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Genetic toxicity in vitro

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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; Thompson, 2013; Klimisch 1), yttrium trinitrate was demonstrated to be negative for mutagenicity with and without metabolic activation under the conditions of the test. 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.

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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:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
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:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
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:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Between 22 August 2012 and 22 January 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Target gene:
Salmonella typhimurium
TA1537: his C 3076; rfa-; uvrB- (frame shift mutations)
TA98: his D 3052; rfa-; uvrB-; R-factor (frame shift mutations)
TA1535: his G 46; rfa-; uvrB- (base-pair mutations)
TA100: his G 46; rfa-; uvrB-; R-factor (base-pair mutations)

Escherichia coli
WP2 uvrA: trp-; uvrA- (base-pair substitution)
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
S9 microsomal fraction from rats induced with phenobarbitone/beta-naphthoflavone
Test concentrations with justification for top dose:
Preliminary toxicity test: 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate (yttrium trinitrate as active ingredient)
Mutation test - experiment 1:
WP2uvrA (with and without S9-mix):
15, 50, 150, 500, 1500 and 5000 µg/plate (yttrium trinitrate as active ingredient)
19, 64, 191, 637, 1500, 5000 µg/plate (based on test item (including a water content of 27.4%))
All Salmonella strains (without S9-mix):
0.15, 0.5, 1.5, 5, 15, 50, 150 µg/plate (yttrium trinitrate as active ingredient)
0.2, 0.6, 2, 6, 19, 64, 191 µg/plate (based on test item (including a water content of 27.4%))
All Salmonella strains (with S9-mix):
0.5, 1.5, 5, 15, 50, 150, 500 µg/plate (yttrium trinitrate as active ingredient)
0.6, 2, 6, 19, 64, 191, 637 µg/plate (based on test item (including a water content of 27.4%))

Mutation test - experiment 2:
WP2uvrA (with and without S9-mix):
15, 50, 150, 500, 1500, 5000 µg/plate (yttrium trinitrate as active ingredient)
19, 64, 191, 637, 1911, 6370 µg/plate (based on test item (including a water content of 27.4%))
All Salmonella strains (without S9-mix):
0.15, 0.5, 1.5, 5, 15, 50, 150 µg/plate (yttrium trinitrate as active ingredient)
0.2, 0.6, 2, 6, 19, 64, 191 µg/plate (based on test item (including a water content of 27.4%))
All Salmonella strains (with S9-mix):
0.5, 1.5, 5, 15, 50, 150, 500 µg/plate (yttrium trinitrate as active ingredient)
0.6, 2, 6, 19, 64, 191, 637 µg/plate (based on test item (including a water content of 27.4%))
Vehicle / solvent:
Vehicle(s)/solvent(s) used: sterile distilled water
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
N-ethyl-N-nitro-N-nitrosoguanidine
Remarks:
2 µg/plate for WP2uvrA, 3 µg/plate for TA100, 5 µg/plate for TA1535; without S9-mix
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
80 µg/plate for TA1537; without S9 mix
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 4-nitroquinoline-1-oxide
Remarks:
0.2 µg/plate for TA98; without S9 mix
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
1 µg/plate for TA100, 2 µg/plate for TA1535 and TA1537, 10 µg/plate for WP2uvrA; with S9-mix
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
5 µg/plate for TA98; with S9-mix
Details on test system and experimental conditions:
METHOD OF APPLICATION: plate incorporation (mutation test - experiment I) and pre-incubation method (mutation test - experiment II)

DURATION
- Pre-incubation period: 20 minutes (mutation test - experiment 2 - pre-incubation method)
- Exposure duration: 48 hours
- Selection time (if incubation with a selection agent): simultaneous with exposure

SELECTION AGENT (mutation assays): trace histidine or tryptophan supplemented

NUMBER OF REPLICATIONS: triplicate

DETERMINATION OF CYTOTOXICITY
- Method: reduction in number of revertant colonies and reduction of bacterial background lawn
- Preliminary toxicity test:
In order to select appropriate dose levels for use in the main test, a preliminary test was carried out to determine the toxicity of the test item. The concentrations tested were 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate (yttrium trinitrate as active ingredient). The test was performed by mixing 0.1 mL of bacterial culture (TA100 or WP2uvrA), 2 mL of molten, trace histidine or tryptophan supplemented, top agar, 0.1 mL of test item formulation and 0.5 mL of S9-mix or phosphate buffer and overlaying onto sterile plates of Vogel-Bonner Minimal agar (30 mL/plate). Ten concentrations of the test item formulation and a vehicle control (sterile distilled water) were tested. In addition, 0.1 mL of the maximum concentration of the test item and 2 mL of molten, trace histidine or tryptophan supplemented, top agar were overlaid onto a sterile nutrient agar plate in order to assess the sterility of the test item. After approximately 48 hours incubation at 37°C the plates were assessed for numbers of revertant colonies using an automated colony counter and examined for effects on the growth of the bacterial background lawn.

Evaluation criteria:
There are several criteria for determining a positive result. Any, one, or all of the following can be used to determine the overall result of the study:
1. A dose-related increase in mutant frequency over the dose range tested
2. A reproducible increase at one or more concentrations
3. Biological relevance against in-house historical control ranges
4. Statistical analysis of data as determined by UKEMS (Mahon et al., 1989)
5. Fold increase greater than two times the concurrent solvent control for any tester strain (especially if accompanied by an out-of-historical range response).

A test item will be considered non-mutagenic (negative) in the test system if the above criteria are not met.

Although most experiments will give clear positive or negative results, in some instances the data generated will prohibit making a definite judgement about test item activity. Results of this type will be reported as equivocal.
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
The test item was tested up to the maximum recommended dose level of 5000 µg/plate or the toxic limit, depending on bacterial strain type and presence or absence of S9-mix.
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
Remarks:
The test item was tested up to the maximum recommended dose level of 5000 µg/plate or the toxic limit, depending on bacterial strain type and presence or absence of S9-mix.
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
The test item was tested up to the maximum recommended dose level of 5000 µg/plate or the toxic limit, depending on bacterial strain type and presence or absence of S9-mix.
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
Remarks:
The test item was tested up to the maximum recommended dose level of 5000 µg/plate or the toxic limit, depending on bacterial strain type and presence or absence of S9-mix.
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
Remarks:
The test item was tested up to the maximum recommended dose level of 5000 µg/plate or the toxic limit, depending on bacterial strain type and presence or absence of S9-mix.
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
The test item caused a visible reduction in the growth of the bacterial background lawns of all of the tester strains (except Escherichia coli strain WP2uvrA dosed in the presence of S9-mix), initially from 50 and 150 µg/plate in the absence and presence of S9-mix respectively in both experiments. The test item was tested up to the maximum recommended dose level of 5000 µg/plate or the toxic limit, depending on bacterial strain type and presence or absence of S9-mix. A test item precipitate (particulate in appearance) was noted at 5000 µg/plate in both the absence and presence of S9-mix in each experiment, this observation did not prevent the scoring of the revertant colonies.

No significant increases in the frequency of revertant colonies were recorded for any of the strains of bacteria, at any dose level either with or without metabolic activation or exposure method.

All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies thus confirming the activity of the S9-mix and the sensitivity of the bacterial strains.
Conclusions:
The test item, yttrium trinitrate, was considered to be non-mutagenic under the conditions of the test, in the absence and presence of metabolic activation.
Endpoint:
in vitro DNA damage and/or repair study
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
no guideline followed
Principles of method if other than guideline:
Rec assay: experimental procedures of the rec assay with cold incubation were mainly based on those already described by Kada et al. (1972). In vitro and host-mediated "rec-assay" procedures for screening chemical mutagens, and phloxine, a mutagenic red dye detected. Mutation Research 16, 164-174. The REC assay may be one of the most sensitive systems to detect metal mutagens.
GLP compliance:
no
Type of assay:
Bacillus subtilis recombination assay
Species / strain / cell type:
bacteria, other: Bacillus subtilis strains H17 (Rec+, arg- try-) and M45 (Rec-, arg-, try-)
Metabolic activation:
without
Test concentrations with justification for top dose:
0.05 mL portion of metal solution (0.005 - 0.5 M)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: distilled water
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
not specified
True negative controls:
no
Positive controls:
other: references to mutagens detected as positive in rec assay
Positive control substance:
no
Details on test system and experimental conditions:
The two strains of B. subtilis H17 and M45 were grown overnight in B-2 broth (meat extract 10 g, polypeptone powder 10 g and NaCl 5 g in 1 L, pH adjusted to 7.0) and conserved at - 80°C with 12.5% glycerol supplementation. On the day of the experiments, each stock was melted and streaked radially from small pipettes onto B-2 agar. A 0.05 mL portion of each metal solution (0.005 - 0.5 M) was dropped onto a filter paper disk (diameter 10 mm), and the disk was placed on the starting point of the streak. The plates were kept at 4°C for 24h and then incubated at 37°C overnight. The above "cold incubation" inserted between drug administration and 37°C incubation increases the assay sensitivity about 20 - 50 times for many drugs. When the DNA damage is produced by a chemical and subjected to cellular recombination-repair function, the growth of recombination-deficient cells is usually inhibited much more than that of the wild cells. Many mutagens were efficiently detected among the rec-assay positive chemicals.
Evaluation criteria:
When a metal compound has the capacity to damage DNA, cellular growth of the rec deficient strain of B. subtilis must be more inhibited than that of the wild strain (rec proficient). Its DNA-damaging capacity can easily be predicted by measuring and comparing the inhibition zones.
Species / strain:
bacteria, other: B. subtilis strains H17 and M45
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not determined
Vehicle controls validity:
not examined
Untreated negative controls validity:
not examined
Positive controls validity:
not examined
Conclusions:
Yttrium trinitrate was observed to be negative in the rec assay with Bacillus subtilis strains H17 and M45, indicating that the substance would not damage DNA.
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 and one supporting study is available.

Bacterial reverse mutation assay

Thompson (2013) 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 caused a visible reduction in the growth of the bacterial background lawns of all the tester strains (except E. coli WP2 uvrA dosed in the presence of S9-mix), initially from 50 and 150 µg/plate with and without metabolic activation and in both experiments. Based on these observations, a suitable dose level series was established for each bacterial strain and each condition of metabolic activation (with or without) such that the test item was tested either up to cytotoxic concentration or up to the limit dose level. Solvent control, negative control and positive controls were also run. The test item did not cause increases in mutation frequency with and without metabolic activation in any bacterial strain at any of the dose levels tested. Solvent, negative and positive controls were valid. This study is considered reliable without restrictions (Klimisch 1) and was assigned key status for endpoint coverage.

Further studies in bacteria

In a supporting study (Klimisch 2), yttrium trinitrate was observed to be negative in the rec assay with Bacillus subtilis strains H17 and M45, indicating that the test substance would not damage DNA (Kanematsu et al., 1980).

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 and according to the criteria of the CLP Regulation, yttrium trinitrate should not be classified for genetic toxicity.