Registration Dossier

Toxicological information

Genetic toxicity: in vitro

Currently viewing:

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:
2003-07-16 to 2003-12-22
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Referenceopen allclose all

Reference Type:
study report
Title:
Unnamed
Year:
2003
Report date:
2003
Reference Type:
publication
Title:
Genotoxicity Evaluation of Sodium Tungstate Dihydrate and Tungsten Powder.
Author:
Reddy G, McCain WC, Leach GJ.
Year:
2007
Bibliographic source:
The Toxicologist, Vol.96, No. 1, March 2007

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (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:
Disodium wolframate
EC Number:
236-743-4
EC Name:
Disodium wolframate
Cas Number:
13472-45-2
Molecular formula:
Na2WO4
IUPAC Name:
disodium dioxotungstenbis(olate)
Details on test material:
- Name of test material (as cited in study report): Sodium tungstate dihydrate
- Molecular weight (if other than submission substance): 329.9
- Substance type: Active
- Physical state: White, crystalline powder
- Storage condition of test material: room temperature

Method

Species / strain
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
- Type and identity of media: The CHO cells were grown in McCoy's 5a culture medium, which was supplemented
with approx. 10% heat-inactivated fetal bovine serum (FBS), L-glutatnine (2mM), penicillin G (100 units/mL), and streptomycin (100 ug/mL).
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
Additional strain / cell type characteristics:
other: permanent cell line with an average cycle time of 12 to 14 hours and a modal chromosome number of 21
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254 induced rat liver S9
Test concentrations with justification for top dose:
Initial Assay (3 hour treatment with and without S9)-23.7, 33.9, 48.4, 69.2, 98.9, 141, 202, 288, 412, 588, 840, 1200, 1720, 2450, and 3500 ug/ml
Confirmatory assay (3 hour treatment with S9)-500, 1000, 1400, 2100, 2880, and 3500 ug/ml
Confirmatory assay (20 hour treatment without S9)-250, 500, 1000, 1400, 2100, 2800, and 3500 ug/ml

Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Cell culture grade water
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:
Non-activated test system Migrated to IUCLID6: 0.750 and 1.50 ug/mL, for the 3-hour treatment, 0.200 and 0.400 ug/mL, for 20-hour treatment
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
Activated test system Migrated to IUCLID6: 7.50 and 12.5 ug/mL, initial assay; 7.50 ug/mL, confirmatory assay
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: Initial assay (3 hours in the presence and absence of S9); Confirmatory assay (3 hours in the presence of S9 and 20 hours in the absence of S9)
- Fixation time (start of exposure up to fixation or harvest of cells): 20 hours with Colcemid present during the last 2 +/- 0.5 hours
SPINDLE INHIBITOR (cytogenetic assays): Colcemid
STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: 100 hundred cells, when possible, from each replicate were anlyzed for the different types of chromosomal aberrations. At least 25 cells were analyzed from those cultures that had greater than 25 % of cells with one or more aberrations.

DETERMINATION OF CYTOTOXICITY
- Method: Prior to the harvest of the cultures, visual observations of cytotoxicity were made. These observations included an assessment of the percent confluence of the cell monolayers within the culture flasks. The cultures were also evaluated for the presence of mitotic (large rounded cells) or dead cells floating in the medium; mitotic index

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: yes



Evaluation criteria:
Evaluation of a Positive Response- The test substance was considered positive for inducing chromosomal aberrations if a significant increase (the difference was considered significant when pEvaluation of a Negative Response- The test substance was considered negative for inducing chromosomal aberrations if no significant increase was observed in the number of cells with chromosomal aberrations at any of the concentrations.
Equivocal Evaluation- Although most assays give clearly positive or negative results, in rare cases the data set would preclude making a definitive judgment about the activity of the test substance. Results might remain equivocal or questionable regardless of the number of times the assay is repeated.
Statistics:
Statistical analysis employed a Cochran-Amitage test for linear trend and Fisher's Exact Test to compare the percentage of cells with aberrations in treated cells to the results obtained for the vehicle controls.
Statistical analysis was also performed for cells exhibiting polyploidy and/or endoreduplication in order to indicate significant (p

Results and discussion

Test results
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
In the 20 hour treatment confirmatory assay.
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: At a dose concentration of 20000 ug/mL, the pH was 8.5 (pH of the culture medium was 8.0) and at 3300 ug/mL, the pH was 8.0.
- Water solubility: In the cell culture grade water, the test substance formed transparent, colorless solutions at 33.0 and 200 mg/mL.
- Precipitation: At a dose concentration of 20000 ug/mL, no precipitate was observed and the culture medium became a slightly darker shade of red. At 3300 ug/mL, no precipitate was observed and no change in the appearance of the culture medium was observed.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
1. Initial assay in the absence of S9 with a 3-hour treatment period: A slight reduction in the number of dividing cells was visible. Reductions of 0 and 8 % were observed in the mitotic indices of the cultures treated with 2450 and 3500 ug/ml, respectively, as compared with the vehicle control cultures.
2. Initial assay in the presence of S9 with a 3-hour treatment period: Reductions of 0 and 6 % were observed in the mitotic indices of the cultures treated with 2450 and 3500 ug/ml, respectively, as compared with the vehicle control cultures.
3. Confirmatory assay in the absence of S9 with a 20-hour treatment period: A slight reduction in dividing cells was visible at 500 and 1000 ug/ml and a reduction was visible at 1400-3500 ug/ml. Monolayer confluence as compared to the vehicle control for 1000, 1400, 2100, 2800, and 3500 ug/ml were 71, 57, 57, 57, and 57 %, respectively. Reductions of 29, 60, 77, 86, 82, 91, and 100 % were observed in the mitotic indices of the cultures treated with 250, 500, 1000, 1400, 2100, 2800, and 3500 ug/ml, respectively, as compared with the vehicle control cultures.
4. Confirmatory assay in the presence of S9 with a 3-hour treatment period- Reductions of 0 and 1 % were observed in the mitotic indices of the cultures treated with 2800 and 3500 ug/ml, respectively, as compared with the vehicle control cultures.

CHROMOSOME ABERRATIONS:
1. Initial assay in the absence of S9 with a 3-hour treatment period: Chromosomal aberrations were analyzed from the cultures treated with 1200, 1720, 2450, and 3500 ug/ml. No significant increase in cells with chromosomal aberrations, polyploidy, or endoreduplication was observed in the cultures analyzed.
2. Initial assay in the presence of S9 with a 3-hour treatment period: Chromosome aberrations were analyzed from the cultures treated with 1200, 1720, 2450, and 3500 ug/ml. No significant increase in cells with chromosome aberrations, polyploidy, or endoreduplication was observed in the cultures analyzed.
3. Confirmatory assay in the absence of S9 with a 20-hour treatment period-Chromosomal aberrations were analyzed from the cultures treated with 250, 500, and 1000 mg/ml. No significant increase in cells with chromosomal aberrations or polyploidy was observed in the cultures analyzed. A weakly significant increase in endoreduplication was observed in the cultures treated with 1000 ug/ml. There was no clear understanding of the mechanism or meaning of this induction. The weak increase was observed at a single toxic dose level and the significance was probably a statistical anomaly due to 0% endoreduplication in the vehicle controls. Thus, the significance of the observation of endoreduplication is debatable since the increase observed was probably related to toxicity and not to any potential of the test substance to inhibit mitotic processes.
4. Confirmatory assay in the presence of S9 with a 3-hour treatment period- Chromosomal aberrations were analyzed from the cultures treated with 1400, 2100, 2800, and 3500 ug/ml. No significant increase in the cells with chromosomal aberrations, polyploidy, or endoreduplication was observed in the cultures analyzed.
5. Positive control- Both positive controls induced chromosomal aberrations.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:
The test substance was considered negative for inducing stuctural chromosomal aberrations in CHO cells with and without metabolic activation.
Executive summary:

The objective of this in vitro assay was to evaluate the ability of sodium tungstate to induce chromosomal aberrations in cultured Chinese hamster ovary (CHO) cells with and without an exogenous metabolic activation system.

The test article was dissolved in cell culture grade water for the assay. The highest concentration tested in the assay was 3500 ug/mL, which is slightly >10 mM of Sodium tungstate dihydrate (molecular weight=329.86; 10 mM=3299 ug/mL), the high dose recommended for this assay by the OECD Testing Guidelines. All dosing was achieved using dosing volumes of 10% (100 uL/mL) and the vehicle control cultures were treated with 100 uL/mL of cell culture grade water.

In the initial chromosomal aberrations assay, the treatment period was for approx. 3 hours without and with metabolic activation. Cultures were harvested approx. 20 hours from the initiation of treatment. Concentrations of 23.7, 33.9, 48.4, 69.2, 98.9, 141, 202, 288, 412, 588, 840, 1200, 1720, 2450, and 3500 ug/mL were tested with and without metabolic activation. Cultures treated with concentrations of 1200, 1720, 2450, and 3500 ug/mL without and with metabolic activation were analyzed for chromosomal aberrations. No significant increase in cells with chromosomal aberrations, polyploidy, or endoreduplication was observed in the cultures analyzed.

In a confirmatory chromosomal aberrations assay, the treatment period was for approx. 20 hours without metabolic activation and approx. 3 hours with metabolic activation and the cultures were harvested approx. 20 hours from the initiation of treatment. Concentrations of 250, 500, 1000, 1400, 2100, 2800, and 3500 ug/mL, were tested without metabolic activation and 500, 1000, 1400, 2100, 2800, and 3500 ug/mL were tested with metabolic activation. Cultures treated with concentrations of 250, 500, and 1000 ug/mL without metabolic activation and 1400, 2100, 2800, and 3500 ug/mL with metabolic activation were analyzed for chromosomal aberrations. No significant increase in cells with chromosomal aberrations or polyploidy, and no biologically relevant increase in endoreduplication was observed in the cultures analyzed.

The test article, sodium tungstate, was considered negative for inducing structural chromosomal aberrations in CHO cells with and without metabolic activation.