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Genetic toxicity in vitro

Description of key information

Genetic toxicity in vitro

Three key studies are available. In a bacterial reverse mutation test (Ames test, performed according to OECD Guideline 471, EU Method B.13/14 and EPA OPPTS 870.5100; Varga-Kanizsai, 2017b; Klimisch 1), yttrium trichloride hexahydrate was demonstrated to be negative for mutagenicity with and without metabolic activation under the conditions of the test. No reliable data have been identified on in vitro cytogenicity and gene mutation of yttrium trichloride in mammalian cells. Therefore, these endpoints have been covered using data from the read across substance yttrium trinitrate, which is an yttrium compound with similar water solubility as yttrium trichloride. In a chromosome aberration test in mammalian cells (performed according to OECD Guideline 473; Ciliutti, 2016; Klimisch 1), yttrium trinitrate was demonstrated to be non-clastogenic with and without metabolic activation under the conditions of the test. Finally, in a Mouse Lymphoma Assay (performed according to OECD Guideline 476 and EU method B.17; Getuli, 2015; Klimisch 1), yttrium trinitrate was demonstrated to be negative for mutagenicity with and without metabolic activation under the conditions of the test. These results are considered relevant for yttrium trichloride as well. The read across justification document is attached to IUCLID Section 13.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
14 September 2016 - 3 August 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
21 July 1997
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Version / remarks:
August 1998
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
30 May 2008
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Based on the results of the solubility test, a 100 mg/mL stock solution was prepared in distilled water, which was diluted by serial dilution to obtain dosing formulations for lower doses, spaced by factors of 2, 2.5 and approximately √10.
- No correction for purity of the test item was applied.
Target gene:
Histidine locus (S. typhimurium strains TA98, TA100, TA1535 and TA1537); tryptophan locus (E. coli strain WP2uvrA)
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
cofactor-supplemented post-mitochondrial S9 fraction (rat liver)
Test concentrations with justification for top dose:
Preliminary Concentration Range Finding Test: 5000, 2500, 1000, 316, 100, 31.6 and 10 μg/plate (TA98 and TA100).
Initial Mutation Test and Confirmatory Mutation Test: 5000, 1581, 500, 158.1, 50, 15.81, 5 and 1.581 µg/plate (all strains).
Complementary Confirmatory Mutation Test in Salmonella typhimurium TA98 without metabolic activation: 15.81, 5, 1.581, 0.5, 0.1581, 0.05, 0.01581, 0.005 and 0.001581.
Complementary Confirmatory Mutation Test in Salmonella typhimurium TA100, TA1535, TA1537 and Escherichia coli WP2 uvrA strains without metabolic activation: 15.81, 5, 1.581, 0.5, 0.1581, 0.05, 0.01581 and 0.005 µg/plate.

The selection of the doses was based on the results of the range finding study. No inhibitory, cytotoxic effect of the test item was observed in the range finding study. Test item precipitate and/or slight precipitate was observed on the plates at 5000, 2500, 1000 and 316 μg/plate concentrations in all examined strains with metabolic activation and at 5000, 2500, 1000, 316 and 100 μg/plate concentrations without metabolic activation in the preliminary experiment.

In the Confirmatory Mutation Test using the pre-incubation method, excessive cytotoxicity was observed in all examined bacterial strains without metabolic activation. In this case, the number of analysable doses did not meet the recommendations of the test guidelines. Therefore, an additional experiment (Complementary Confirmatory Mutation Test) was performed in these strains to complete the data. The experimental conditions were the same as in the Confirmatory Mutation Test, except of the tested concentrations.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Distilled water
- Justification for choice of solvent/vehicle: The solubility of the test item was examined using distilled water, dimethyl sulfoxide (DMSO) and N,N-dimethylformamide (DMF). At a concentration of 100 mg/mL a clear solution was observed using distilled water as vehicle and after approximately 3 minutes vortex a clear solution was observed using DMSO and DMF as vehicles. Due to the better biocompatibility, distilled water was selected as vehicle (solvent) for the study.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
sodium azide
other: 4-nitro-1,2-phenylenediamine
Remarks:
Salmonella TA98, non-activation, 4 µg
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
distilled water
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
Salmonella TA100 and TA1535, non-activation, 2 µg
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
Salmonella TA1537, non-activation, 50 µg
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
distilled water
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
E. coli WP2 uvrA, non-activation, 2 µL
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
All Salmonella strains, activation, 2 µg
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
E. coli WP2 uvrA, activation, 50 µg
Details on test system and experimental conditions:
METHOD OF APPLICATION
- Preliminary concentration range finding test / Initial mutation test: in agar (plate incorporation)
Bacteria (cultured in Nutrient Broth) were exposed to the test item both in the presence and absence of an appropriate metabolic activation system. Molten top agar was prepared and kept at 45°C. The equivalent number of minimal glucose agar plates (3 per concentration and for each control) was properly labelled. The test item and other components were prepared freshly and added to the overlay (45°C). The content of the tubes: top agar 2000 μL; vehicle or test item formulation (or reference controls) 50 μL; overnight culture of test strain 100 μL; phosphate buffer (pH 7.4) or S9 mix 500 μL. This solution was mixed and poured on the surface of minimal agar plates. For activation studies, instead of phosphate buffer, 0.5 mL of the S9 mix was added to each overlay tube. The entire test consisted of non-activated and activated test conditions, with the addition of untreated, negative (vehicle/solvent) and positive controls. After preparation, the plates were incubated at 37°C for 48 ± 1 hours.
- Confirmatory mutation test and complementary confirmatory mutation test: pre-incubation method
Bacteria (cultured in Nutrient Broth) were exposed to the test item both in the presence and absence of an appropriate metabolic activation system. The equivalent number of minimal glucose agar plates was properly labelled. Molten top agar was prepared and kept at 45°C. Before the overlaying, the test item formulation (or vehicle/solvent or reference control), the bacterial culture and the S9 mix or phosphate buffer were added into appropriate tubes to provide direct contact between bacteria and the test item. The tubes (3 tubes per control and 3 tubes for each concentration level) were gently mixed and incubated for 20 min at 37ºC in a shaking incubator. After the incubation period, 2 mL of molten top agar was added to the tubes, and then the content mixed and poured on the surface of minimal glucose agar plates. The entire test consisted of non-activated and activated test conditions, with the addition of untreated, negative and positive controls. After preparation, the plates were incubated at 37°C for 48 ± 1 hours.

DURATION
- Pre-incubation period: 48 h (confirmatory mutation test and complementary confirmatory mutation test)
- Exposure duration: 48 h
- Selection time (if incubation with a selection agent): 48 h (simultaneously with exposure duration)
- Fixation time (start of exposure up to fixation or harvest of cells): 48 h

SELECTION AGENT (mutation assays): histidine (S. typhimurium strains); tryptophan (E. coli strains)

NUMBER OF REPLICATIONS: triplicate

DETERMINATION OF CYTOTOXICITY
- Method: bacterial background inhibition; decrease in the number of revertant colonies
Evaluation criteria:
Criteria for a Positive Response:
A test item was considered mutagenic if:
- a concentration-related increase in the number of revertants occurs and/or;
- a reproducible biologically relevant positive response for at least one of the dose groups occurs in at least one strain with or without metabolic activation.

An increase was considered biologically relevant if:
- the number of reversions is more than two times higher than the reversion rate of the negative (solvent) control in Salmonella typhimurium TA98, TA100 and Escherichia coli WP2 uvrA bacterial strains;
- the number of reversions is more than three times higher than the reversion rate of the negative (solvent) control in Salmonella typhimurium TA1535 and TA1537 bacterial strains.

Criteria for a Negative Response:
A test article was considered non-mutagenic if:
- the total number of revertants in tester strain Salmonella typhimurium TA98, TA100 or Escherichia coli WP2 uvrA is not greater than two times the concurrent vehicle control, and the total number of revertants in tester strain Salmonella typhimurium TA1535 or TA1537 is not greater than three times the concurrent vehicle control;
- the negative response should be reproducible in at least one follow up experiment.
Statistics:
According to the guidelines, statistical methods may be used as an aid in evaluating the test results. However, statistical significance should not be the only determining factor for a positive response.
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
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:
cytotoxicity
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:
cytotoxicity
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:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation:
Test item precipitate and/or slight precipitate was detected in the Initial Mutation in Salmonella typhimurium TA98, TA100 and TA1537 at 5000, 1581, 500 and 158.1 μg/plate concentrations without metabolic activation, in Salmonella typhimurium TA1535 and Escherichia coli WP2 uvrA at 5000, 1581, 500 μg/plate concentrations without metabolic activation, in Salmonella typhimurium TA98, TA1537 and E. coli WP2 uvrA at 5000, 1581 and 500 μg/plate concentrations with metabolic activation, and in Salmonella typhimurium TA100 and TA1535 at 5000 and 1581 μg/plate concentrations with metabolic activation.
In the Confirmatory Mutation Test test item precipitate and/or slight precipitate was observed in Salmonella typhimurium TA1537 and E. coli WP2 uvrA with metabolic activation at 5000, 1581 and 500 μg/plate concentrations. In addition to these concentrations this effect was observed in Salmonella typhimurium TA100 and TA1535 on the plates at 158.1 μg/plate concentration and in Salmonella typhimurium TA98 on the plates at 158.1 and 50 μg/plate concentrations. In the Complementary Confirmatory Mutation Test the same effect was detected in all examined bacterial strains without metabolic activation on the plates at 15.81 and 5 μg/plate concentrations. In addition to these concentrations, this effect was observed at 1.581 μg/plate concentration in Salmonella typhimurium TA98 and TA1535.

CYTOTOXICITY
- In the Initial Mutation Test reduced and/or slightly reduced background lawn development was detected in Salmonella typhimurium TA98 and TA100 with and without metabolic activation on the plates at 5000 μg/plate concentration and on one plate at 1581 μg/plate concentration in Salmonella typhimurium TA100 with metabolic activation. Slightly reduced background lawn development was detected in Salmonella typhimurium TA1535 and TA1537 on the plates at 5000 μg/plate concentration with metabolic activation. No inhibitory, cytotoxic effect of the test item was observed on E. coli WP2 uvrA strain.
- The same effect was observed in the Confirmatory Mutation Test with metabolic activation in E. coli WP2 uvrA on the plates at 5000 μg/plate concentration, in Salmonella typhimurium TA100 and TA1535 on the plates at 5000 and 1581 μg/plate concentrations, and in Salmonella typhimurium TA98 and TA1537 on the plates at 5000, 1581 and 500 μg/plate concentrations.
- In the Complementary Confirmatory Mutation Test the same effect was detected without metabolic activation in Salmonella typhimurium TA100 and TA1537 and in E. coli WP2 uvrA on the plates at 15.81 and 5 μg/plate concentrations, in Salmonella typhimurium TA1535 on the plates at 15.81, 5 and 1.581 μg/plate concentrations, and in Salmonella typhimurium TA98 on the plates at 15.81, 5, 1.581 and 0.5 μg/plate concentrations.

RESULTS
- In the Initial Mutation Test, the highest revertant rate was observed in Salmonella typhimurium TA1537 bacterial strain at 50 μg/plate concentration with metabolic activation (the observed mutation factor value was 1.91). However, there was no clear dose-response relationship, the observed
mutation factor values were below the biologically relevant threshold limit and the numbers of revertant colonies were within the historical control range.
- In the Confirmatory Mutation Test and in the Complementary Confirmatory Mutation Test, the highest revertant rate was observed in Salmonella typhimurium TA1535 at 1581, 50 and 5 μg/plate concentrations with metabolic activation (the observed mutation factor value was 1.95). However, there was no clear dose-response relationship, the observed mutation factor values were below the biologically relevant threshold limit and the numbers of revertant colonies were within the historical control range.
- Higher numbers of revertant colonies compared to the vehicle (solvent) control were detected in the main tests in some other sporadic cases. However, no dose-dependence was observed in those cases and they were below the biologically relevant threshold value. The numbers of revertant colonies were within the historical control range in each case, so they were considered as reflecting the biological variability of the test.
- Sporadically, lower revertant counts compared to the vehicle (solvent) control were observed in the main tests at some non-cytotoxic concentrations. However, no background inhibition was recorded and the mean numbers of revertant colonies were in the historical control range in all cases, thus they were considered as biological variability of the test system.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%))
- Positive historical control data: The reference mutagens showed a distinct increase of induced revertant colonies. The viability of the bacterial cells was checked by a plating experiment in each test. The tests were considered to be valid.
- Untreated, negative (solvent/vehicle) and positive control plates were run concurrently. The mean values of revertant colony numbers of untreated, negative (solvent) and positive control plates were within the historical control range.

Validity of the tests

Untreated, negative (solvent) and positive controls were run concurrently. The mean values of revertant colony numbers of untreated, negative (solvent) and positive control plates were within the historical control range. At least five analysable concentrations were presented in all strains of the main tests. The selected dose range exhibited limited solubility as demonstrated by the preliminary range finding test and extended to 5 mg/plate. No more than 5% of the plates were lost through contamination or some other unforeseen event. The tests were considered to be valid.

Conclusions:
The reported data of this mutagenicity assay show that under the experimental conditions applied the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.
In conclusion, the test item yttrium trichloride hexahydrate had no mutagenic activity in the applied bacterium tester strains under the test conditions used in this study.
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:
No reliable studies have been identified on in vitro cytogenicity of yttrium trichloride in mammalian cells. Therefore, the endpoint is covered using data from the read across substance yttrium trinitrate, i.e. an yttrium compound with similar water solubility as yttrium trichloride. The read across justification document is attached to IUCLID Section 13.
Reason / purpose for cross-reference:
read-across source
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
severe toxicity at 2000 µg/mL; marked toxicity at 1000 µg/mL (relative PD 39%)
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
severe toxicity at 2000 and 1000 µg/mL, moderate toxicity at 500 µg/mL (relative PD 49%)
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
severe toxicity at 1500 and 1000 µg/mL; mild toxicity at 667 and 444 µg/mL (PD 68% and 71%)
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
severe toxicity at 1200 µg/mL; adequate toxicity at 1000 µg/mL (PD 42%)
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Experiment 1 / 3h-exposure
Remarks:
These results are obtained in an in vitro chromosome aberration test performed with yttrium trinitrate (Ciliutti, 2015). Yttrium trinitrate is an yttrium compound with similar water solubility as yttrium trichloride and therefore these results were considered relevant for yttrium trichloride as well.
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
Justification for type of information:
No reliable studies have been identified on in vitro gene mutation in mammalian cells for yttrium trichloride. Therefore, the endpoint is covered using data from the read across substance yttrium trinitrate, i.e. an yttrium compound with similar water solubility as yttrium trichloride. The read across justification document is attached to IUCLID Section 13.
Reason / purpose for cross-reference:
read-across source
Key result
Species / strain:
mouse lymphoma L5178Y cells
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: These results are obtained in an in vitro mouse lymphoma assay performed with yttrium trinitrate (Getuli, 2015).
Remarks:
Yttrium trinitrate is an yttrium compound with similar water solubility as yttrium trichloride and therefore these results were considered relevant for yttrium trichloride as well.
Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2015-07-14 to 2016-06-15
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:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
in vitro mammalian chromosome aberration test
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
- Chinese hamster ovary cells were obtained from Dr. A.T. Natarajan (State University of Leiden). This cell line derives from the CHO isolate originally described by Kao and Puck (1968).
- Type and identity of media: Culture medium: Ham's F10 (1X) 499 mL, Antibiotic solution 1.0 mL, Foetal Calf Serum 55.6 mL
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: no data
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital/5,6-benzoflavone induced rat liver S9
Test concentrations with justification for top dose:
Main experiment 1: 0, 7.81, 15.6, 31.3, 62.5, 125, 250*, 500*, 1000*, 2000 µg/mL (3h treatment; without S9); 0, 7.81, 15.6, 31.3, 62.5, 125*, 250*, 500*, 1000, 2000 µg/mL (3h treatment; with S9)
Main experiment 2: 0, 26.0, 39.0, 58.5, 87.8, 132, 198, 296*, 444*, 667*, 1000, 1500 µg/mL (20h treatment; without S9)
Main experiment 3: 0, 137, 206, 309, 463, 694, 833, 1000, 1200 µg/mL (20h treatment; without S9)
*Dose levels selected for scoring chromosome aberrations
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: A preliminary solubility trial was performed using DMSO concurrently with RTC Study No. A0646. This solvent was selected since it is compatible with the survival of the cells and the S9 metabolic activity. The test item was found soluble in DMSO at the concentration of 275 mg/mL.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Remarks:
CPA
Positive control substance:
cyclophosphamide
Remarks:
Main experiment 1, with S9, 15 µg/mL and 23 µg/mL, dissolved in sterile water
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Remarks:
MMC
Positive control substance:
mitomycin C
Remarks:
Main experiment 2 and 3, without S9, 0.100 µg/mL and 0.150 µg/mL, dissolved in sterile water
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium
- In the first experiment, both in the absence and presence of S9 mix, the cultures were incubated for 3 hours. At the end of treatment, the medium was removed and the flasks were washed twice with Phosphate Buffered Solution (PBS). Mitotic cells in the treatment medium were collected by centrifugation and added back to cultures in fresh medium.
- In the second and third experiment, in the absence of S9 metabolic activation, the treatment media were added to the flasks.

DURATION
- Exposure duration: 3h (experiment 1); 20h (experiment 2 and 3)
- Expression time (cells in growth medium): 14h (experiment 1); 0h (experiment 2 and 3)
- Selection time (if incubation with a selection agent): Not applicable
- Fixation time (start of exposure up to fixation or harvest of cells): 20h (approx. 1.5 cell cycle length, all experiments)

SPINDLE INHIBITOR (cytogenetic assays): Colcemid (0.2 µg/mL final concentration) was added for the last three hours of the recovery period

STAIN (for cytogenetic assays): 3% Giemsa in tap water, rinsed in tap and distilled water, and then made permanent with Eukitt

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: One thousand metaphases were examined for each replicate culture (2000 metaphases per experimental point). For each culture, 150 metaphase spreads per cell culture were scored to asses the frequency of aberrant cells.

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; Population doubling (relative % compared to concurrent control). Population doubling is the log of the ratio of the final cell count (N) to the starting (baseline) count (X0) divided by the log of 2. Required level of toxicity for scoring chromosome aberrations: reduction of PD to 45+/- 5% over the concurrent control.

OTHER EXAMINATIONS
- Determination of polyploidy: yes
- Determination of endoreplication: Yes. Polyploid and endoreduplicated cells encountered were recorded, but not included in the count of eligible metaphases.

OTHER: Harvesting was done by removing the medium from the flasks and bringing the cells into suspension by trypsinisation. The cell suspension was centrifuged and the cell pellet was resuspended in hypotonic solution. The cells were then fixed in freshly prepared methanol:acetic acid fixative and washed two times with fixative. A few drops of the cell suspension obtained in this way were dropped onto slides to produce metaphase chromosome spreads. For each culture three slides were prepared.
Evaluation criteria:
In this assay, the test item is considered as clearly positive if the following criteria are met:
- Any dose level shows a statistically significant increase in aberration-bearing cells (excluding gaps)
- The incidence of cells bearing aberrations is outside the normal distribution of historical control values
- The increase of cells bearing aberration is dose-related when evaluated with an appropriate trend test
The test item is considered clearly negative in this assay if none of the above criteria is met.
Statistics:
For the statistical analysis, Fisher’s Exact Test was used to compare the number of cells bearing aberrations (assumed to be Poisson distributed) in control and treated cultures. Bonferroni’s corrections were applied for multiple comparisons. The analysis was performed using sets of data either including or excluding gaps. Cochran-Armitage trend test (one-sided) was performed to aid determination of concentration response relationship. The percentage of cells bearing aberrations excluding gaps was considered for the evaluation of the outcome of the study.
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
severe toxicity at 2000 µg/mL; marked toxicity at 1000 µg/mL (relative PD 39%)
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
severe toxicity at 2000 and 1000 µg/mL, moderate toxicity at 500 µg/mL (relative PD 49%)
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
severe toxicity at 1500 and 1000 µg/mL; mild toxicity at 667 and 444 µg/mL (PD 68% and 71%)
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
severe toxicity at 1200 µg/mL; adequate toxicity at 1000 µg/mL (PD 42%)
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Following treatment with the test item, for the first main experiment, an increase of cells bearing aberrations, mainly represented by chromatid breaks, was observed in the presence of S9 mix at the intermediate dose level selected for scoring. A marked increase in the number of endoreduplicated cells was observed in one replicate culture from the highest dose level selected for scoring in the absence of S9. In order to better evaluate this result, an additional scoring was performed by examining slides from the solvent control and the high dose. One thousand metaphases were examined for each replicate culture (2000 metaphases per experimental point). Results obtained using a larger sample size confirmed an increase of endoreduplicated cells over the concurrent negative controls, at the highest dose level. Marked increases in the incidence of cells bearing aberrations were observed following treatments with the positive controls cyclophosphamide and mitomycin-C, indicating the correct functioning of the test system.

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: Following treatment with the test item, for all treatment series, a dose-related reduction of pH was observed at higher dose levels. However, pH values at the dose levels selected for metaphase analysis were deemed adequate since only pH changes higher than one unit are considered culture conditions leading to artifactual positive results.
- Effects of osmolality: No remarkable variation of osmolality was observed at any dose level, in the absence or presence of S9 mix.
- Precipitation: Opacity of the medium was observed when adding solutions at 275, 138, and 68.8 mg/mL (final concentrations of 2750, 1380 and 688 mg/mL, respectively). A clear medium was observed when adding a solution at 22.2 mg/mL.

RANGE-FINDING/SCREENING STUDIES
- Based on a preliminary solubility assay, dose levels of 2000, 1000, 500, 250, 125, 62.5, 31.3, 15.6 and 7.81 µg/mL were used for the short treatment time, both in the absence and presence of S9 metabolic activation (main experiment 1).
- On the basis of cytotoxicity observed in the first main experiment, dose levels selected for the second experiment were 1500, 1000, 667, 444, 296, 198, 132, 87.8, 58.5, 39.0 and 26.0 µg/mL.
- An additional experiment (main experiment 3) was performed using the continuous treatment and testing a narrow space interval in order to obtain the required level of toxicity for scoring chromosome aberrations (reduction of PD to 45 5% over the concurrent control). The following dose levels were used: 1200, 1000, 833, 694, 463, 309, 206 and 137 µg/mL.

COMPARISON WITH HISTORICAL CONTROL DATA
- Results show that the proportion of cells with structural aberrations (excluding gaps) in vehicle control cultures fell within the normal range based on historical control data.
- The positive control items, mitomycin-C and cyclophosphamide, induced statistically significant increases in the incidence of cells with structural aberrations compared with the concurrent negative control and the responses were compatible with the historical control range.
- A statistically significant increase (p < 0.05) in the incidence of cells bearing structural aberrations was observed at the intermediate dose level selected for scoring in the presence of S9 mix. However, it should be noted that the aberration frequency of the concurrent vehicle control was zero and the incidences observed at this dose level fell within the normal distribution of historical control values, therefore the increases were not considered biologically relevant.

ADDITIONAL INFORMATION ON CYTOTOXICITY: It should be noted that in both experiments 2 and 3, cytotoxic effects were more evident if measured as reduction of Mitotic Index rather than Population Doubling. This could be attributable to an effect on cell cycle progress at higher dose levels with a consequential cell synchronisation induced by the test item.
Remarks on result:
other: Experiment 1
Remarks:
3h-exposure

- Due to the high incidence of aberrant cells (excluding gaps), scoring was terminated at 75 metaphases for the cultures treated with the positive control mitomycin-C.

- The statistically significant increase in endoreduplicated cells at the highest dose level in the absence of S9 metabolism, may indicate that the test item has the potential to inhibit cell cycle progress.

- Foetal calf serum at a final concentration of 10% was used instead of 15% in order to improve cell growth. This did not influence the test reliability.

Conclusions:
On the basis of these results, it is concluded that yttrium trinitrate does not induce structural chromosome aberrations in Chinese hamster ovary cells after in vitro treatment, with and without metabolic activation, under the reported experimental conditions.
Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2015-03-30 to 2015-10-01
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
Qualifier:
according to guideline
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
in vitro mammalian cell gene mutation tests using the thymidine kinase gene
Target gene:
Thymidine kinase (TK) locus of L5178Y TK+/- mouse lymphoma cells
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
- Type and identity of media: see "Any other information on materials and methods incl. tables"
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically "cleansed" against high spontaneous background: yes, prior to use cells were cleansed of pre-existing mutants.

Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital/5,6-benzoflavone induced rat liver S9 tissue homogenate
Test concentrations with justification for top dose:
Cytotoxicity test:
10.7, 21.5, 43.0, 85.9, 172, 344, 688, 1380, 2750 µg/mL (with and without S9; both 3h and 24h treatments)

Mutation assay 1:
5.38, 10.8, 21.5, 43.0, 86.0, 172 µg/mL (with and without S9; 3h treatment)
Mutation assay 2:
5.38, 10.8, 21.5, 43.0, 86.0, 172 µg/mL (without S9; 24h treatment)
5.38, 10.8, 21.5, 43.0, 86.0, 172 µg/mL (with S9; 3h treatment)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: DMSO was selected since it is compatible with cell survival and S9 metabolic activity. In addition, many historical control data demonstrate no mutagenic effects induced by the chosen solvent.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Remarks:
MMS
Positive control substance:
methylmethanesulfonate
Remarks:
without S9, dissolved in sterile injectable water
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Remarks:
B(a)P
Positive control substance:
benzo(a)pyrene
Remarks:
with S9, dissolved in DMSO
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 3h and 24h
- Expression time (cells in growth medium): 8 days (cytotoxicity test); 2 days (mutation assay; plating for 5-TFT); 16 days (mutation assay; plating for viability)
- Selection time (if incubation with a selection agent): 14 days (mutation assay; plating for 5-TFT)
- Fixation time (start of exposure up to fixation or harvest of cells): 8-9 days (cytotoxicity test); 16-17 days (mutation assay)

SELECTION AGENT (mutation assays): 5-trifluorothymidine

NUMBER OF REPLICATIONS: A single culture was used at each test point (Cytotoxicity assay); duplicate cultures were prepared at each test point, with the exception of the positive controls which were prepared in a single culture (Mutation Assays).

NUMBER OF CELLS EVALUATED: During the expression period (two days after treatment), the cell populations were subcultured in order to maintain them in exponential growth. At the end of this period, the cell densities of each culture were determined and adjusted to give 2x10^5 cells/mL.
Wells containing clones were identified by eye using background illumination and counted. In addition, the number of wells containing large colonies as well as the number of those containing small colonies were scored.

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency; relative total growth

OTHER:
- Since negative results were obtained without metabolic activation, the second experiment in the absence of S9 metabolism was performed, using a longer treatment time (24 hours).
- The number of wells containing large colonies as well as the number of those containing small colonies were scored.
Evaluation criteria:
For a test item to be considered mutagenic in this assay, it is required that:
1. The induced mutant frequency (IMF) is higher than the global evaluation factor (GEF) suggested for the microwell method (126x10^-6) at one or more doses.
2. There is a significant dose-relationship as indicated by the linear trend analysis.
Results which only partially satisfy the above criteria will be dealt with on a case-by-case basis. Similarly, positive responses seen only at high levels of cytotoxicity will require careful interpretation when assessing their biological significance. Any increase in mutant frequency should lie outside the historical control range to have biological relevance.
Statistics:
Statistical analysis was performed according to UKEMS guidelines (Robinson W.D., 1990).
Key result
Species / strain:
mouse lymphoma L5178Y cells
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
- Effects of pH: A slight decrease of pH was observed at 172 µg/mL (without S9) and at 86 and 172 µg/mL (with S9). However, since this decrease is within the physiological values, it is not considered to affect the test system.
- Effects of osmolality: The addition of the test item solution did not have any obvious effect on the osmolality of the treatment medium.
- Precipitation:
*Cytotoxicity Test: dose-related cloudy appearance starting from 43.0 μg/mL (3h and 24h), dose-related precipitation starting from 85.9 µg/mL (3h treatment) and particles in suspension starting from 85.9 µg/mL (24h treatment)
*Mutation Assay 1: dose-related precipitation at 43, 86 and 172 µg/mL (with and without S9; 3h)
*Mutation Assay 2: dose-related precipitation at 86 and 172 µg/mL (without S9; 24h) and dose-related precipitation at 72 µg/mL (with S9; 3h)

RANGE-FINDING/SCREENING STUDIES: Upon addition of the test item to the cultures, a dose-related cloudy appearance of the treatment medium was noted starting from 43.0 μg/mL for all treatment series. At the end of the 3 hour treatment period, dose-related precipitation was observed starting from 85.9 μg/mL, while by the end of the 24 hour treatment period, at the same dose levels, particles in suspension were observed.
In the absence of S9 metabolic activation, using the 3 hour treatment time, no cells survived to treatment at the highest dose level tested (2750 μg/mL).
At the next lower dose level (1380 μg/mL) slight toxicity was observed reducing relative survival (RS) to 71% of the concurrent negative control value, while no toxicity was noted over the remaining concentrations tested. Using the 24 hour treatment time, no cells survived to treatment at 2750 μg/mL and moderate toxicity was observed at 1380 μg/mL reducing RS to 16% of the concurrent negative control value. Slight toxicity was observed at the two lower dose levels. Following treatment in the presence of S9 metabolic activation, using the short treatment time (3 hours), severe toxicity was seen at 2750 μg/mL (7% RS), while no toxicity was observed over the remaining dose levels tested.
In order to assay the test item beyond its limit of solubility, 172 μg/mL was selected as the highest concentration to be used in Mutation Assays.

COMPARISON WITH HISTORICAL CONTROL DATA
- The positive control items induced clear increases in mutant frequency (the difference between the positive and negative control mutant frequencies was greater than half the historical mean value).
- The mutant frequencies in the solvent control cultures fell within the historical control range.

MUTATION RESULTS
- No increases in mutant frequency were observed in the absence or presence of S9 metabolic activation, following treatment with the test item at any concentration level.
- Using the long treatment time in the absence of S9 metabolism, a linear trend was indicated, however no statistically significant and biologically relevant increase in mutant frequency was observed at any dose level. Hence, the linear trend indicated in both treatment series was considered to be attributable to a chance event.

ADDITIONAL INFORMATION ON CYTOTOXICITY
In the Cytotoxicity Test, no cells survived to treatment at the highest dose level in the absence of S9 (3h and 24h treatment) and severe toxicity was observed at the highest dose level in the presence of S9. Slight toxicity was observed at 1380 µg/mL (without S9, 3h treatment), 344 and 688 µg/mL (without S9, 24h treatment). In the presence of S9, only at the highest dose level toxicity was observed. In order to assay the test item beyond its limit of solubility, 172 μg/mL was selected as the highest concentration to be used in Mutation Assays.
In the Mutation Assays, no relevant toxicity was observed in the absence or presence of S9 metabolic activation, following treatment with the test item at any concentration level, in any experiment.

Colony sizing:

The small and large colony mutant frequencies were estimated and the proportion of small mutant colonies was calculated. An adequate recovery of small colony mutants was observed following treatment with the positive controls.

Validity

- The mutant frequencies in the solvent control cultures fell within the normal range (50-200 x 10^6 viable cells)

- The positive control items induced clear increases in mutant frequency (the difference between the positive and negative control mutant frequencies was greater than half the historical mean value)

- The cloning efficiencies at Day 2 in the negative control cultures fell within the range of 65 - 120%.

- The control growth factor over 2 days fell within the range of 8 - 32 in both experiments.

- Higher than acceptable heterogeneity of viability counts was observed between replicate cultures at the top concentration tested in Mutation Assay 2 in the presence of S9 metabolic activation, hence this dose level was excluded from statistical analysis. However, this did not affect the validity of the study.

The study was accepted as valid.

Conclusions:
It is concluded that yttrium trinitrate does not induce mutation at the TK locus of L5178Y mouse lymphoma cells in vitro in the absence or presence of S9 metabolic activation, under the reported experimental conditions.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Genetic toxicity in vitro

Three key studies are available. For in vitro cytogenicity and gene mutation in mammalian cells, no reliable data are available for yttrium trichloride. Therefore these endpoints have been covered using key information available for the read across substance yttrium trinitrate, which is an yttrium compound with similar water solubility as yttrium trichloride. The read across justification document is attached to IUCLID Section 13.

Bacterial reverse mutation assay

Varga-Kanizsai (2017b) performed an Ames test with Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2 uvrA with and without metabolic activation. The test item yttrium trichloride hexahydrate had no mutagenic activity in the applied bacterium tester strains under the test conditions used in this study. Solvent, negative and positive controls were valid. This study is considered reliable without restrictions (Klimisch 1) and was assigned key status for endpoint coverage.

In vitro chromosome aberration in mammalian cells

The test item yttrium trinitrate was investigated in a GLP-compliant chromosomal aberration test in Chinese hamster ovary cells (according to OECD Guideline 473; Ciliutti, 2016), following in vitro treatment in the presence and absence of S9 metabolic activation.

In the first experiment, the cells were treated with test item dissolved in DMSO at concentrations 2000, 1000, 500, 250, 125, 62.5, 31.3, 15.6 and 7.81 µg/mL for 3 hours in the presence and absence of S9 metabolic activation, respectively. The harvest time of 20 hours, corresponding to approximately 1.5 cell cycle, was used. In the second experiment, the cells were treated in the absence of S9 metabolic activation using a continuous treatment until harvest at 20 hours, at concentrations 1500, 1000, 667, 444, 296, 198, 132, 87.8, 58.5, 39.0 and 26.0 µg/mL.

Dose levels selected for scoring were as follows:

- 3-hour treatment without S9: 1000, 500 and 250 µg/mL.

- 3-hour treatment with S9: 500, 250 and 125 µg/mL.

- 20-hour treatment without S9: 667, 444 and 296 µg/mL.

Following treatment with the test item, a statistically significant increase in the incidence of cells bearing aberrations, including and excluding gaps (p<0.05) over the vehicle control values, was observed at the intermediate dose level selected for scoring in the presence of S9 metabolic activation. On the basis of the criteria for the outcome of the study, the increase was not considered biologically meaningful. A statistically significant increase in endoreduplicated cells was observed at the highest dose level using the 3-hour treatment time in the absence of S9 metabolic activation. This result may indicate that the test item has the potential to inhibit cell cycle progress. Statistically significant increases in the number of cells bearing aberrations (including and excluding gaps) were observed following treatments with the positive controls cyclophosphamide and mitomycin-C, indicating the correct functioning of the test system.

It is concluded that yttrium trinitrate does not induce structural chromosome aberrations in Chinese hamster ovary cells after in vitro treatment, under the reported experimental conditions.

This study was considered reliable without restrictions (Klimisch 1) and was assigned key status for endpoint coverage.

 

Mouse lymphoma assay

A key study (Getuli, 2015) investigated the induction of 5 trifluorothymidine resistant mutants in mouse lymphoma L5178Y cells after in vitro treatment with yttrium trinitrate, in the absence and presence of S9 metabolic activation, using a fluctuation method. The study was GLP compliant and performed according to OECD Guideline 476.

The test item, diluted in DMSO, was tested in two independent assays:

- 3h-treatment time with and without metabolic activation

- 24h-treatement time without metabolic activation and 3h-treatment with metabolic activation

The following dose levels were tested for the two assays: 172, 86.0, 43.0, 21.5, 10.8 and 5.38 µg/mL.

The expression period for the mutation assays was 2 days.

No increases in mutant frequencies were observed following treatment with the test item, in the absence or presence of S9 metabolic activation.

Negative and positive control treatments were included in each mutation experiment in the absence and presence of S9 metabolic activation. The mutant frequencies in the solvent control cultures fell within the historical control range. Marked increases were obtained with the positive control treatments indicating the correct functioning of the assay system.

It is concluded that yttrium trinitrate does not induce mutation at the TK locus of L5178Y mouse lymphoma cells in vitro in the absence or presence of S9 metabolic activation, under the reported experimental conditions.

This study was considered reliable without restrictions (Klimisch 1) and was assigned key status for endpoint coverage.

Justification for classification or non-classification

Based on the available in vitro data for yttrium trichloride and the read across substance yttrium trinitrate and according to the criteria of the CLP Regulation, yttrium trichloride should not be classified for genetic toxicity.