Ammonium wolframate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

read-across based on grouping of substances (category approach)

Adequacy of study:

key study

Study period:

2003-07-16 to 2003-12-22

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

The reliability of this study for the substance tested is a K1, but in application of read-across to a different substance ECHA’s guidance specifies that the score can be a maximum of K2. Due to similar water solubility and lower toxicity for the target substance (ammonium wolframate) compared to the source substance (sodium tungstate), the resulting read across from the source substance to the target substance is appropriate as a conservative estimate of potential toxicity for this endpoint. In addition, read across is appropriate because the classification and labeling is the more protective for the source substance than the target substance, the PBT/vPvB profile is the same, and the dose descriptors are, or are expected to be, conservative for the target substance. For more details refer to the attached description of the read across approach on Annex 3 in the CSR.

Justification for type of information:

1. HYPOTHESIS FOR THE CATEGORY APPROACH: The hypothesis is that properties are likely to be similar or follow a similar pattern because of the presence of a common metal ion, in this case tungstate.
2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES):
Source: Sodium tungstate
Target: Ammonium paratungstate
3. CATEGORY APPROACH JUSTIFICATION: See Annex 3 in CSR
4. DATA MATRIX: See Annex 3 in CSR

Cross-reference

Reason / purpose for cross-reference:

read-across: supporting information

Data source

Reference

Reference 1

Reference Type:

study report

Title:

Unnamed

Year:

2003

Report date:

2003

Reference 2

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

Test material information

Constituent 1

Reference substance name:

Disodium wolframate

EC Number:

236-743-4

EC Name:

Disodium wolframate

Cas Number:

13472-45-2

Molecular formula:

Na2O4W

IUPAC Name:

Disodium dioxido(dioxo)tungsten

Constituent 2

Reference substance name:

Sodium Tungstate

IUPAC Name:

Sodium Tungstate

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

Controls

Controls 1

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

Controls 2

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 monolayes 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

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

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:

No genetic toxicity data of sufficient quality were available specifically on ammonium paratungstate (target substance). However, genetic toxicity data is available on sodium tungstate (source substance), which are used for read-across.

Due to similar water solubility and lower toxicity for the target substance compared to the source substance, the resulting read-across from the source substance to the target substance is appropriate as a conservative estimate of potential toxicity for this endpoint. In addition, read-across is appropriate because the classification and labelling is more protective for the source substance than the target substance, the PBT/vPvB profile is the same, and the dose descriptors are, or are expected to be, lower for the source substance. For more details, refer to the read-across category approach description in the Category section of this IUCLID submission or Annex 3 of the CSR.