |
PE/NE ratio (mean ± standard deviation) |
24 |
|
|
|
|
Vehicle |
1.9 ± 1.0 |
1.0 ± 0.2 |
|
Test substance |
2.4 ± 1.3 |
1.1 ± 0.2 |
|
Cyclophosphamide |
56.6 ± 10.4*** |
0.8 ± 0.1* |
48 |
|
|
|
|
Vehicle |
1.7 ± 1.6 |
1.0 ± 0.2 |
|
Test substance |
2.9 ± 1.3 |
1.1 ± 0.3 |
*** p < 0.001 * p < 0.05
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Additional information
One key in vitro study (Ames test) is available on the reaction mass of cerium dioxide and zirconium dioxide. Other studies (Ames test - OECD 471, in vitro gene mutation assay in mammalian cells - OECD 476, in vivo mouse bone marrow micronucleus assay - OECD 474) are available on cerium dioxide, one of the constituents of the reaction mass. In addition, reliable in vitro studies are available on the other constituent zirconium dioxide (Ames test - OECD 471, in vitro cytogenicity assay - OECD 473, in vitro gene mutation assay - OECD 476). As these constituents show similar physicochemical, toxicological, ecotoxicological and environmental properties, results of studies performed with cerium dioxide and zirconium dioxide are used as supporting information (in vitro gene mutation in bacteria) or, in the absence of information on the reaction mass itself (i.e. for all other endpoints on genetic toxicity), in a weight-of-evidence approach to draw conclusions on the endpoint.
Further genetic toxicity studies on the reaction mass of cerium dioxide and zirconium dioxide are therefore not regarded as scientifically necessary according to section 1 of Reach Annex XI and is not recommended under animal protection considerations.
In vitro gene mutation in bacteria
The potential of the reaction mass of cerium dioxide and zirconium dioxide to induce reverse gene mutations in Salmonella typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102 was evaluated in a study performed according to OECD guideline 471 and in compliance with the GLP. This study (Haddouk, 2007; Klimisch 1) was used as key study.
The test item, which was a representative nanoform of the reaction mass, was studied in two independent experiments, with and without a metabolic activation system. A third experiment was performed with S9 mix. Each strain was exposed to at least five dose levels (from 312.5 to 5000 µg/plate) of the test item, solvent control (DMSO) and positive controls (three plates/dose-level) with and without metabolic activation.
No noteworthy increase in the number of revertants was observed following treatment with the test substance, either with or without metabolic activation, in any of the five tester strains. A moderate to strong precipitate was observed in the Petri plates when scoring the revertants at dose levels >= 312.5 µg/plate. No noteworthy toxicity was induced in any of the five tester strains.
The number of revertants for the vehicle and positive controls was as specified in the acceptance criteria. The study was therefore considered valid.
Under these experimental conditions, the reaction mass of cerium dioxide and zirconium dioxide did not show any mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium.
The observation made for the reaction mass was supported by the results of a number of Ames studies performed with cerium dioxide, zirconium dioxide, or zirconium dioxide with a small w/w % of yttrium in it:
- Two bacterial reverse mutation assays (Shimizu, 1985, Klimisch 1; Park et al., 2007; Klimisch 2) are available for bulk cerium dioxide, and yielded negative results up to the limit concentration of 5000 µg/plate with or without exogenous metabolic activation.
- The study of Park et al. (2007; Klimisch 2) also performed an Ames test on a nanoparticular form of cerium dioxide and yielded similar results as for the bulk form (negative, with and without metabolic activation).
- For (bulk) zirconium dioxide (LAUS, 2008, Klimisch 1) as well as for zirconium dioxide with a small w/w % of yttrium in it (Chemicals Inspection and Testing Institute, 1997), the Ames tests yielded negative results up to the limit concentration of 5000 µg/plate with or without exogenous metabolic activation.
In vitro gene mutation in mammalian cells
No studies are available for the reaction mass of cerium dioxide and zirconium dioxide. Therefore, the endpoint is covered using the results of studies performed with (bulk) cerium dioxide and (bulk) zirconium dioxide:
- In a gene mutation assay at the hprt locus of V79 cells (Wollny, 2006; Klimisch 1), (bulk) cerium dioxide induced no mutations up to 1800 µg/mL with or without metabolic activation, under the conditions of the test.
- In a gene mutation assay at the TK locus of mouse lymphoma L5178Y cells (NOTOX, 2010b; Klimisch 1), (bulk) zirconium dioxide did not induce mutations up to 100 µg/L with and without metabolic activation, under the conditions of the test.
Based on the results of these studies, it can safely be concluded that no mutagenic activity is expected from the reaction mass of cerium dioxide and zirconium dioxide either.
In vitro and in vivo cytogenicity
No studies are available for the reaction mass of cerium dioxide and zirconium dioxide. Therefore, the endpoint is covered using the results of an in vitro chromosome aberration study performed with (bulk) zirconium dioxide and - in the absence of such in vitro studies for cerium dioxide - an in vivo mouse micronucleus test performed with (bulk) cerium dioxide:
- In an in vitro chromosome aberration test in cultured peripheral human lymphocytes (NOTOX, 2010a; Klimisch 1), (bulk) zirconium dioxide did not show any clastogenicity in the absence and presence of metabolic activation, under the conditions of the test.
- In vivo, (bulk) cerium dioxide was tested in a mouse bone marrow micronucleus assay (Molinier, 1993; Klimisch 1). There was no indication of clastogenic effects at 24 h or 48 h following a single administration of a limit dose level of 2000 mg/kg bw.
Based on the results of these studies, it can safely be concluded that no clastogenicity is expected from the reaction mass of cerium dioxide and zirconium dioxide either.
Genetic Toxicity |
Reaction mass |
Cerium dioxide |
Zirconium dioxide |
In vitro Ames test |
Negative +/- metabolic activation (nano) |
Negative +/- metabolic activation (bulk and nano) |
Negative +/- metabolic activation (bulk) |
In vitro gene mutation assay in mammalian cells |
- |
Negative +/- metabolic activation (bulk) |
Negative +/- metabolic activation (bulk) |
In vivo mouse bone marrow micronucleus assay |
- |
Negative at 2000 mg/kg bw (oral route) (bulk) |
- |
In vitro cytogenicity assay |
- |
- |
Negative +/- metabolic activation (bulk) |
Justification for classification or non-classification
Based on the CLP classification criteria, and considering the negative results in all in vitro and in vivo genetic toxicity tests using either a representative nanoform of the reaction mass of cerium dioxide and zirconium dioxide (Ames) or its nano or bulk constituents cerium dioxide (Ames: nano; other test: bulk) and zirconium dioxide (bulk form tested in all available studies), no classification for genetic toxicity is required for the reaction mass of cerium dioxide and zirconium dioxide.
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Reproduction or further distribution of this information may be subject to copyright protection. Use of the information without obtaining the permission from the owner(s) of the respective information might violate the rights of the owner.
