Yttrium trinitrate

Genetic toxicity: in vitro

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

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

Test material

Test material information

Constituent 1

Reference substance name:

Yttrium trinitrate

EC Number:

233-802-6

EC Name:

Yttrium trinitrate

Cas Number:

10361-93-0

Molecular formula:

N3O9Y

IUPAC Name:

yttrium trinitrate

Test material form:

solid: particulate/powder

Details on test material:

- Name of test material: yttrium trinitrate hexahydrate
- Molecular formula: Y(NO3)3.6H2O
- Molecular weight: 383.01 g/mol
- Appearance: powder
- Storage condition of test material: room temperature (20 ± 5°C) in the dark under dry conditions
- Further information on test material confidential.

Method

Species / strain

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.

Controls

Controls 1

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

Controls 2

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.

Results and discussion

Test results

Test results 1

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

Test results 2

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

Test results 3

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

Test results 4

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

Any other information on results incl. tables

- 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.

Applicant's summary and conclusion

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.