Disodium wolframate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

A summary of on the in vitro genotoxicity evidence is discussed below.

A. Bacterial Mutagenicity
Sodium tungstate in non-standard genotoxicity methods (e.g., bacterial bioluminescence) or in E. coli an accepted testing strain tested in bacterial SOS system (both studies scored a Klimisch scoring of 3) produces mutagenicity activity ranging from weakly positive to positive (Ulitzur S and Barak M 1988; Rossman, 1991). Whereas in more scientifically reliable in vitro studies (K1 score) conducted under GLP, following OECD 471 testing guidelines reported negative mutagenicity results in S. typhimurium and E. coli tests (Covance Laboratories Inc., 2004; Reddy et al. 2007). These negative results are supported by a limited bacterial mutagenicity study (K4) in three strains (two Salmonella strains and one E. coli) (US NTP, 2021). Overall, based on the results reported on higher quality studies, the bacterial mutagenic activity is negative.

B. Mammalian Mutagenicity
Sodium tungstate was weekly mutagenic in eucaryotic cells (yeast) (Singh,1983), but not in mammalian cells as it was negative in the mouse lymphoma assay (K1 score, OECD Guideline 476) (Covance Laboratories Inc., 2004; Reddy et al. 2007). Therefore, mammalian mutagenic activity is negative.

C. In vitro DNA damage and/or repair studies
Sodium tungstate produced disomic and/or diploid spores during meiosis of Saccharomyces cerevisiae (K3 scoring) (Sora et al., 1986) but did not cause sister chromatid exchanges in two Syrian hamster embryo cell studies or in human blood cells exposed up to 10 μg/mL sodium tungstate for 48-hrs (all these two studies received a K3 scoring) (Larramendy et al., 1981). There is one positive in vitro Comet assay study (K2 scoring) in primary murine bone marrow cell cultures (5–500 μg/mL) after 6 h exposure to sodium tungstate (Guilbert et al. 2011). Overall, the in vitro DNA damage and/or repair studies in mammalian cells is positive based on the higher reliable study.

D. In vitro cytogenicity-chromosomal aberration
No significant increase (with and without an exogenous metabolic activation system) in Chinese hamster ovary (CHO) cells (OECD 473, K1 score) with chromosomal aberrations or polyploidy, and no biologically relevant increase in endoreduplication was observed in the cultures analyzed after sodium tungstate exposure (highest concentration tested 3500 mg/L). Overall, the in vitro cytogenicity-chromosomal aberration in mammalian cells is negative.

 

Link to relevant study records

Reference

Reference 1

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:

test procedure in accordance with national standard methods with acceptable restrictions

Remarks:

No GLP Information or testing guideline employed. There is no OECD guideline for in vitro Comet assay

Qualifier:

no guideline available

Principles of method if other than guideline:

The extent of DNA strand breaks is a reflection of the Comet tail moment, a calculation that
takes into account the length, width and intensity of the tail with respect to the head of the Comet.

GLP compliance:

not specified

Type of assay:

comet assay

Target gene:

Alkaline Comet assay (single cell gel electrophoresis)

Species / strain / cell type:

primary culture, other: primary murine bone marrow-derived developing B lymphocytes

Details on mammalian cell type (if applicable):

85% of the non-adherent cells stained positive for the B-cell marker B220/CD45R

Additional strain / cell type characteristics:

other: BU-11 cells cultured on BMS2, or primary developing B cells

Species / strain / cell type:

other: BU-11 cells cultured on BMS2

Details on mammalian cell type (if applicable):

BU-11 cells cultured in rIL-7 in the absence of stromal cells.

Additional strain / cell type characteristics:

other: murine co-culture model composed of the BU-11 early pro/preB-cell line grown upon the BMS2 bone marrow stromal cell line

Cytokinesis block (if used):

Not specified

Metabolic activation:

not specified

Test concentrations with justification for top dose:

50, 100, 250 or 500 mg/ml tungsten 6 h

Vehicle / solvent:

Cultures were treated with vehicle control

Details on test system and experimental conditions:

Comet assays were performed, and the Comet tail moment was assessed in >50 cells per treatment

Evaluation criteria:

The alkaline Comet assay (single cell gel electrophoresis), where the extent of DNA strand breaks is a reflection of the Comet tail moment, a calculation that takes into account the length, width and intensity of the tail with respect to the head of the Comet.

Key result

Species / strain:

other: All cells tested

Metabolic activation:

not specified

Genotoxicity:

positive

Remarks:

In all cells tested, tungsten induced a significant increase in the Comet tail moment (DNA breaks). In primary proB cells, treatment with as low as 10 mg/ml tungsten for 6 h significantly increased the extent of DNA damage.

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Bu-11/BM52 Cytotoxicity at 250 and 500 mg/L BU-11/rIL-7 Cytotoxicity at 100, 250, 500 mg/L

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

True negative controls validity:

not specified

Positive controls validity:

not specified

Additional information on results:

Comet results were confirmed with western blotting experiments using antibodies against gH2AX, a phosphorylated histone variant that is induced upon DNA damage. Tungsten treatment for 6 h increased the expression of gH2AX in a dose-dependent manner in all cell types tested. Same low doses used in the Comet assays induced gH2AX expression in the primary proB cells.

Conclusions:

In vitro exposure to tungsten for 6 results in DNA damage.

Reference 2

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

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

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

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

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'.

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.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2003-07-17 to 2004-01-07

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

yes

Remarks:

The top concentration used in the assay (3500 ug/mL) is slightly above the recommended testing limit (10 mM) for the assay.

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Target gene:

Thymidine kinase locus

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media: The medium used for the study was RPMI 1640 supplemented with horse serum (10 % by volume), pluronic F68, L-glutamine, sodium pyruvate, penicillin and streptomycin. Treatment medium was Fischer's medium with the same medium supplements used in the culture medium except that the horse serum concentration was reduced to 5 % by volume. Cloning medium consisted of the RPMI 1640 culture medium with up to 20 % horse serum, without Pluronic F68 and with the addition of 0.24 % Noble agar to achieve a semi-solid state. Selection medium was cloning medium that contained 3 ug/mL of 5-trifluorothymidine.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes

Additional strain / cell type characteristics:

other: heterozygous at the TK locus

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254 induced rat liver S9

Test concentrations with justification for top dose:

62.5, 125, 250, 500, 1000, 1500, 2000, 2500, 3000 and 3500 ug/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Water

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

Non-activation assay Migrated to IUCLID6: 13 ug/mL

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: Methylcholanthrene-2 and 4 ug/mL

Remarks:

Activation assay

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium


DURATION
- Exposure duration: 4 hours
- Expression time (cells in growth medium): 2 days
- Selection time (if incubation with a selection agent): 14 days


SELECTION AGENT (mutation assays): 5-trifluorothymidine


NUMBER OF REPLICATIONS: 3


NUMBER OF CELLS EVALUATED: 3 x 10^6 cells


DETERMINATION OF CYTOTOXICITY
- Method: relative total growth


OTHER: Sizing Analysis- Both the small and large colonies were quantified for all cultures. A bimodal curve was generated and small and large colonies were quantitated by the areas under the curves. The large colonies presumably arose from point mutations and the small colonies from chromosome changes.

Evaluation criteria:

See below

Statistics:

no data

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS:
- Precipitation: The test substance formed a transparent, colorless solution in water at 35 mg/mL, the highest concentration prepared for use in the assay. The test substance remained soluble at all concentrations tested.

RANGE-FINDING/SCREENING STUDIES: Cells were treated with the test substance for approximately 4 hours in the presence and absence of S9 activation at concentrations ranging from 6.90-3500 ug/mL. Test substance concentration for the main gene mutation assay were chosen to cover a toxicity range from 10 % to 20 % survival to no apparent effect on growth compared to the vehicle control. If little or no toxicity was observed and solubility was maintained, the mutation experiment was initiated with a maximum concentration of 5 mg/mL or 10 mM (whichever was lowest). If precipitation of the test substance occurred in the culture medium, the maximum applied dose was at least twice the solubility limit in culture medium. In this assay, the high dose was slightly higher than the OECD recommended guideline of 10 mM.
In the non-activation and activation assays, the test substance induced no cytotoxicity to weak cytotoxicity up to and including 1750 ug/mL and moderate cytotoxicity at 3500 ug/mL.


COMPARISON WITH HISTORICAL CONTROL DATA: One of the vehicle control cultures for the initial assay in the absence of metabolic activation (144.8 x 10^-6) was slightly above historical data range (36.4 to 135.7 x 10^-6) for mutant frequency. In this same assay one of the positive control cultures (522.0 x 10^-6) was also above the historical data range (227.0 to 487.4 x 10^-6) for mutant frequency. In addition, one vehicle control culture (139.1 x 10^-6) in the initial mutation assay with activation was slightly above the historical data range (34.0 to 123.9 x 10^-6) for mutant frequency.


ADDITIONAL INFORMATION ON CYTOTOXICITY:
1. Initial Non-activation Mutation Assay-Concentrations at 62.5 and 125 ug/mL were discarded because a sufficient number of higher concentrations were available. The remaining eight treatments induced no cytotoxicity to moderate cytotoxicity (88.3 % (250 ug/mL) to 34.0 % (3000 ug/mL) relative growths).
2. Confirmatory Non-activation Mutation Assay- Concentrations at 62.5 and 125 ug/mL were terminated because there were sufficient higher concentrations available for analysis. The remaining eight concentrations induced no cytotoxicity to high cytotoxicity (120.3 % (250 ug/mL) to 13 % (3500 ug/mL) relative growth).
3. Initial Activation Mutation Assay- Concentrations at 62.5 and 125 ug/mL were terminated because there were sufficient higher concentrations available for analysis. The remaining eight concentrations induced no cytotoxicity (117.2 % to 81.8 % relative growths).
4. Confirmatory Activation Mutation Assay- Concentrations at 62.5 and 125 ug/mL were terminated because there were sufficient higher concentrations available for analysis. The remaining eight concentrations induced no cytotoxicity to moderate cytotoxicity (95.5 % (250 ug/mL) to 39.9 % (3500 ug/mL) relative growths).


OTHER: The average cloning efficiencies for the vehicle control were 91.4 % and 110.9 % without metabolic activation and 96.6 % and 109.5 % with metabolic activation, which demonstrated acceptable cloning conditions for the assays. The positive control cultures induced large increases in mutant frequencies that were greatly in excess of the minimum criteria.
Mutant colonies from all the cultures showed the expected bimodal distribution, and mutant colonies from the positive control cultures showed both small and large colonies.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

The test substance was reported as negative for inducing forward mutations at the TK locus in L5178Y mouse lymphoma cells in the presence and absence of Aroclor 1254 induced rat liver S9.

Executive summary:

The objective of thisin vitroassay was to evaluate the ability of sodium tungstate to induce forward mutations at the thymidine kinase (TK) locus in the mouse lymphoma L5178Y cell line. The test article, Sodium Tungstate Dihydrate, formed a transparent, colorless solution in the vehicle (water) at 35 mg/mL. Thirty-five mg/mL was the highest concentration prepared for use in the assay and is slightly greater than the OECD Testing Guidelines of 10 mM (molecular weight=329.86; 10 mM=3299 ug/mL). The test article remained soluble at all concentrations tested.

The test article, sodium tungstate dihydrate, was tested in a preliminary dose range finding assay with a treatment period of approximately 4 hours both with and without S9 metabolic activation. Ten dose levels were used in each case that ranged from 6.90 to 3500 ug/mL; a vehicle control was included under each activation condition.

In both the nonactivation and activation dose range finding assays, sodium tungstate dihydrate induced no cytotoxicity to weak cytotoxicity up to and including 1750 ug/mL, and moderate cytotoxicity at 3500 ug/mL. The test article concentrations chosen for the mutation assays were based on these results.

In the initial nonactivation mutation assay, eight doses ranging from 250 to 3500 ug/ml were analyzed for mutant induction and no cytotoxicity to moderate cytotoxicity was induced. None of the concentrations induced a mutant frequency that exceeded the minimum criteria for a positive response. A confirmatory assay was performed. In the confirmatory nonactivation mutation assay, eight doses ranging from 250 to 3500 ug/mL were also analyzed and no cytotoxicity to high cytotoxicity was induced. None of the concentrations induced a mutant frequency that exceeded the minimum criteria for a positive response. Therefore, the test article was evaluated as negative without metabolic activation.

In the initial activation mutation assay, eight doses ranging from 250 to 3500 ug/mL were analyzed and no cytotoxicity was induced. None of the concentrations induced a mutant frequency that exceeded the minimum criteria for a positive response. A confirmatory assay was performed.

In the confirmatory activation mutation assay, eight doses ranging from 250 to 3500 ug/mL were also analyzed and no cytotoxicity to moderate cytotoxicity was induced. None of the concentrations induced a mutant frequency that exceeded the minimum criteria for a positive response. Therefore, the test article was evaluated as negative without metabolic activation.

The test article, sodium tungstate, was evaluated as negative for inducing forward mutations at the TK locus in L5178Y mouse lymphoma cells, under activation and nonactivation conditions used for this assay.

Reference 4

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2003-07-16 to 2004-01-02

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial forward mutation assay

Target gene:

Histidine locus and tryptophan locus

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:

Aroclor 1254 induced rat liver S9

Test concentrations with justification for top dose:

33.3, 100, 333, 1000, 3330, and 5000 ug/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: deionized water
- Justification for choice of solvent/vehicle: In solubility testing, the test substance was observed to form a transparent, colorless solution in deionized water at concentrations of 200 and 33.0 mg/ml.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

Activated test system Migrated to IUCLID6: Used for strain TA98 at 2.5 ug/plate

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

Non-activated test system Migrated to IUCLID6: Used for strain TA98 at 1.0 ug/plate

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene-used for strains TA100, TA1535, TA1537 and WP2uvrA at 2.5, 2.5, 2.5, and 25.0 ug/plate, respectively.

Remarks:

Activated test system

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

Non-activated test system Migrated to IUCLID6: Used for strains TA100 and TA1535 at 2.0 ug/plate.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: ICR-191-used for strain TA1537 at 2.0 ug/plate.

Remarks:

Non-activated test system

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

Non-activated test system Migrated to IUCLID6: Used for strain WP2uvrA at 1.0 ug/plate.

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)


DURATION
- Exposure duration: 52 +/- 4 hours
- Selection time (if incubation with a selection agent):

SELECTION AGENT (mutation assays): histidine (Salmonella strains) and trypophan (E. coli strain)

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: Evaluation of the bacterial background lawn

Evaluation criteria:

Tester Strains TA98, TA100, and WP2uvrA- For the test substance to be considered positive, it had to produce at least a 2-fold increase in the mean revertants per plate of at least one of the tester strains over the mean revertants per plate of the appropriate vehicle control. This increase in the mean number of revertants per plate had to be accompanied by a dose response to increasing concentrations of the test substance.

Tester Strains TA1535 and TA1537-For the test substance to be considered positive, it had to produce at least a 3-fold increase in the mean revertants per plate of at least one of the tester strains over the mean revertant per plate of the appropriate vehicle control. This increase in the mean number of revertants per plate had to be accompanied by a dose response to increasing concentrations of the test substance.

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

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:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: The test substance was observed to form a transparent, colorless, non-viscous solution in deionized water at 100 mg/ml, and remained in solution at all succeeding dilutions prepared.

RANGE-FINDING/SCREENING STUDIES: The test substance was tested for toxicity using tester strains TA100 and WP2uvrA in the presence and absence of metabolic activation at concentrations ranging from 6.67-5000 ug/plate. No indications of cytotoxicity were observed at any concentration in the presence or absence of metabolic activation, therefore, 5000 ug/plate was chosen as the high dose for the mutagenicity assays.

COMPARISON WITH HISTORICAL CONTROL DATA: All values were within historical control range.

OTHER: In the initial mutagenicity assay, tester strain TA98 did not exhibit the characteristic mean number of spontaneous revertants per plate in the absence of S9 as specified in the protocol, and for this reason the data was not used to evaluate the test substance. The test substance was retested in TA98 in the absence of S9. All other data generated in the initial mutagenicity assay were acceptable, and no positive increases were observed in the mean number of revertants per plate with any of the tester strains in the presence or absence of S9.
In the confirmatory mutagenicity assay, tester strain TA100 did not exhibit the characteristic number of mean spontaneous revertants per plate in the absence of S9 mix as specified in the protocol, and for this reason the data was not used to evaluate the test substance. The test substance was re-tested in TA100 in the absence of S9. All other data generated in the confirmatory assay were acceptable, and no positive increases were observed in the mean number of revertants per plate with any of the tester strains in either the presence or absence of S9.
In the repeat assays with TA100 and TA98, no positive increases in the mean number of revertants were observed.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

Under the conditions of the study, the test substance did not cause a positive increase in the mean number of revertants per plate with any of the tester strains either in the presence or absence of metabolic activation.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

A summary of on the in vivo genotoxicity evidence is discussed below.

A. In vivo cytogenicity-chromosomal aberration

In an in vivo gavage micronucleus study (OCED 474, K1 study) in male mice, sodium tungstate did not induce any statistically significant increases in micronucleated PCEs at any dose level examined (250, 500, and 750 mg/kg).

Sodium tungstate did not show an increase in micronucleated cells following 3 months of exposure via drinking water up to 2,000mg/L (approximately 157.2-160.5 mg/kg-d). Significant increases in the percent reticulocytes were seen in both male and female rats, suggesting that sodium tungstate could have stimulated erythropoiesis in the bone marrow; however, the absolute increases in the percentages were small compared to the vehicle control animals. In male and female mice, there were no significant increases in micronucleated reticulocytes or in micronucleated erythrocytes in either sex following 3 months of exposure to sodium tungstate via drinking water up to 2,000 mg/L (202-212 mg/kg-d). Therefore, mammalian in vivo mammalian cytogenicity is negative.

B. In vivo DNA damage and/or repair studies

There is a positive in vivo Comet assay study in bone marrow of mice exposed to 15 or 200 mg sodium tungstate/mL for 8 weeks.

There are several Comet assays in different tissues from rats and mice exposed to sodium tungstate up to concentration of 2,000 mg/L (approximately 157.2-160.5 mg/kg-d in rats and 202-212 mg/kg-d in mice) (US NTP, 2021). Significant increases in DNA damage, measured as percent tail DNA, were observed in liver cells from male and female rats. Increase in DNA damage were not observed for peripheral blood leukocytes in male or female rats, or for ileum cells in female rats. In male mice, significant increases in DNA damage were observed in liver and ileum cells, but not in kidney cells or peripheral blood leukocytes. No increases in percent tail DNA were observed in female mice for liver, kidney, ileum, or peripheral blood leukocyte cells. Therefore, mammalian in vivo DNA damage and/or repair is equivocal.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study without detailed documentation

Reason / purpose for cross-reference:

reference to same study

Remarks:

In vivo micronucleus animals were a subgroup part of a 90-day toxicity study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

GLP compliance:

yes

Type of assay:

mammalian erythrocyte micronucleus test

Specific details on test material used for the study:

Stability studies conducted on the 20 mg/L formulation by the analytical chemistry laboratory found that the formulation was stable when sealed and stored in Nalgene bottles for 42 days at 5°C and at room temperature (approximately 25°C). An animal room simulation was conducted using the 20 mg/L formulation stored in a drinking water bottle with aliquots periodically removed to simulate animal drinking. There was no significant loss in tungsten over 7 days at room temperatures.

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Route of administration:

oral: drinking water

Vehicle:

Deionized water

Details on exposure:

Animals were exposed for 3-months

Duration of treatment / exposure:

90-days

Frequency of treatment:

Daily: Drinking water

Dose / conc.:

0 mg/L drinking water

Remarks:

Deionized water

Dose / conc.:

125 mg/L drinking water

Remarks:

Average daily sodium tungstate doses of approximately 11.8 mg/kg/day for males and 14.0 mg/kg/day for females.

Dose / conc.:

250 mg/L drinking water

Remarks:

Average daily sodium tungstate doses of approximately 24.3 mg/kg/day for males and 26.1 mg/kg/day for females.

Dose / conc.:

500 mg/L drinking water

Remarks:

Average daily sodium tungstate doses of approximately 48.9 mg/kg/day for males and 54.4 mg/kg/day for females.

Dose / conc.:

1 000 mg/L drinking water

Remarks:

Average daily sodium tungstate doses of approximately 91.8 mg/kg/day for males and 101.4, and mg/kg/day for females.

Dose / conc.:

2 000 mg/L drinking water

Remarks:

Average daily sodium tungstate doses of approximately 157.2 mg/kg/day for males and 160.5 mg/kg/day for females.

No. of animals per sex per dose:

5 animals per sex per dose

Control animals:

yes, concurrent no treatment

Positive control(s):

None

Tissues and cell types examined:

Micronucleated immature erythrocytes (polychromatic erythrocytes [PCEs], reticulocytes)

Details of tissue and slide preparation:

At termination of the 3-month toxicity studies of sodium tungstate, blood samples (approximately 200 μL) were collected from male and female rats and mice, placed in ethylenediaminetetraacetic acid (EDTA)-coated tubes, and shipped overnight to the testing laboratory. Upon arrival, blood samples were fixed in ultracold methanol using a MicroFlowPLUS Kit (Litron Laboratories, Rochester, NY) according to the manufacturer’s instructions. Fixed samples were stored in a −80°C freezer until analysis. Thawed blood samples were analyzed for frequency of micronucleated immature erythrocytes (polychromatic erythrocytes [PCEs], reticulocytes) and mature erythrocytes (normochromatic erythrocytes, NCEs) using a flow cytometer; both the mature and the immature erythrocyte populations can be analyzed separately by employing special cell surface markers to differentiate the two cell types. Because the very young reticulocyte subpopulation (CD71-positive cells) can be targeted using this technique, rat blood samples can be analyzed for damage that occurred in the bone marrow within the past 24–48 hours, before the rat spleen appreciably alters the percentage of micronucleated reticulocytes in circulation.

Evaluation criteria:

In the micronucleus test, it is preferable to base a positive result on the presence of both a significant trend as well as at least one significantly elevated exposure group compared with the corresponding control group. In addition, historical control data are used to evaluate the biological significance of any observed response. Both statistical significance and biological significance are considered when arriving at a call. The presence of either a significant trend or a single significant exposure group generally results in an equivocal call. The absence of both a trend and a significant exposure group results in a negative call. Ultimately, the scientific staff determines the final call after considering the results of statistical analyses, reproducibility of any effects observed (in acute studies), and the magnitudes of those effects.

Statistics:

Using flow cytometric scoring techniques suggests it is reasonable to assume that the proportion of micronucleated reticulocytes is approximately normally distributed. The statistical tests selected for trend and for pairwise comparisons with the control group depend on whether the variances among the groups are equal. The Levene test at α = 0.05 is used to test for equal variances. In the case of equal variances, linear regression is used to test for a linear trend with exposure concentration and the Williams test is used to test for pairwise differences between each exposed group and the control group. In the case of unequal variances, the Jonckheere test is used to test for linear trend and the Dunn test is used for pairwise comparisons of each exposed group with the control group. To correct for multiple pairwise comparisons, the p value for each comparison with the control group is multiplied by the number of comparisons made. In the event that this product is >1.00, it is replaced with 1.00. Trend tests and pairwise comparisons with the controls are considered significant at p ≤ 0.025.

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

Exposure-related histological lesions were limited to the kidneys and included increased incidences of renal tubule regeneration in the 1,000 and 2,000 mg/L males and females; the increases in the 2,000 mg/L groups were significant relative to control.

Vehicle controls validity:

valid

Positive controls validity:

not applicable

Additional information on results:

There were no early deaths during the 3-month study. When compared to the vehicle control group, final mean body weights were lower for the 1,000 and 2,000 mg/L males and 2,000 mg/L females. Water consumption was lower for the 1,000 and 2,000 mg/L males and females. The urine xanthine/creatinine ratios were significantly increased in all male and female exposed groups. Serum insulin concentrations were significantly decreased in the 2,000 mg/L males relative to the vehicle control males. Significantly decreased absolute weights were observed in several organs but were considered secondary to body weights reductions.

Conclusions:

At the end of the 3-month studies, peripheral blood samples were obtained from male and female rats and mice and analyzed for the frequency of micronucleated reticulocytes and erythrocytes. In male and female rats, the reticulocyte population (PCEs), which is the only red blood cell population that can be accurately assessed for micronucleus frequency in peripheral blood of rats due to efficient splenic scavenging of damaged erythrocytes, did not show an increase in micronucleated cells following 3 months of exposure to sodium tungstate via drinking water (0, 125, 250, 500, 1,000, and 2,000 mg/L). Significant increases in the percent reticulocytes were seen in both male and female rats, suggesting that sodium tungstate could have stimulated erythropoiesis in the bone marrow; however, the absolute increases in the percentages were small compared to the vehicle control animals.

Reference 2

Endpoint:

in vivo mammalian cell study: DNA damage and/or repair

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Reason / purpose for cross-reference:

reference to same study

Remarks:

In vivo micronucleus animals were a subgroup part of a 90-day toxicity study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)

GLP compliance:

yes

Type of assay:

mammalian comet assay

Specific details on test material used for the study:

Stability studies conducted on the 20 mg/L formulation by the analytical chemistry laboratory found that the formulation was stable when sealed and stored in Nalgene bottles for 42 days at 5°C and at room temperature (approximately 25°C). An animal room simulation was conducted using the 20 mg/L formulation stored in a drinking water bottle with aliquots periodically removed to simulate animal drinking. There was no significant loss in tungsten over 7 days at room temperatures.

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Route of administration:

oral: drinking water

Vehicle:

Deionized water

Details on exposure:

Animals were exposed for 3-months

Duration of treatment / exposure:

90-days

Frequency of treatment:

Daily: Drinking water

Dose / conc.:

0 mg/L drinking water

Remarks:

Deionized water

Dose / conc.:

125 mg/L drinking water

Remarks:

Average daily sodium tungstate doses of approximately 11.8 mg/kg/day for males and 14.0 mg/kg/day for females.

Dose / conc.:

250 mg/L drinking water

Remarks:

Average daily sodium tungstate doses of approximately 24.3 mg/kg/day for males and 26.1 mg/kg/day for females.

Dose / conc.:

500 mg/L drinking water

Remarks:

Average daily sodium tungstate doses of approximately 48.9 mg/kg/day for males and 54.4 mg/kg/day for females.

Dose / conc.:

1 000 mg/L drinking water

Remarks:

Average daily sodium tungstate doses of approximately 91.8 mg/kg/day for males and 101.4, and mg/kg/day for females.

Dose / conc.:

2 000 mg/L drinking water

Remarks:

Average daily sodium tungstate doses of approximately 157.2 mg/kg/day for males and 160.5 mg/kg/day for females.

No. of animals per sex per dose:

5 animals per sex per dose

Control animals:

yes, concurrent no treatment

Positive control(s):

None

Tissues and cell types examined:

Small portions (3 to 4 mm) of the ileum, liver, and kidney

Details of tissue and slide preparation:

For preparation of samples for the comet assay, a 50 μL sample of blood was transferred to a tube containing 1 mL of freshly prepared cold mincing buffer [Mg+2, Ca+2, and phenol-free Hank’s Balanced Salt Solution (Life Technologies, Carlsbad, CA) with 20 mM EDTA, pH 7.3 to 7.5, and 10% v/v fresh dimethyl sulfoxide (DMSO)]. The ileum, liver, and kidney were rinsed with cold mincing buffer to remove residual blood and were held on ice briefly (≤5 minutes) until processed. Small portions (3 to 4 mm) of the ileum, liver, and kidney were placed in tubes containing cold mincing solution and rapidly minced until finely dispersed. All samples prepared for the comet assay were immediately flash frozen in liquid nitrogen and subsequently transferred to a −80°C freezer for storage until shipment by overnight courier on dry ice to the analytical laboratory. Upon receipt, all samples were immediately placed in a −80°C freezer for storage until further processing.
Blood and tissue samples were thawed on ice and maintained on ice during slide preparation. Just before use, each cell suspension was shaken gently to mix the cells and placed back on ice for 15 to 30 seconds to allow clumps to settle. A portion of the supernatant was empirically diluted with 0.5% low melting point agarose (Lonza, Walkersville, MD) dissolved in Dulbecco’s phosphate buffer (Ca+2, Mg+2, and phenol-free) at 37°C and layered onto each well of a 2-well CometSlide™ (Trevigen, Gaithersburg, MD). Slides were immersed in cold lysing solution [2.5 M NaCl, 100 mM Na2EDTA, 10 mM tris(hydroxymethyl)aminomethane (Tris), pH 10, containing freshly added 10% DMSO (Fisher Scientific, Pittsburgh, PA), and 1% Triton X-100] overnight in a refrigerator, protected from light. The following day, the slides were rinsed in 0.4 M Trizma base (pH 7.5), randomly placed onto the platform of a horizontal electrophoresis unit, and treated with cold alkali solution (300 mM NaOH, 1 mM Na2EDTA, pH > 13) for 20 minutes to allow DNA unwinding, then electrophoresed at 4°C to 9°C for 20 minutes at 25 V (0.7 V/cm), with a current of approximately 300 mA. After electrophoresis, slides were neutralized with 0.4 M Trizma base (pH 7.5) for 5 minutes and then dehydrated by immersion in absolute ethanol (Pharmco-AAPER, Shelbyville, KY) for at least 5 minutes and allowed to air dry. Slides were prepared in a laboratory with a relative humidity no more than 60% and stored at room temperature in a desiccator with a relative humidity of no more than 60% until stained and scored; stained slides were stored in a desiccator. NaCl, Na2EDTA, Triton X-100, and Trizma base were purchased from Sigma-Aldrich (St. Louis, MO); NaOH was purchased from Fisher Scientific (Pittsburgh, PA).
After staining with SYBR®Gold (Molecular Probes, Life Technologies, Grand Island, NY), the slides, independently coded to mask treatment, were scored using Comet Assay IV Imaging Software, Version 4.3.1 (Perceptive Instruments, Ltd., Suffolk, UK),

Evaluation criteria:

In the alkaline (pH > 13) comet assay, damaged nuclear DNA fragments undergo unidirectional migration through the agarose gel within an electrical field, forming an image that resembles a comet, and the greater the amount of fragmentation, the greater the amount of DNA migration that will occur. The image analysis software partitions the intensity of the fluorescent signal of the DNA in the entire comet image into the percent that is attributable to the comet head and the percent attributable to the tail. Manual adjustment of the automated detection of head and tail features is sometimes required. To evaluate DNA damage levels, the extent of DNA migration was characterized for 100 scorable comet figures per animal/tissue as percent tail DNA (intensity of all tail pixels divided by the total intensity of all pixels in the comet, expressed as a percentage).

Key result

Sex:

female

Genotoxicity:

negative

Remarks:

Ileum cells

Toxicity:

yes

Remarks:

Exposure-related histological lesions were limited to the kidneys and included increased incidences of renal tubule regeneration in the 1,000 and 2,000 mg/L females; the increases in the 2,000 mg/L groups were significant relative to the control.

Key result

Sex:

male/female

Genotoxicity:

negative

Remarks:

Peripheral blood leukocytes

Toxicity:

yes

Remarks:

Exposure-related histological lesions were limited to the kidneys and included increased incidences of renal tubule regeneration in the 1,000 and 2,000 mg/L males and females; the increases in the 2,000 mg/L groups were significant relative to the control

Key result

Sex:

male/female

Genotoxicity:

positive

Remarks:

Liver cells

Toxicity:

yes

Remarks:

Exposure-related histological lesions were limited to the kidneys and included increased incidences of renal tubule regeneration in the 1,000 and 2,000 mg/L males and females; the increases in the 2,000 mg/L groups were significant relative to the control

Vehicle controls validity:

valid

Additional information on results:

There were no early deaths during the 3-month study. When compared to the vehicle control group, final mean body weights were lower for the 1,000 and 2,000 mg/L males and 2,000 mg/L females. Water consumption was lower for the 1,000 and 2,000 mg/L males and females. The urine xanthine/creatinine ratios were significantly increased in all male and female exposed groups. Serum insulin concentrations were significantly decreased in the 2,000 mg/L males relative to the vehicle control males. Significantly decreased absolute weights were observed in several organs but were considered secondary to body weights reductions.

Although cells from kidney tissue were evaluated from male and female rats, and ileum tissue was evaluated from male rats, results from these tissues were considered invalid due to unusually high levels of percent tail DNA in the control group.

Conclusions:

DNA damage from exposure to sodium tungstate was assessed in liver, ileum, and kidney cell samples, and in blood leukocyte. Significant increases in DNA damage, measured as percent tail DNA, were observed in liver cells from male and female rats. Increases in DNA damage were not observed for peripheral blood leukocytes in male or female rats, or for ileum cells in female rats. Although cells from kidney tissue were evaluated from male and female rats, and ileum tissue was evaluated from male rats, results from these tissues were considered invalid due to unusually high levels of percent tail DNA in the control group.

Reference 3

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study without detailed documentation

Reason / purpose for cross-reference:

reference to same study

Remarks:

In vivo micronucleus animals were a subgroup part of a 90-day toxicity study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

GLP compliance:

yes

Type of assay:

mammalian erythrocyte micronucleus test

Specific details on test material used for the study:

Stability studies conducted on the 20 mg/L formulation by the analytical chemistry laboratory found that the formulation was stable when sealed and stored in Nalgene bottles for 42 days at 5°C and at room temperature (approximately 25°C). An animal room simulation was conducted using the 20 mg/L formulation stored in a drinking water bottle with aliquots periodically removed to simulate animal drinking. There was no significant loss in tungsten over 7 days at room temperatures.

Species:

mouse

Strain:

B6C3F1

Sex:

male/female

Route of administration:

oral: drinking water

Vehicle:

Deionized water

Details on exposure:

Animals were exposed for 3-months

Duration of treatment / exposure:

90-days

Frequency of treatment:

Daily: Drinking water

Dose / conc.:

0 mg/L drinking water

Remarks:

Deionized water

Dose / conc.:

125 mg/L drinking water

Remarks:

Average daily sodium tungstate doses of approximately 14 mg/kg/day for males and 14 mg/kg/day for females.

Dose / conc.:

250 mg/L drinking water

Remarks:

Average daily sodium tungstate doses of approximately 27 mg/kg/day for males and 29 mg/kg/day for female.

Dose / conc.:

500 mg/L drinking water

Remarks:

Average daily sodium tungstate doses of approximately 57 mg/kg/day for males and 58 mg/kg/day for female.

Dose / conc.:

1 000 mg/L drinking water

Remarks:

Average daily sodium tungstate doses of approximately 108 mg/kg/day for males and 113 mg/kg/day for female.

Dose / conc.:

2 000 mg/L drinking water

Remarks:

Average daily sodium tungstate doses of approximately 212 mg/kg/day for males and 202 mg/kg/day for female

No. of animals per sex per dose:

5 animals per sex per dose

Control animals:

yes, concurrent no treatment

Positive control(s):

None

Tissues and cell types examined:

Micronucleated immature erythrocytes (polychromatic erythrocytes [PCEs], reticulocytes)

Details of tissue and slide preparation:

At termination of the 3-month toxicity studies of sodium tungstate, blood samples (approximately 200 μL) were collected from male and female rats and mice, placed in ethylenediaminetetraacetic acid (EDTA)-coated tubes, and shipped overnight to the testing laboratory. Upon arrival, blood samples were fixed in ultracold methanol using a MicroFlowPLUS Kit (Litron Laboratories, Rochester, NY) according to the manufacturer’s instructions. Fixed samples were stored in a −80°C freezer until analysis. Thawed blood samples were analyzed for frequency of micronucleated immature erythrocytes (polychromatic erythrocytes [PCEs], reticulocytes) and mature erythrocytes (normochromatic erythrocytes, NCEs) using a flow cytometer; both the mature and the immature erythrocyte populations can be analyzed separately by employing special cell surface markers to differentiate the two cell types. In mice, both the immature and mature erythrocyte populations can be evaluated for micronucleus frequency because the mouse spleen does not sequester and eliminate damaged erythrocytes. Damaged erythrocytes achieve steady state in the peripheral blood of mice following 4 weeks of continuous exposure. Approximately 20,000 reticulocytes and 1×10^6 erythrocytes were analyzed per animal for frequency of micronucleated cells, and the percentage of immature erythrocytes (% PCE) was calculated as a measure of bone marrow toxicity resulting from sodium tungstate exposure.

Evaluation criteria:

In the micronucleus test, it is preferable to base a positive result on the presence of both a significant trend as well as at least one significantly elevated exposure group compared with the corresponding control group. In addition, historical control data are used to evaluate the biological significance of any observed response. Both statistical significance and biological significance are considered when arriving at a call. The presence of either a significant trend or a single significant exposure group generally results in an equivocal call. The absence of both a trend and a significant exposure group results in a negative call. Ultimately, the scientific staff determines the final call after considering the results of statistical analyses, reproducibility of any effects observed (in acute studies), and the magnitudes of those effects.

Statistics:

Using flow cytometric scoring techniques suggests it is reasonable to assume that the proportion of micronucleated reticulocytes is approximately normally distributed. The statistical tests selected for trend and for pairwise comparisons with the control group depend on whether the variances among the groups are equal. The Levene test at α = 0.05 is used to test for equal variances. In the case of equal variances, linear regression is used to test for a linear trend with exposure concentration and the Williams test is used to test for pairwise differences between each exposed group and the control group. In the case of unequal variances, the Jonckheere test is used to test for linear trend and the Dunn test is used for pairwise comparisons of each exposed group with the control group. To correct for multiple pairwise comparisons, the p value for each comparison with the control group is multiplied by the number of comparisons made. In the event that this product is >1.00, it is replaced with 1.00. Trend tests and pairwise comparisons with the controls are considered significant at p ≤ 0.025.

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

The only histological lesion associated with exposure was in the kidney. The incidences of renal tubule regeneration were higher in the 1,000 and 2,000 mg/L male and female groups compared to the respective vehicle control groups.

Vehicle controls validity:

valid

Positive controls validity:

not applicable

Additional information on results:

All mice survived to the end of the study. Over the course of the study, mean body weights were below 90% of the vehicle control group for the 250, 1,000, and 2,000 mg/L females and the 2,000 mg/L males. At study termination, the mean body weights of all exposed groups of males and females were within 10% of the vehicle control groups. Weekly mean water consumption values were <90% of the vehicle control groups in the 1,000 and 2,000 mg/L males and the 2,000 mg/L females. Lower absolute organ weights were attributed to body weight reductions.

Conclusions:

In male and female mice, there were no significant increases in micronucleated reticulocytes or in micronucleated erythrocytes in either sex following 3 months of exposure to ST via drinking water (0, 125, 250, 500, 1,000, and 2,000 mg/L). A significant increase in the percent reticulocytes was seen in male mice suggesting that ST could have stimulated erythropoiesis in the bone marrow; however, the absolute increase was small compared to the vehicle control group.

Reference 4

Endpoint:

in vivo mammalian cell study: DNA damage and/or repair

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Reason / purpose for cross-reference:

reference to same study

Remarks:

In vivo micronucleus animals were a subgroup part of a 90-day toxicity study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)

GLP compliance:

yes

Type of assay:

mammalian comet assay

Specific details on test material used for the study:

Stability studies conducted on the 20 mg/L formulation by the analytical chemistry laboratory found that the formulation was stable when sealed and stored in Nalgene bottles for 42 days at 5°C and at room temperature (approximately 25°C). An animal room simulation was conducted using the 20 mg/L formulation stored in a drinking water bottle with aliquots periodically removed to simulate animal drinking. There was no significant loss in tungsten over 7 days at room temperatures.

Species:

mouse

Strain:

B6C3F1

Sex:

male/female

Route of administration:

oral: drinking water

Vehicle:

Deionized water

Details on exposure:

Animals were exposed for 3-months

Duration of treatment / exposure:

90-days

Frequency of treatment:

Daily: Drinking water

Dose / conc.:

0 mg/L drinking water

Remarks:

Deionized water

Dose / conc.:

125 mg/L drinking water

Remarks:

Average daily sodium tungstate doses of approximately 14 mg/kg/day for males and females

Dose / conc.:

250 mg/L drinking water

Remarks:

Average daily sodium tungstate doses of approximately 27 mg/kg/day for males and 29 mg/kg/day for females

Dose / conc.:

500 mg/L drinking water

Remarks:

Average daily sodium tungstate doses of approximately 57 mg/kg/day for males and 58 mg/kg/day for females

Dose / conc.:

1 000 mg/L drinking water

Remarks:

Average daily sodium tungstate doses of approximately 108 for males and 113 mg/kg/day for females

Dose / conc.:

2 000 mg/L drinking water

Remarks:

Average daily sodium tungstate doses of approximately 212 mg/kg/day for males and 202 mg/kg/day for females

No. of animals per sex per dose:

5 animals per sex per dose

Control animals:

yes, concurrent no treatment

Positive control(s):

None

Tissues and cell types examined:

Small portions (3 to 4 mm) of the ileum, liver, and kidney

Details of tissue and slide preparation:

For preparation of samples for the comet assay, a 50 μL sample of blood was transferred to a tube containing 1 mL of freshly prepared cold mincing buffer [Mg+2, Ca+2, and phenol-free Hank’s Balanced Salt Solution (Life Technologies, Carlsbad, CA) with 20 mM EDTA, pH 7.3 to 7.5, and 10% v/v fresh dimethyl sulfoxide (DMSO)]. The ileum, liver, and kidney were rinsed with cold mincing buffer to remove residual blood and were held on ice briefly (≤5 minutes) until processed. Small portions (3 to 4 mm) of the ileum, liver, and kidney were placed in tubes containing cold mincing solution and rapidly minced until finely dispersed. All samples prepared for the comet assay were immediately flash frozen in liquid nitrogen and subsequently transferred to a −80°C freezer for storage until shipment by overnight courier on dry ice to the analytical laboratory. Upon receipt, all samples were immediately placed in a −80°C freezer for storage until further processing.
Blood and tissue samples were thawed on ice and maintained on ice during slide preparation. Just before use, each cell suspension was shaken gently to mix the cells and placed back on ice for 15 to 30 seconds to allow clumps to settle. A portion of the supernatant was empirically diluted with 0.5% low melting point agarose (Lonza, Walkersville, MD) dissolved in Dulbecco’s phosphate buffer (Ca+2, Mg+2, and phenol-free) at 37°C and layered onto each well of a 2-well CometSlide™ (Trevigen, Gaithersburg, MD). Slides were immersed in cold lysing solution [2.5 M NaCl, 100 mM Na2EDTA, 10 mM tris(hydroxymethyl)aminomethane (Tris), pH 10, containing freshly added 10% DMSO (Fisher Scientific, Pittsburgh, PA), and 1% Triton X-100] overnight in a refrigerator, protected from light. The following day, the slides were rinsed in 0.4 M Trizma base (pH 7.5), randomly placed onto the platform of a horizontal electrophoresis unit, and treated with cold alkali solution (300 mM NaOH, 1 mM Na2EDTA, pH > 13) for 20 minutes to allow DNA unwinding, then electrophoresed at 4°C to 9°C for 20 minutes at 25 V (0.7 V/cm), with a current of approximately 300 mA. After electrophoresis, slides were neutralized with 0.4 M Trizma base (pH 7.5) for 5 minutes and then dehydrated by immersion in absolute ethanol (Pharmco-AAPER, Shelbyville, KY) for at least 5 minutes and allowed to air dry. Slides were prepared in a laboratory with a relative humidity no more than 60% and stored at room temperature in a desiccator with a relative humidity of no more than 60% until stained and scored; stained slides were stored in a desiccator. NaCl, Na2EDTA, Triton X-100, and Trizma base were purchased from Sigma-Aldrich (St. Louis, MO); NaOH was purchased from Fisher Scientific (Pittsburgh, PA).
After staining with SYBR®Gold (Molecular Probes, Life Technologies, Grand Island, NY), the slides, independently coded to mask treatment, were scored using Comet Assay IV Imaging Software, Version 4.3.1 (Perceptive Instruments, Ltd., Suffolk, UK),

Evaluation criteria:

In the alkaline (pH > 13) comet assay, damaged nuclear DNA fragments undergo unidirectional migration through the agarose gel within an electrical field, forming an image that resembles a comet, and the greater the amount of fragmentation, the greater the amount of DNA migration that will occur. The image analysis software partitions the intensity of the fluorescent signal of the DNA in the entire comet image into the percent that is attributable to the comet head and the percent attributable to the tail. Manual adjustment of the automated detection of head and tail features is sometimes required. To evaluate DNA damage levels, the extent of DNA migration was characterized for 100 scorable comet figures per animal/tissue as percent tail DNA (intensity of all tail pixels divided by the total intensity of all pixels in the comet, expressed as a percentage).

Key result

Sex:

female

Genotoxicity:

negative

Remarks:

For liver, kidney, ileum, or peripheral blood leukocyte cells

Toxicity:

yes

Remarks:

Exposure-related histological lesions were limited to the kidneys and included increased incidences of renal tubule regeneration in the 1,000 and 2,000 mg/L females; the increases in the 2,000 mg/L groups were significant relative to the control.

Vehicle controls validity:

valid

Key result

Sex:

male

Genotoxicity:

negative

Remarks:

For kidney cells or peripheral blood leukocytes

Toxicity:

yes

Remarks:

Exposure-related histological lesions were limited to the kidneys and included increased incidences of renal tubule regeneration in the 1,000 and 2,000 mg/L males; the increases in the 2,000 mg/L groups were significant relative to the control.

Vehicle controls validity:

valid

Key result

Sex:

male

Genotoxicity:

positive

Remarks:

For liver and ileum cells

Toxicity:

yes

Remarks:

Exposure-related histological lesions were limited to the kidneys and included increased incidences of renal tubule regeneration in the 1,000 and 2,000 mg/L males, the increases in the 2,000 mg/L groups were significant relative to the control.

Vehicle controls validity:

valid

Additional information on results:

There were no early deaths during the 3-month study. When compared to the vehicle control group, final mean body weights were lower for the 1,000 and 2,000 mg/L males and 2,000 mg/L females. Water consumption was lower for the 1,000 and 2,000 mg/L males and females. The urine xanthine/creatinine ratios were significantly increased in all male and female exposed groups. Serum insulin concentrations were significantly decreased in the 2,000 mg/L males relative to the vehicle control males. Significantly decreased absolute weights were observed in several organs but were considered secondary to body weights reductions.

Conclusions:

In male mice, significant increases in DNA damage were observed in liver and ileum cells, but not in kidney cells or peripheral blood leukocytes. No increases in percent tail DNA were observed in female mice for liver, kidney, ileum, or peripheral blood leukocyte cells.

Reference 5

Endpoint:

in vivo mammalian cell study: DNA damage and/or repair

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 available

Principles of method if other than guideline:

The In Vivo Mammalian Alkaline Comet Assay (OECD TG 489) was published on July 29, 2016, and the study was conducted in 2011.

GLP compliance:

not specified

Type of assay:

mammalian comet assay

Species:

mouse

Strain:

C57BL

Sex:

not specified

Details on test animals or test system and environmental conditions:

Not specified

Route of administration:

oral: drinking water

Vehicle:

Tap water

Details on exposure:

57BL/6J mice were treated for 8 weeks with tap water or water containing 15 mg/ml or 200 mg/ml tungsten

Duration of treatment / exposure:

8-weeks

Frequency of treatment:

Daily

Dose / conc.:

0 mg/L drinking water

Remarks:

Vehicle control

Dose / conc.:

15 mg/L drinking water

Remarks:

These doses correspond to the lower and higher doses used in in vitro work where DNA damage was observed

Dose / conc.:

200 mg/L drinking water

Remarks:

These doses correspond to the lower and higher doses used in in vitro work where DNA damage was observed.

No. of animals per sex per dose:

Five mice were used for each treatment

Control animals:

yes, concurrent no treatment

Positive control(s):

None

Tissues and cell types examined:

Bone marrow

Details of tissue and slide preparation:

Not specified

Evaluation criteria:

DNA damage was assessed in non-adherent bone marrow cells using Comet assay. Comet tail moment was assessed for >50 cells per mouse.

Statistics:

Not described, but p values were reported

Key result

Genotoxicity:

positive

Remarks:

Tungsten exposure significantly increased DNA damage

Toxicity:

not specified

Vehicle controls validity:

valid

Positive controls validity:

not applicable

Conclusions:

In vivo exposure to 15 mg/ml tungsten induces DNA damage.

Reference 6

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2003-07-16 to 2004-01-06

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Study was conducted according to OECD guideline 474 and GLP.

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

mammalian erythrocyte micronucleus test

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Remarks:

Clinical signs included slightly hypoactive, soft feces, rough haircoat, recumbent, cold to touch, opaque eyes, and hypoactive.

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: 500-2000 mg/kg
- Clinical signs of toxicity in test animals: Clinical signs included slightly hypoactive, soft feces, rough haircoat, recumbent, cold to touch, opaque eyes, and hypoactive.
- Harvest times: Animals were analyzed at 1 hour, 4 hours, 6 hours, 1 day, and 2 days after dosing.
- High dose with and without activation: 2000 mg/kg
- Other: 3 males and 3 females per group were used in this study, but since no relevant differences in toxicity between the sexes were observed, only males were used in the micronucleus assay. Two males and 1 female died in the 1500 mg/kg group, and 3 males and 2 females in the 2000 mg/kg died. Based on these results, the maximum tolerated dose was estimated to be 1000 mg/kg.

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): The test substance did not induce any statistically significant increases in micronucleated PCEs at any dose level examined (250, 500, and 750 mg/kg). The vehicle control group had less than approximately 0.4% micronucleated PCEs and the group mean was within the historical control range. The positive control induced a statistically significant increase in micronucleated PCEs as compared to that of the vehicle control, with means and standard errors of 3.95 +/- 0.33 % and 2.37 +/- 0.32 %, for the initial and repeat micronucleus assays, respectively.
- Ratio of PCE/NCE (for Micronucleus assay): The test substance was not cytotoxic to the bone marrow (i.e., no statistically significant decrease in the PCE:NCE ratio) at any dose level of the test substance.

Toxic Signs: Toxic signs were observed at the 1000 mg/kg dose level including soft feces, hypoactivity, rough haircoat and death at both the 24 and 48 hour timepoints (5 out of 12 died). Based on the high mortality rate, the rest of the animals in this group were euthanized and the bone marrow was not analyzed. One animal at the 500 mg/kg dose developed soft feces, and animals at the 750 mg/kg dose level developed soft feces, hypoactivity, rough haircoats, irregular respiration, and/or recumbency. In addition, one animal died in the 750 mg/kg group.

Conclusions:

The test substance was reported as negative in the mice bone marrow micronucleus assay, under the conditions of this study.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Justification for classification or non-classification

Sodium tungstate was negative for genotoxicity in an in vitro bacterial reverse mutation assay in five strains conducted according to OECD 471, an in vitro mammalian chromosome aberration assay conducted according to OECD 473, an in vitro L5178Y TK +/- Mouse Lymphoma Forward Mutation Assay conducted according to OECD 476, and several in vivo erythrocyte micronucleus assays (in rats and mice) conducted according or equivalent to OECD 474. DNA damage and/or repair was observed in murine bone marrow in the in vitro and in vivo Comet Assay . However, longer term studies in rats and mice tested with higher exposure concentrations showed only equivocal results (not tissue or sex specific) in a few tissues (kidney, liver, ileum, peripheral blood leukocytes) in the Comet Assay.
Therefore, based on the weight-of-evidence from the available data, sodium tungstate does not warrant classification.