Registration Dossier
Registration Dossier
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EC number: 231-174-8 | CAS number: 7440-65-5
Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
No genetic toxicity study is available with the test substance yttrium metal. Therefore, read across is performed using three in vitro studies performed according to OECD Guidelines and in compliance with GLP with the related substance yttrium oxide. The justification for read across is attached in IUCLID Section 13.
In vitro mutagenicity in bacteria
One key study is available (Klimisch 1; Sokolowski A, 2006), performed according to EU Method B.13/14 guideline and conform GLP requirements. In this study, yttrium oxide did not show mutagenic activity in the applied bacterium tester strains in the absence or presence of metabolic activation under the conditions of the test system.
In vitro mammalian chromosome aberration test
One key study is available (Klimisch 1; Hofman-Hüther, 2014). This GLP study was performed according to OECD guideline 473. In this study, yttrium oxide did not induce structural chromosomal aberrations in human lymphocyte cells with and/or without metabolic activation.
In vitro gene mutation study in mammalian cells (HPRT assay)
One key study is available (Klimisch 1; Wallner, 2013). This GLP study was performed according to OECD guideline 476. In this study, no mutagenic effect of yttrium oxide was observed either in the presence or absence of metabolic activation system under the conditions of this HPRT assay in CHO K1 Chinese hamster ovary cells.
Link to relevant study records
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- weight of evidence
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Justification for type of information:
- Data from the related substance yttrium oxide is used to cover this endpoint. The justification for read across is attached in IUCLID Section 13.
- Reason / purpose for cross-reference:
- read-across source
- Key result
- Species / strain:
- S. typhimurium TA 98
- 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
- Key result
- Species / strain:
- S. typhimurium 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
- Key result
- 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
- Key result
- Species / strain:
- S. typhimurium TA 1535
- 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
- Key result
- Species / strain:
- S. typhimurium TA 1537
- 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
- Conclusions:
- No study is available on in vitro genetic toxicity of yttrium metal. Therefore, read across is performed using a study from the related substance yttrium oxide. In this reverse gene mutation assay in bacteria, strains TA1535, TA1537, TA98, TA100 and TA102 of S. typhimurium were exposed to yttrium oxide at concentrations of 0 to 5000 µg/plate in the presence and absence of mammalian metabolic activation [plate co-incubation and pre-incubation]. The positive controls induced the appropriate responses in the corresponding strains. There was no evidence of induced mutant colonies over background in each strain with and without metabolic activation. The same is assumed for yttrium metal. The read across justification is attached to IUCLID Section 13.
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- from 2005-10-21 to 2006-01-06
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
- Version / remarks:
- Cited as Directive 2000/32/EC, B.13/14
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- bacterial reverse mutation assay
- Target gene:
- histidine
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
- Metabolic activation:
- with and without
- Metabolic activation system:
- Liver S9 fraction of rats induced with phenobarbital/ß-naphthoflavone
- Test concentrations with justification for top dose:
- Pre-experiment (Experiment I): 3, 10, 33, 100, 333, 1000, 2500, and 5000 µg/plate
Experiment II: 33, 100, 333, 1000, 2500, and 5000 µg/plate
See table 1 in "Any other information on materials and methods incl. tables". - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The solvent was chosen because of its solubility properties and its relative non-toxicity to bacteria, and allows to obtain an homogeneous suspension.
- Vehicle controls tested: medium with solvent or vehicle alone
- Volume of vehicle/solvent in the medium: 100 µL/2600 µL medium - Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- Remarks:
- distilled water
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- sodium azide
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: 4-nitro-o-phenylenediamine
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- Remarks:
- distilled water
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- methylmethanesulfonate
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: 2-aminoanthracene
- Details on test system and experimental conditions:
- METHOD OF APPLICATION
- in agar (plate incorporation) in experiment I
- pre-incubation in experiment II
DURATION
- Pre-incubation period (experiment II): 60 minutes at 37°C
- Exposure duration: 48 hours at 37°C
NUMBER OF REPLICATES PER CONCENTRATION: 3
DETERMINATION OF CYTOTOXICITY: Toxicity of the test item can be evident as a reduction in the number of spontaneous revertants or a clearing of the bacterial background lawn.
OTHER
-Scoring method: The colonies were counted using Petri Viewer Mk2 (Perceptive Instruments Ltd, Suffolk CB 7BN, UK) with the software program Ames Study Manager. - Evaluation criteria:
- A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA98, TA100 and TA102) or thrice (strains TA1535 and TA1537) the colony count of the corresponding solvent control is observed.
A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.
An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.
A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant. - Statistics:
- not mandatory
- Key result
- Species / strain:
- S. typhimurium TA 98
- 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
- Key result
- Species / strain:
- S. typhimurium 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
- Key result
- 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
- Remarks:
- a minor toxic effect (below the indication factor of 0.5) was observed at 5000 µg/plate with S9 mix in Experiment I
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1535
- 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
- Key result
- Species / strain:
- S. typhimurium TA 1537
- 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 at 2500 and 5000 µg/plate, except in experiment II with metabolic activation, in which precipitation was observed only at 5000 µg/plate in strains TA1537, TA98, TA100 and TA102.
ADDITIONAL INFORMATION ON CYTOTOXICITY:
A minor toxic effect (below the indication factor of 0.5) was observed in strain TA102 at 5000 µg/plate with metabolic activation in experiment I. No toxic effects were observed in experiment II.
COMPARISON WITH HISTORICAL CONTROL DATA:
The laboratory´s historical control range was exceeded in the untreated and solvent control of strain TA102 without metabolic activation in experiment I and with metabolic activation in experiment II. These deviations are judged to be based on biologically irrelevant fluctuations in the number of colonies and have no impact on the outcome of the study.
See detailed results in Table 2 and 3 in the field "Any other information on results incl. tables". - Conclusions:
- In this reverse gene mutation assay in bacteria, strains TA1535, TA1537, TA98, TA100 and TA102 of S. typhimurium were exposed to yttrium oxide at concentrations of 0 to 5000 µg/plate in the presence and absence of mammalian metabolic activation [plate co-incubation and pre-incubation]. The positive controls induced the appropriate responses in the corresponding strains. There was no evidence of induced mutant colonies over background in each strain with and without metabolic activation.
- Endpoint:
- in vitro cytogenicity / chromosome aberration study in mammalian cells
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- key study
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Justification for type of information:
- Data from the related substance yttrium oxide is used to cover this endpoint. The justification for read across is attached in IUCLID Section 13.
- Reason / purpose for cross-reference:
- read-across source
- Key result
- Species / strain:
- lymphocytes:
- 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
- Remarks on result:
- other: not clastogenic
- Conclusions:
- No study is available on in vitro genetic toxicity of yttrium metal. Therefore, read across is performed using a study from the related substance yttrium oxide. In this in vitro cytogenicity / chromosome aberration study in mammalian cells the test item yttrium oxide did not induce structural chromosomal aberrations in human lymphocyte cells. Therefore, yttrium oxide is considered to be non-clastogenic in this chromosome aberration test. The same is assumed for yttrium metal. The read across justification is attached to IUCLID Section 13.
- Endpoint:
- in vitro cytogenicity / chromosome aberration study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 2013-02-13 - 2014-07-03
- 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:
- Blood samples were obtained from healthy donors not receiving medication. For this study (in each experiment) blood was collected from two donors. Due to organizational purposes. This deviation did not influence the quality or integrity of the study.
- GLP compliance:
- yes
- Type of assay:
- in vitro mammalian chromosome aberration test
- Species / strain / cell type:
- lymphocytes:
- Details on mammalian cell type (if applicable):
- Blood samples were obtained from healthy donors not receiving medication. For this study (in each experiment) blood was collected from two donors.
Blood samples were drawn by venous puncture and collected in heparinized tubes. Before use the blood was stored under sterile conditions at 4 °C for a maximum of 4 h. - Metabolic activation:
- with and without
- Metabolic activation system:
- Male Wistar or male Sprague Dawley rats were induced with phenobarbital and b-naphthoflavone
- Test concentrations with justification for top dose:
- The cultures were treated at each concentration. The following concentrations were used in the main experiments:
Experiment I:
Without and with metabolic activation, 4 h treatment, 24 h preparation interval:
0.3, 0.6, 1.2, 2.5, 5.0, 8.0 and 10.0 mM
Experiment II:
Without metabolic activation, 24 h treatment, 24 h preparation interval:
0.6, 1.2, 2.5, 5.0, 7.5 and 10.0 mM
With metabolic activation, 4 h treatment, 24 h preparation interval:
0.5, 1.0, 2.0, 4.0, 7.0 and 10.0 mM - Vehicle / solvent:
- The test item was suspended in DMSO (1.0% v/v) and diluted in RPMI prior to treatment.
- Untreated negative controls:
- yes
- Remarks:
- treatment medium
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- Positive controls:
- yes
- Remarks:
- EMS: Ethylmethanesulfonate 400 and 600 µg/mL respectively, diluted in nutrient media without metabolic activation. CPA: cyclophosphamide 5 µg/mL diluted in nutrient media with metabolic activation.
- Positive control substance:
- cyclophosphamide
- ethylmethanesulphonate
- Details on test system and experimental conditions:
- Exposure:
Cells were exposed by a suspension of the tested substance.
Pre-experiment:A pre-experiment was conducted at 10 mM of Yttrium Oxide and the relative mitotic index was assessed as parameter for toxicity. The concentrations evaluated in the main experiment are based on the results obtained in the pre-experiment
Preparation of the cultures:
At least 2 h before harvesting, Colcemid was added to the cultures as spindle inhibitor.
The cultures were harvested by centrifugation 24 h after beginning of treatment.
The supernatant was discarded and the cells were resuspended in hypotonic solution). The cell suspension was incubated at room temperature for 20 min.
Fixation and stained:
After removal of the hypotonic solution cells were fixed with 3+1 methanol + glacial acetic acid. The fixation procedure was repeated twice.
Slides were prepared by dropping the cell suspension onto a clean microscopic slide.
The cells were stained with Giemsa and according to the Fluorescent plus Giemsa technique, respectively.
Scoring:
At least, if available, 200 well spread metaphases per concentration and validity controls were scored for cytogenetic damage.
All slides, including those of positive and negative controls were independently coded before microscopic analysis. Evaluation of the cultures was performed using microscopes with 100x oil immersion objectives.
Cytotoxicity effect determination:
To describe a cytotoxic effect the mitotic index (% cells in mitosis) is determined. Additionally the number of polyploid cells was scored. - Evaluation criteria:
- The chromosomal aberration assay is considered acceptable if it meets the following criteria:
- the number of aberration found in the negative and/or solvent controls falls within the range of historical laboratory control data: 0.0% - 4.0% (without and with metabolic activation)
- the positive control substance should produce biologically relevant increases in the number of cells with structural chromosome aberrations.
There are several criteria for determining a positive result:
- a clear and dose-related increase in the number of cells with aberrations,
- a biologically relevant response for at least one of the dose groups, which is higher than the laboratory negative control range (0.0% - 4.0% aberrant cells (without and with metabolic activation)).
According to the OECD guidelines, the biological relevance of the results is the criterion for the interpretation of results, a statistical evaluation of the results is not regarded as necessary. However, for the interpretation of the data both, biological and if evaluated statistical significance should be considered together.
A test item is considered to be negative if there is no biologically relevant increase in the percentages of aberrant cells above concurrent control levels, at any dose group. - Key result
- Species / strain:
- lymphocytes:
- 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:
- Precipitation of the test item was noted in experiment I and II without and with metabolic activation in all evaluated concentrations.
In both experiments no biologically relevant increase of the aberration rates was noted after treatment with the test item without and with metabolic activation. The aberration rates of all dose groups treated with the test item were within the historical control data of the negative control - Remarks on result:
- other: not clastogenic
- Conclusions:
- In conclusion, it can be stated that during the described in vitro chromosomal aberration test and under the experimental conditions reported, the test item yttrium oxide did not induce structural chromosomal aberrations in human lymphocyte cells.
Therefore, yttrium oxide is considered to be non-clastogenic in this chromosome aberration test. - Executive summary:
A chromosome aberration assay was carried out in order to investigate a possible potential of yttrium oxide for its ability to induce structural chromosome aberrations in Human lymphocytes.
The metaphases were prepared 24 h after start of treatment with the test item. The treatment interval was 4 h with and without metabolic activation (experiment I) and 4 h with and 24 h without metabolic activation (experiment II). Two parallel cultures were set up. Per culture 100 metaphases were scored for structural chromosomal aberrations (for exceptions see Tables).
The following concentrations were evaluated:
Experiment I:
Without and with metabolic activation, 4 h treatment, 24 h preparation interval:
2.5, 5.0, and 10.0 mM
Experiment II:
Without metabolic activation, 24 h treatment, 24 h preparation interval:
0.6, 1.2, and 2.5 mM
With metabolic activation, 4 h treatment, 24 h preparation interval:
1.0, 2.0, and 4.0 mM
Precipitation of the test item was noted in experiment I and II without and with metabolic activation in all evaluated concentrations.
In experiment I and II no biologically relevant decreases of proliferation index were observed.
In experiment I and II with and without metabolic activation, the number of aberrant cells found in the cultures treated with the test item did not show a biologically relevant increase compared to the corresponding negative control. In addition, no dose-response relationship was observed.
In experiment II with metabolic activation only 140 instead of 200 cells could be evaluated due to precipitation at a concentration of 2 mM. However, the assessment of genotoxicity was still possible as at the higher concentration of 4 mM 200 cells could be evaluated.
EMS (400 and 600 µg/mL, respectively) and CPA (5 µg/mL) were used as positive controls and induced distinct and biologically relevant increases in cells with structural chromosomal aberrations, thus proving the ability of the test system to indicate potential clastogenic effects.
No biologically relevant increase in the frequencies of polyploid cells was found after treatment with the test item.
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- key study
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Justification for type of information:
- Data from the related substance yttrium oxide is used to cover this endpoint. The justification for read across is attached in IUCLID Section 13.
- Reason / purpose for cross-reference:
- read-across source
- Key result
- 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:
- not applicable
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Conclusions:
- No study is available on in vitro genetic toxicity of yttrium metal. Therefore, read across is performed using a study from the related substance yttrium oxide. In this described study (Klimisch 1) in vitro cell gene mutagenicity test under the experimental conditions reported, the item yttrium oxide is considered to be non-mutagenic in the HPRT locus using V79 cells of the chinese hamster. The same is assumed for yttrium metal. The read across justification is attached to IUCLID Section 13.
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 2012-06-21 to 2013-07-15
- 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:
- Due to the relative insolubility of the test item and the assumption of a related not or low toxic effect no pre-experiment was performed. The deviation did not influence the quality or integrity of the study.
- GLP compliance:
- yes
- Type of assay:
- mammalian cell gene mutation assay
- Target gene:
- HPRT
- Species / strain / cell type:
- Chinese hamster lung fibroblasts (V79)
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9 liver microsomal fraction
- Test concentrations with justification for top dose:
- Exposure concentrations
The test item was investigated at the following concentrations:
Experiment 1:
without metabolic activation:
0.00316 / 0.0100 / 0.0316 / 0.100 / 0.316 / 1.0 / 2.5 /5.0 / 7.5 and 10 mM
and with metabolic activation:
0.00316 / 0.0100 / 0.0316 / 0.100 / 0.316 / 1.0 / 2.5 / 7.5 and 10 mM
Experiment II:
without metabolic activation:
0.00316 / 0.0100 / 0.0316 / 0.100 / 0.316 / 1.0 / 2.5 / 5.0 and 7.5 mM
with metabolic activation:
0.025 / 0.05 / 0.1 / 0.2 / 0.4 / 0.8 / 1.6 / 3.2 / 6.4 and 10 mM
According to OECD guidelines at least 8 concentrations of the test item were set up in experiments with and without metabolic activation. - Vehicle / solvent:
- The substance was suspended at the right concentration in the cell culture medium.
- Untreated negative controls:
- yes
- Remarks:
- treatment medium, duplicate cultures
- Negative solvent / vehicle controls:
- no
- Remarks:
- as no solvent was used no additional solvent control was necessary
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 7,12-dimethylbenzanthracene
- ethylmethanesulphonate
- Remarks:
- The dilution of the stock solution of the positive controls were prepared on the day of the experiment and used immediately. The stability of both positive control substances in solution is proven by the mutagenic response in the expected range.
- Details on test system and experimental conditions:
- - METHOD OF APPLICATION: in suspension
- DURATION
Experiment I with and without metabolic activation and experiment II with metabolic activation were performed as 4h00 short term exposure assay. Experiment II without metabolic activation was performed as 20h00 long-term exposure assay.
- Expression time: 7 days
- Selection time: 7 to 10 days in selection medium (6-TG)
- Fixation and staining time: Day 13 to 18
- SELECTION AGENT: thioguanine (6-TG) 11 µm/mL
- FIXATION: in methanol,
- STAIN: in Giemsa.
- DETERMINATION OF CYTOTOXICITY: cloning efficiency
The cloning efficiency is calculated as follows:
CE [50 %]= [mean of colonies (dose group)/200] x 100
The mutation rate is calculated as follows:
Mutants per 10^6 cells= [mean number of mutants (dose group) / (400000 * CE[%] / 100 (dose group)] * 10^6 - Evaluation criteria:
- Acceptability of the assay:
A mutation assay is considered acceptable if meets the following criteria:
- negative and/or solvent controls fall within the performing laboratories historical control data range: 1 - 39 mutants/10^6 cells.
-S9 +S9
Mean 15 13
Min 1 2
Max 39 39
SD 9,9 8,8
RSD [%] 68 66
n= 52 51
S9: metabolic activation
Mean: mean of mutants/10^6 cells
Min: minimum of mutants/10^6 cells
Max.: maximum of mutants/10^6 cells
SD: standard deviation
RSD: relative standard deviation
n: number of control values
- The absolute cloning efficiency:
({number of positive cultures x 100] / total number of seeded cultures) of the negative and/or solvent control is above 50 %.
- The positive controls (EMS and DMBA) induce significant increases (at least 3-fold increase of mutant frequencies related to the comparable negative control values and higher than the historical range of negative controls) in the mutant frequencies.
Evaluation of results:
A test is considered to be negative if there is no biological relevant increase in the number of mutants:
There are several criteria for determining a positive result:
- a reproducible three times higher mutation frequency than the solvent control for at least one of the concentrations
- a concentration related increase of the mutation frequency; such an evaluation may be considered also in the case of three-fold increase of the mutant frequency is not observed
- if there is by chance a low spontaneous mutation rate in the corresponding negative and solvent controls a concentration related increase of the mutations within their range has to be discussed. - Statistics:
- According to the OECD guidelines, the biological relevance of the results is the criterion for the interpretation of results, a statistical evaluation of the results is not regarded as necessary.
- Key result
- 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:
- not applicable
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- The test item Yttrium Oxide was assessed for its potential to induce gene mutations at the HPRT locus using V79 cells of the Chinese hamster. The main experiments were carried out without and with metabolic activation.
In experiment I: (with and without metabolic activation) 10 mM was selected as the highest concentrations.
In experiment II : 7.5 mM (without metabolic activation) and 10 mM (with metabolic activation) were selected as the highest concentrations.
Most of the experiments were performed as 4h short-time exposure only experiment II was performed as 20 h long time exposure assay (without metabolic activation).
Precipitation: Precipitation of the test item was noted in the whole experiment I at concentrations of 1.0 mM and higher and in experiment II at concentrations of 1.0 mM and higher (without metabolic activation) and at concentrations of 0.8 mM and higher (with metabolic activation).
Toxicity (Experiment II results presented in Table 1 and 3): A biologically relevant growth inhibition (reduction of relative growth below 70%) was observed after the treatment with the test item in experiment I and II without metabolic activation. In experiment I without metabolic activation the relative growth was 41.6% for the highest concentration (10 mM) evaluated. The highest biologically relevant concentration evaluated with metabolic activation was 10 mM with a relative growth of 90.0%. In experiment II without metabolic activation the relative growth was 10.8% for the highest concentration (7.5 mM) evaluated. The highest concentration evaluated with metabolic activation was 10 mM with a relative growth of 79.2%.
Mutagenicity (Experiment II results presented in Table 2 and 4): In experiment I without metabolic activation most mutant values of the negative controls and test item concentrations found were within the historical control data of the laboratory test facility (about 1-39 mutants per 106cells). No dose-response relationship could be observed. The mutation frequencies found in the groups treated with the test item did not show a biologically relevant increase as compared to the negative controls. The highest mutation rate (compared to the negative control values) of 1.58 was found at a concentration of 5.0 mM with a relative growth of 60.7%.With metabolic activation all mutant values of the negative controls, and test item concentrations found were within the historical control data of the laboratory test facility (about 2-39 mutants per 106cells). No dose-response relationship could be observed. The mutation frequencies found in the groups treated with the test item did not show a biologically relevant increase as compared to the negativecontrols. The highest mutation rate (compared to the negative control values) of 1.12 was found at a concentration of 0.100 mM with a relative growth of 96.7%.
In experiment II without metabolic activation most mutant values of the negative controls andtest item concentrations found were within the historical control data of the laboratory test facility (about 1-39 mutants per 106cells). No dose-response relationship could be observed. The mutation frequencies found in the groups treated with the test item did not show a biologically relevant increase as compared to the negative controls. The highest mutation rate (compared to the negative control values) of 1.79 was found at a concentration of 0.00316 mM with a relative growth of 106.5%.
In experiment II with metabolic activation all mutant values of the negative controls and test item concentrations found were within the historical control data of the laboratory test facility (about 2-39 mutants per 106cells). No dose-response relationship could be observed. The mutation frequencies found in the groups treated with the test item did not show a biologically relevant increase as compared to the negative controls. The highest mutation rate (compared to the negative control values) of 1.11 was found at a concentration of 0.05 mM with a relative growth of 95.6%. - Conclusions:
- In conclusion, in this described study (Klimisch 1) in vitro cell gene mutagenicity test under the experimental conditions reported, the item yttrium oxide is considered to be non-mutagenic in the HPRT locus using V79 cells of the chinese hamster.
- Executive summary:
The test item yttrium oxide was assessed for its potential to induce mutations at the HPRT locus using V79 cells of the Chinese Hamster.
The test item was grinded and suspended in cell culture medium and processed by ultrasound for 2 min and diluted prior to treatment.
Experiment I with and without metabolic activation and experiment II with metabolic activation were performed as a 4 h short-term exposure assay. Experiment II without metabolic activation was performed as 20 h long time exposure assay.
Test item was tested from 0.00316 to 7.5 mM with and without metabolic activation in experiment I and from 0.00316 to 7.5 mM without metabolic activation and from 0.025 to 10 mM with metabolic activation in experiment II.
Precipitation of the item was observed from 0.8 mM or 1 mM in each study.
DMBA and EMS were used as positive controls and showed distinct and biologically relevant effects in mutation frequency.
In both experiments no biologically relevant increase of mutants was found after treatment with the test item (with and without metabolic activation). No dose-response relationship was observed.
Referenceopen allclose all
Table 2: Number of revertants per plate in experiment I (mean of 3 plates) (plate incorporation)
|
TA 1535 |
TA 1537 |
TA 98 |
TA 100 |
TA 102 |
|||||||||||||||
Conc. |
- MA |
+ MA |
Precipit. |
Cytotox. (yes/no) |
- MA |
+ MA |
Precipit. |
Cytotox. (yes/no) |
- MA |
+ MA |
Precipit. |
Cytotox. (yes/no) |
- MA |
+ MA |
Precipit. |
Cytotox. (yes/no) |
- MA |
+ MA |
Precipit. |
Cytotox. (yes/no) |
0* |
21 |
33 |
no |
no |
12 |
16 |
no |
no |
32 |
42 |
no |
no |
127 |
152 |
no |
no |
495 |
539 |
no |
no |
Untreated |
17 |
25 |
no |
no |
7 |
16 |
no |
no |
34 |
39 |
no |
no |
136 |
165 |
no |
no |
536 |
593 |
no |
no |
3 |
19 |
34 |
no |
no |
7 |
19 |
no |
no |
35 |
40 |
no |
no |
140 |
147 |
no |
no |
463 |
585 |
no |
no |
10 |
25 |
33 |
no |
no |
14 |
18 |
no |
no |
33 |
37 |
no |
no |
171 |
151 |
no |
no |
443 |
561 |
no |
no |
33 |
28 |
25 |
no |
no |
12 |
21 |
no |
no |
36 |
41 |
no |
no |
143 |
151 |
no |
no |
465 |
557 |
no |
no |
100 |
23 |
25 |
no |
no |
12 |
19 |
no |
no |
34 |
40 |
no |
no |
146 |
163 |
no |
no |
451 |
553 |
no |
no |
333 |
20 |
28 |
no |
no |
12 |
22 |
no |
no |
31 |
43 |
no |
no |
135 |
151 |
no |
no |
456 |
552 |
no |
no |
1000 |
24 |
30 |
no |
no |
12 |
22 |
no |
no |
36 |
42 |
no |
no |
139 |
148 |
no |
no |
494 |
452 |
no |
no |
2500 |
19 |
25 |
yes |
no |
13 |
12 |
yes |
no |
26 |
26 |
yes |
no |
128 |
102 |
yes |
no |
468 |
273 |
yes |
no |
5000 |
19 |
18 |
yes |
no |
10 |
9 |
yes |
no |
23 |
21 |
yes |
no |
110 |
90 |
yes |
no |
346 |
219 |
yes |
yes |
NaN3 |
1446 |
1925 |
||||||||||||||||||
4-NOPD |
119 |
444 |
||||||||||||||||||
MMS |
4779 |
|||||||||||||||||||
2-AA |
318 |
387 |
2306 |
3229 |
2600 |
|||||||||||||||
*solvent control with DMSO
MA : metabolic activation
Table 3: Number of revertants per plate in experiment II (mean of 3 plates) (preincubation)
|
TA 1535 |
TA 1537 |
TA 98 |
TA 100 |
TA 102 |
|||||||||||||||
Conc. |
- MA |
+ MA |
Precipit. |
Cytotox. (yes/no) |
- MA |
+ MA |
Precipit. |
Cytotox. (yes/no) |
- MA |
+ MA |
Precipit. |
Cytotox. (yes/no) |
- MA |
+ MA |
Precipit. |
Cytotox. (yes/no) |
- MA |
+ MA |
Precipit. |
Cytotox. (yes/no) |
0* |
17 |
25 |
no |
no |
8 |
17 |
no |
no |
29 |
30 |
no |
no |
124 |
158 |
no |
no |
446 |
536 |
no |
no |
Untreated |
25 |
28 |
no |
no |
9 |
13 |
no |
no |
26 |
36 |
no |
no |
139 |
178 |
no |
no |
446 |
543 |
no |
no |
33 |
17 |
26 |
no |
no |
12 |
13 |
no |
no |
19 |
36 |
no |
no |
130 |
150 |
no |
no |
455 |
495 |
no |
no |
100 |
19 |
25 |
no |
no |
10 |
15 |
no |
no |
23 |
30 |
no |
no |
131 |
162 |
no |
no |
485 |
546 |
no |
no |
333 |
19 |
24 |
no |
no |
9 |
14 |
no |
no |
24 |
29 |
no |
no |
122 |
141 |
no |
no |
471 |
486 |
no |
no |
1000 |
25 |
26 |
no |
no |
10 |
16 |
no |
no |
23 |
33 |
no |
no |
125 |
140 |
no |
no |
452 |
447 |
no |
no |
2500 |
21 |
25 |
yes |
no |
6 |
9 |
yes/no |
no |
25 |
36 |
yes/no |
no |
122 |
153 |
yes/no |
no |
473 |
443 |
yes/no |
no |
5000 |
19 |
32 |
yes |
no |
6 |
10 |
yes |
no |
21 |
22 |
yes |
no |
118 |
128 |
yes |
no |
439 |
402 |
yes |
no |
NaN3 |
1393 |
1944 |
||||||||||||||||||
4-NOPD |
101 |
364 |
||||||||||||||||||
MMS |
1597 |
|||||||||||||||||||
2-AA |
223 |
191 |
1154 |
1938 |
2533 |
|||||||||||||||
*solvent control with DMSO
MA : metabolic activation
Summary: Experiment I and II, without metabolic activation
| Dose Group | Concentration [mM] | Relative mitotic Index [%] | Proliferation Index | Mean % Aberrant cells incl. Gaps | Mean % Aberrant cells excl. Gaps | Historical Laboratory Negative Control Range | |
| Experiment I 4h treatment 24h preparation interval | C | 0 | 97 | 1.22 | 5.0 | 3.0 | 0.0 - 4.0 % aberrant cells |
| S | 0 | 100 | 1.16 | 3.0 | 2.5 | 0.0 - 4.0 % aberrant cells | |
| 4 | 2.5 | 86 | 1.16 | 3.0 | 2.0 | 0.0 - 4.0 % aberrant cells | |
| 5 | 5.0 | 75 | 1.12 | 3.0 | 1.0 | 0.0 - 4.0 % aberrant cells | |
| 7 | 10.0 | 66 | 1.08 | 1.0 | 0.5 | 0.0 - 4.0 % aberrant cells | |
| EMS | 600 µg/mL | 90 | - | 9.5 | 7.0 | 0.0 - 4.0 % aberrant cells | |
| Experiment II 4h treatment 24h preparation interval | C | 0 | 130 | 1.85 | 2.5 | 0.5 | 0.0 - 4.0 % aberrant cells |
| S | 0 | 100 | 1.82 | 2.0 | 2.0 | 0.0 - 4.0 % aberrant cells | |
| 1 | 0.6 | 98 | 1.81 | 0.5 | 0.5 | 0.0 - 4.0 % aberrant cells | |
| 2 | 1.2 | 88 | 1.72 | 1.0 | 0.0 | 0.0 - 4.0 % aberrant cells | |
| 3 | 2.5 | 83 | 1.74 | 1.0 | 0.5 | 0.0 - 4.0 % aberrant cells | |
| EMS | 400 µg/mL | 65 | - | 11.0 | 9.5 | 0.0 - 4.0 % aberrant cells |
The mitotic index was determined in 1000 cells per culture of each test group.
The relative values of the mitotic index are related to the solvent controls.
C: negative control (Culture medium)
S: Solvent control (DMSO)
EMS: Ethylmethanesulfonate
Summary: Experiment I and II, with metabolic activation
| Dose Group | Concentration [mM] | Relative mitotic Index [%] | Proliferation Index | Mean % Aberrant cells incl. Gaps | Mean % Aberrant cells excl. Gaps | Historical Laboratory Negative Control Range | |
| Experiment I 4h treatment 24h preparation interval | C | 0 | 113 | 1.18 | 4.0 | 2.0 | 0.0 - 4.0 % aberrant cells |
| S | 0 | 100 | 1.09 | 3.0 | 1.5 | 0.0 - 4.0 % aberrant cells | |
| 4 | 2.5 | 95 | 1.13 | 1.0 | 0.5 | 0.0 - 4.0 % aberrant cells | |
| 5 | 5.0 | 79 | 1.22 | 1.5 | 0.0 | 0.0 - 4.0 % aberrant cells | |
| 7 | 10.0 | 65 | 1.15 | 1.0 | 0.5 | 0.0 - 4.0 % aberrant cells | |
| EMS | 5 µg/mL | 101 | - | 12.5 | 11.5 | 0.0 - 4.0 % aberrant cells | |
| Experiment II 4h treatment 24h preparation interval | C | 0 | 83 | 1.53 | 2.0 | 0.5 | 0.0 - 4.0 % aberrant cells |
| S | 0 | 100 | 1.57 | 2.0 | 1.05 | 0.0 - 4.0 % aberrant cells | |
| 2 | 1.0 | 66 | 1.59 | 2.5 | 0.5 | 0.0 - 4.0 % aberrant cells | |
| 3 | 2.0 | 66 | 1.59 | 0.7 | 0.0 | 0.0 - 4.0 % aberrant cells | |
| 4 | 4.0 | 41 | 1.57 | 1.5 | 0.5 | 0.0 - 4.0 % aberrant cells | |
| EMS | 5 µg/mL | 69 | - | 11.0 | 10.5 | 0.0 - 4.0 % aberrant cells |
The mitotic index was determined in 1000 cells per culture of each test group.
The relative values of the mitotic index are related to the solvent controls.
C: negative control (Culture medium)
S: Solvent control (DMSO)
EMS: Ethylmethanesulfonate
Table 1:Experiment II - Toxicity, without metabolic activation
Dose Group |
Concen-tration [mM] |
Cell Density [cells/mL]a |
Relative Growth [%]a |
Number of cells per flask |
Cloning Efficiencyb [%] |
||
I |
II |
mean |
|||||
NC1 |
0 |
1230000 |
100 |
138 |
115 |
127 |
63 |
NC2 |
1250000 |
140 |
147 |
144 |
72 |
||
1 |
0.00316 |
1320000 |
106.5 |
136 |
145 |
141 |
70 |
2 |
0.0100 |
1320000 |
106.5 |
113 |
128 |
121 |
60 |
3 |
0.0316 |
1180000 |
95.2 |
133 |
139 |
136 |
68 |
4 |
0.100 |
1130000 |
91.1 |
111 |
131 |
121 |
61 |
5 |
0.316 |
942000 |
76.0 |
114 |
117 |
116 |
58 |
6 |
1.0 |
641000 |
51.7 |
126 |
126 |
126 |
63 |
7 |
2.5 |
384000 |
31.0 |
116 |
122 |
119 |
60 |
8 |
5.0 |
194000 |
15.6 |
112 |
128 |
120 |
60 |
9 |
7.5 |
134000 |
10.8 |
108 |
90 |
99 |
50 |
EMS |
300 µg/mL |
832000 |
67.1 |
96 |
111 |
104 |
52 |
NC: negative control / medium control
a: cell density and relative growth at 1stsubcultivation
b: mean value of cells per flask/200
EMS: Ethylmethanesulfonate [300 µg/ml]
Table 2: Experiment II – Mutagenicity, without metabolic activation
Dose Group |
Concen-tration [mM] |
Number of mutant colonies per flaska |
Mean |
SD |
Mutant colonies per 106cellsb |
Mutation factor |
||||
I |
II |
III |
IV |
V |
||||||
NC1 |
0 |
2 |
3 |
4 |
6 |
11 |
5.2 |
3.19 |
20.55 |
|
NC2 |
7 |
7 |
10 |
10 |
12 |
9.2 |
1.94 |
32.06 |
||
1 |
0.00316 |
11 |
12 |
15 |
11 |
17 |
13.2 |
2.40 |
46.98 |
1.79 |
2 |
0.0100 |
9 |
10 |
10 |
12 |
13 |
10.8 |
1.47 |
44.81 |
1.70 |
3 |
0.0316 |
5 |
7 |
8 |
8 |
8 |
7.2 |
1.17 |
26.47 |
1.01 |
4 |
0.100 |
4 |
7 |
5 |
9 |
12 |
7.4 |
2.87 |
30.58 |
1.16 |
5 |
0.316 |
2 |
3 |
4 |
5 |
8 |
4.4 |
2.06 |
19.05 |
0.72 |
6 |
1.0 |
4 |
6 |
9 |
9 |
11 |
7.8 |
2.48 |
30.95 |
1.18 |
7 |
2.5 |
1 |
5 |
5 |
6 |
7 |
4.8 |
2.04 |
20.17 |
0.77 |
8 |
5.0 |
0 |
1 |
2 |
4 |
4 |
2.2 |
1.60 |
9.17 |
0.35 |
9 |
7.5 |
0 |
3 |
5 |
5 |
5 |
3.6 |
1.96 |
18.18 |
0.69 |
EMS |
300 µg/mL |
211 |
191 |
187 |
181 |
186 |
191.2 |
10.40 |
923.67 |
35.11 |
NC: negative control / medium control
a: number of mutant colonies in flask I to V
b: mean mutant colonies x 106/ (400000 x Cloning Efficiency/100)
EMS: Ethylmethanesulfonate [300 µg/ml]
Table 3: Experiment II - Toxicity, with metabolic activation
Dose Group |
Concen-tration [mM] |
Cell Density [cells/mL]a |
Relative Growth [%]a |
Number of cells per flask |
Cloning Efficiencyb [%] |
||
I |
II |
mean |
|||||
NC1 |
0 |
865000 |
100 |
129 |
143 |
136 |
68 |
NC2 |
932000 |
138 |
149 |
144 |
72 |
||
1 |
0.025 |
801000 |
89.1 |
110 |
140 |
125 |
63 |
2 |
0.05 |
859000 |
95.6 |
135 |
145 |
140 |
70 |
3 |
0.1 |
787000 |
87.6 |
134 |
151 |
143 |
71 |
4 |
0.2 |
795000 |
88.5 |
154 |
168 |
161 |
81 |
5 |
0.4 |
751000 |
83.6 |
128 |
140 |
134 |
67 |
6 |
0.8 |
739000 |
82.2 |
146 |
152 |
149 |
75 |
7 |
1.6 |
869000 |
96.7 |
113 |
124 |
119 |
59 |
8 |
3.2 |
825000 |
91.8 |
116 |
122 |
119 |
60 |
9 |
6.4 |
759000 |
84.5 |
147 |
150 |
149 |
74 |
10 |
10 |
712000 |
79.2 |
145 |
150 |
148 |
74 |
DMBA |
1.0 µg/mL |
547000 |
60.9 |
126 |
136 |
131 |
66 |
DMBA |
1.5 µg/mL |
398000 |
44.3 |
104 |
120 |
112 |
56 |
NC: negative control / medium control
a: cell density and relative growth at 1stsubcultivation
b: mean value of cells per flask/200
DMBA: 7,12-Dimethylbenz(a)anthracene
Table 4: Experiment II – Mutagenicity, with metabolic activation
Dose Group |
Concen-tration [mM] |
Number of mutant colonies per flaska |
Mean |
SD |
Mutant colonies per 106cellsb |
Mutation factor |
||||
I |
II |
III |
IV |
V |
||||||
NC1 |
0 |
3 |
3 |
5 |
7 |
9 |
5.4 |
2.33 |
19.85 |
|
NC2 |
6 |
7 |
8 |
8 |
13 |
8.4 |
2.42 |
29.27 |
||
1 |
0.025 |
0 |
1 |
3 |
5 |
5 |
2.8 |
2.04 |
11.20 |
0.46 |
2 |
0.05 |
3 |
6 |
8 |
8 |
13 |
7.6 |
3.26 |
27.14 |
1.11 |
3 |
0.1 |
1 |
3 |
3 |
7 |
9 |
4.6 |
2.94 |
16.14 |
0.66 |
4 |
0.2 |
3 |
5 |
6 |
9 |
10 |
6.6 |
2.58 |
20.50 |
0.83 |
5 |
0.4 |
0 |
1 |
1 |
2 |
3 |
1.4 |
1.02 |
5.22 |
0.21 |
6 |
0.8 |
1 |
1 |
3 |
5 |
4 |
2.8 |
1.60 |
9.40 |
0.38 |
7 |
1.6 |
0 |
1 |
2 |
3 |
4 |
2.0 |
1.41 |
8.44 |
0.34 |
8 |
3.2 |
0 |
2 |
7 |
10 |
12 |
6.2 |
4.58 |
26.05 |
1.06 |
9 |
6.4 |
4 |
7 |
8 |
9 |
10 |
7.6 |
2.06 |
25.59 |
1.04 |
10 |
10 |
2 |
5 |
7 |
7 |
7 |
5.6 |
1.96 |
18.98 |
0.77 |
DMBA |
1.0 µg/mL |
78 |
91 |
94 |
75 |
106 |
88.8 |
11.27 |
338.93 |
13.80 |
DMBA |
1.5 µg/mL |
108 |
137 |
121 |
121 |
104 |
118.2 |
11.62 |
527.68 |
21.48 |
NC: negative control / medium control
a: number of mutant colonies in flask I to V
b: mean mutant colonies x 106/ (400000 x Cloning Efficiency/100)
DMBA: 7,12-Dimethylbenz(a)anthracene
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Genetic toxicity in vivo
Endpoint conclusion
- Endpoint conclusion:
- no study available
Additional information
No genetic toxicity study with the test substance yttrium metal. Data from the related substance yttrium oxide was used for endpoint coverage in a read across approach justification for this read across approach is included in section 13). The potential of yttrium oxide (source substance for the read-across) to induce genetic toxicity was assessed in three in vitro studies performed according to OECD Guidelines and in compliance with GLP. All studies are used as Key studies and scored Klimisch 1.
In vitro mutagenicity in bacteria
A reverse gene mutation assay in bacteria was conducted according to EU Method B.13/14 guideline and in compliance with GLP (Sokolowski A, 2006). This study was scored as reliability 1 according to Klimisch criteria. Strains TA1535, TA1537, TA98, TA100 and TA102 of S. typhimurium were exposed to yttrium oxide (99.36 %), at concentrations of 0 - 5000 µg/plate in the presence and absence of mammalian metabolic activation [plate co-incubation and pre-incubation].
Yttrium oxide was tested up to limit concentration (5000 µg/plate). The positive controls induced the appropriate responses in the corresponding strains. There was no evidence of induced mutant colonies over background in each strain with and without metabolic activation.
In vitro mammalian chromosome aberration test
The test was conducted according to OECD 473 and with GLP compliance and was scored 1 according to Klimisch criteria. Blood samples were obtained from healthy donors not receiving medication. For this study (in each experiment) blood was collected from two donors. Blood samples were drawn by venous puncture and collected in heparinized tubes. The cultures were treated at each concentration. The following concentrations were used in the main experiments:
- Experiment I: Without and with metabolic activation, 4 h treatment, 24 h preparation interval: 0.3, 0.6, 1.2, 2.5, 5.0, 8.0 and 10.0 mM
- Experiment II:
Without metabolic activation, 24 h treatment, 24 h preparation interval:
0.6, 1.2, 2.5, 5.0, 7.5 and 10.0 mM
With metabolic activation, 4 h treatment, 24 h preparation interval:
0.5, 1.0, 2.0, 4.0, 7.0 and 10.0 mM
The cells were treated in experiment I (without and with metabolic activation) for 4 h with the test item. The metaphases were prepared 24 h after the treatment. In experiment II without metabolic activation the cells were treated for 24 h and prepared at the end of the treatment. In experiment II with metabolic activation the cells were treated for 4 h and prepared 24 h after the treatment. The dose group selection for microscopic analyses of chromosomal aberrations are based on the mitotic index in accordance with the guidelines.
In experiment I and II no biologically relevant decreases of proliferation index were observed.
In experiment I and II with and without metabolic activation, the number of aberrant cells found in the cultures treated with the test item did not show a biologically relevant increase compared to the corresponding negative control. In addition, no dose-response relationship was observed.
In experiment II with metabolic activation only 140 instead of 200 cells could be evaluated due to precipitation at a concentration of 2 mM. However, the assessment of genotoxicity was still possible as at the higher concentration of 4 mM 200 cells could be evaluated.
EMS (400 and 600 µg/mL, respectively) and CPA (5 µg/mL) were used as positive controls and induced distinct and biologically relevant increases in cells with structural chromosomal aberrations, thus proving the ability of the test system to indicate potential clastogenic effects.
No biologically relevant increase in the frequencies of polyploid cells was found after treatment with the test item.
In vitro gene mutation study in mammalian cells (HPRT assay)
This test was scored 1 according to Klimisch criteria as it was conducted according to OECD 476 and in compliance with GLP. The test item yttrium oxide was assessed for its potential to induce mutations at the HPRT locus using V79 cells of the Chinese Hamster. Experiment I with and without metabolic activation and experiment II with metabolic activation were performed as a 4 h short-term exposure assay. Experiment II without metabolic activation was performed as 20 h long time exposure assay.
The test item was investigated at the following concentrations as single cultures:
- Experiment I
without metabolic activation:
0.00316, 0.0100, 0.0316, 0.100, 0.316, 1.0, 2.5, 5.0, 7.5 and 10 mM
and with metabolic activation:
0.00316, 0.0100, 0.0316, 0.100, 0.316, 1.0, 2.5, 7.5 and 10 mM
- Experiment II
without metabolic activation:
0.00316, 0.0100, 0.0316, 0.100, 0.316, 1.0, 2.5, 5.0 and 7.5 mM
and with metabolic activation:
0.025, 0.05, 0.1, 0.2, 0.4, 0.8, 1.6, 3.2, 6.4 and 10 mM
Precipitation of the test item was noted in experiment I at concentrations of 1.0 mM and higher (with and without metabolic activation). In experiment II precipitation was found at concentrations of 1.0 mM and higher (without metabolic activation) and at concentrations of 0.8 mM and higher (with metabolic activation). Biologically relevant growth inhibition was observed in experiment I and II without metabolic activation. Inexperiment I without metabolic activation the relative growth was 41.6% for the highest concentration (10 mM) evaluated. The highest biologically relevant concentration evaluated with metabolic activation was 10 mM with a relative growth of 90.0%. In experiment II without metabolic activation the relative growth was 10.8% for the highest concentration (7.5 mM) evaluated. The highest concentration evaluated with metabolic activation was 10 mM with a relative growth of 79.2%.
In both experiments no biologically relevant increase of mutants was found after treatment with the test item (with and without metabolic activation). No dose-response relationship was observed.
Justification for classification or non-classification
No reliable genetic toxicity study is available with yttrium metal. Data from three reliable in vitro studies with the related substance yttrium oxide are used for endpoint coverage. The justification for read across is attached in Section 13.
Based on the negative results of the three mentioned genetic toxicity tests, yttrium oxide does not need to be classified according to GHS (Regulation (EU) 1272/2008). The same is assumed for yttrium metal.
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