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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 19 February 2013 to 21 March 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Well documented GLP study performed according to OECD Guideline 473 and EU Method B.10.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report date:
2013

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test

Test material

Constituent 1
Chemical structure
Reference substance name:
[μ-[carbonato(2-)-O:O']]dihydroxydioxodizirconium
EC Number:
260-633-5
EC Name:
[μ-[carbonato(2-)-O:O']]dihydroxydioxodizirconium
Cas Number:
57219-64-4
Molecular formula:
CH2O7Zr2
IUPAC Name:
[μ-[carbonato(2-)-O:O']]dihydroxydioxodizirconium
Details on test material:
- Name of test material (as cited in study report): zirconium basic carbonate

Method

Species / strain
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
- Type and identity of media: Cultures were grown in Ham's F10 medium supplemented with 15% foetal bovine serum. All incubations were at 37°C in a 5% carbon dioxide atmosphere (100% humidity nominal).
- Properly maintained: yes
- Periodically "cleansed" against high spontaneous background: yes
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital and 5,6-Benzoflavone induced rat liver S9
Test concentrations with justification for top dose:
Main experiment 1: 5.35, 2.68, 1.34, 0.669, 0.334, 0.167, 0.0836, 0.0418, 0.0209 and 0.0104 mM (corresponding to 3080, 1540, 770, 385, 193, 96.3, 48.1, 24.1, 12.0 and 6.02 µg/mL of zirconium basic carbonate hydrate; corresponding to 1650, 825, 413, 206, 103, 51.6, 25.8, 12.9, 6.44 and 3.22 µg/mL of zirconium basic carbonate anhydrous)
Main experiment 2: 5.35, 3.06, 1.75, 0.998, 0.570, 0.326, 0.186, 0.106, 0.0608 and 0.0348 mM (corresponding to 3080, 1760, 1010, 575, 328, 188, 107, 61.3, 35.0 and 20.0 µg/mL zirconium basic carbonate hydrate; corresponding to 1650, 943, 539, 308, 176, 101, 57.4, 32.8, 18.8 and 10.7 µg/mL zirconium basic carbonate anhydrous)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
Controlsopen allclose all
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
without S9 at 0.450 and 0.300 µg/mL in experiment 1 and without S9 at 0.150 and 0.100 µg/mL in experiment 2
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
23.0 and 15.0 µg/mL with S9 in experiment 1
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 3 hours in experiment 1 with and without S9; 20 hours in experiment 2
- Expression time (cells in growth medium): No data
- Harvest of cells at 20 hours (approximately 1.5 cell cycle length)
- Fixation time (start of exposure up to fixation or harvest of cells): No data, two cultures were prepared at each test point. Air-dried slides were prepared from each culture and stained with 3% Giemsa.

STAIN (for cytogenetic assays): Giemsa 3%

NUMBER OF REPLICATIONS: duplicate

NUMBER OF CELLS EVALUATED: One hundred metaphase spreads were scored for chromosomal aberrations from each culture with the exception of replicate cultures treated with the positive control cyclophosphamide from the first experiment in the presence of S9 metabolism, where, due to the high incidence of aberrant cells (excluding gaps), scoring was terminated at 50 metaphases.

DETERMINATION OF CYTOTOXICITY
- Method: reduction of population doubling

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: yes
Evaluation criteria:
In this assay, the test item was considered to have clastogenic properties if the following criteria were all fulfilled:
- Statisticallly significant increases in the incidence of cells bearing aberrations were observed at any dose level over the current control.
- The increases exceeded the historical control values.
- The increases were reproduced in both replicate cultures.
The evaluation was based on the set of results, which excluded gaps.
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. The analysis was performed using sets of data either including or excluding gaps.

Results and discussion

Test results
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Details are specified in the field 'Additional information on results'
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: Following treatment with the test item, no remarkable variation of pH over the vehicle control values were observed at any dose level, in the absence or presence of S9 metabolism.
- Effects of osmolality: Following treatment with the test item, no remarkable variation of osmolality over the vehicle control values was observed at any dose level, in the absence and presence of S9 metabolism. It should be noted that an increase in osmolality was determined by the presence of DMSO in the treatment medium since the solvent depresses the freezing point. However, it passed freely in and out of cells, hence the increases were only apparent. A slight dose related reduction of the osmolality values over the vehicle control was observed at higher dose levels. These reductions were attributable to the DMSO displacement in the test item suspensions.
- Water solubility: Solubility of the test item was evaluated in a preliminary trial using sterile distilled water, culture medium, dimethylsulfoxide (DMSO), ethanol and acetone. These solvents were selected since they were compatible with the survival of the cells and the S9 metabolic activity. In addition, there were many historical control data demonstrating that no mutagenic effects were induced by the chosen solvents. The test item was not found to be soluble in any solvent. Solubility at low pH conditions was evaluated without better results. On the basis of these results and in agreement with the Study Monitor, it was decided to use the test item as homogeneous suspensions in DMSO obtaining the maximum final concentration of 5.35 mM.
- Precipitation: During the first main experiment, dose related precipitation was observed at the three highest dose levels at the beginning and by the end of treatment. During the second main experiment, upon addition of the test item to the cultures, slight dose related precipitation and opacity were observed at concentration levels between 5.35 and 0.570 mM. By the end of treatment, opacity of the treatment medium was observed at the three highest dose levels, while few particles of test item adherent to the flask surfaces were noted at all concentrations tested.

COMPARISON WITH HISTORICAL CONTROL DATA:
The frequency of cells bearing chromosomal aberrations in untreated and vehicle control cultures was within the laboratory historical control data.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
In the first main experiment, following treatment in the absence of S9 metabolism, mild toxicity was observed at the highest dose level (5.35 mM) where the relative population doubling was 63% of the concurrent vehicle control value. No relevant toxicity was observed over the remaining dose range.
In the presence of S9 metabolism, the highest dose level tested yielded slight toxicity reducting the relative population doubling value to 72% of the concurrent negative control. No toxicity was observed over the remaining dose range.

In the second main experiment, the maximum dose level of 5.35 mM yielded a marked cytotoxicity reducing the population doubling value to 17% of the control, while no relevant toxicity was noted at lower dose levels.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Following treatment with zirconium basic carbonate, no relevant increases over the concurrent vehicle control in the incidence of cells bearing aberrations were observed in any experiment.

A few polyploid or endoreduplicated cells were randomly observed in treated and control cultures. The incidences however were within the range of the laboratory historical control values for negative control.

Marked and biologically relevant increases in the frequency of cells bearing aberrations (including and excluding gaps) were seen in the cultures treated with the positive control substances, indicating the correct functioning of the assay system.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

On the basis of the results, it is concluded that zirconium basic carbonate does not induce structural chromosomal aberrations in Chinese hamster ovary cells after in vitro treatment with and without metabolic activation (S9), under the reported experimental conditions.