Hexaammonium wolframate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

No in vitro mutagenicity data of sufficient quality are available on ammonium metatungstate. However, in vitro data are available on sodium metatungstate and sodium tungstate (source substances), which were used for read-across. Sodium metatungstate was negative for mutagenicity in an in vitro bacterial reverse mutation assay (Ames assay) conducted according to OECD 471. In addition, sodium tungstate was negative for mutagenicity in an Ames assay conducted according to OECD 471, an in vitro chromosome aberration assay conducted according to OECD 473 and an in vitro L5178Y TK+/- mouse lymphoma forward mutation assay conducted according to OECD 476. Based on the lack of mutagenicity of the read-across substances reported in all three in vitro assays, ammonium metatungstate is not considered a mutagen.

Link to relevant study records

Reference

Reference 1

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 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.

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 metatungstate
3. CATEGORY APPROACH JUSTIFICATION: See Annex 3 in CSR
4. DATA MATRIX: See Annex 3 in CSR

Reason / purpose for cross-reference:

read-across: supporting information

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 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

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:

No in vitro mutagenicity data of sufficient quality were available specifically on ammonium metatungstate (target substance). However, in vitro mutagencity data are available on sodium tungstate (source substance), which are used for read-across. Due to similar water solubility and toxicity for the target substance compared to the source substance, the resulting read-across from the source substance to the target substance is appropriate for this endpoint. In addition, read-across is appropriate because the classification and labelling is similar for the source substance and 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 read-across category approach description in the Category section of this IUCLID submission or Annex 3 of the CSR.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

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

Adequacy of study:

key study

Study period:

2003-07-17 to 2004-01-07

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.

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 metatungstate
3. CATEGORY APPROACH JUSTIFICATION: See Annex 3 in CSR
4. DATA MATRIX: See Annex 3 in CSR

Reason / purpose for cross-reference:

read-across: supporting information

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:

The test substance was evaluated as positive, negative, or equivocal in the assay.
Positive Response- The test substance was evaluated as positive if dose-dependent increases of 2-fold or greater in mutant frequency were obtained over the concurrent background mutant frequency. The background mutant frequency was defined as the average mutant frequency of the vehicle control cultures. The 2-fold or greater increase was based on extensive experience, which indicated such responses were repeatable in additional trials. It was desirable to obtain this relationship for at least three doses, but this goal depended on the dose steps chosen for the assay and toxicity at which mutagenic activity appeared. The dose-dependent requirement was waived if a large increase in mutant frequency (4-fold or higher) was obtained for a single dose at or near the highest testable toxicity. However, for the test substance to be evaluated as positive, any increase had to be repeatable in a second trial.
Negative Response- The test substance was evaluated as negative if a 2-fold increase in mutant frequency was not observed for (1) a range of doses that extended to toxicities causing 10 % to 20 % relative total growth, or (2) for a relatively nontoxic test substance, a range of doses extending to the maximum concentration of 5 mg/mL or 10 mM (whichever was lower), or (3) a range of doses that extended to a level approximately twice the solubility limit in culture medium or (4) the increase(s) were not repeatable in a confirmatory trial.
Equivocal Response- The test substance was evaluated as equivocal in the test system if there was no consistent evidence for either a positive or negative evaluation.

Statistics:

no data

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

No in vitro mutagenicity data of sufficient quality were available specifically on ammonium metatungstate (target substance). However, in vitro mutagencity data are available on sodium tungstate (source substance), which are used for read-across. Due to similar water solubility and toxicity for the target substance compared to the source substance, the resulting read-across from the source substance to the target substance is appropriate for this endpoint. In addition, read-across is appropriate because the classification and labelling is similar for the source substance and 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 read-across category approach description in the Category section of this IUCLID submission or Annex 3 of the CSR.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

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

Adequacy of study:

supporting study

Study period:

1989-02-13 to 1989-03-22

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

Well documented, scientifically sound study conducted according to OECD Guideline 471 (May 1983) following GLP. A S. typhimurium strain TA102 or an E. coli strain was not included as specified in the current guideline.

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 Metatungstate
Target: Ammonium metatungstate
3. CATEGORY APPROACH JUSTIFICATION: See Annex 3 in CSR
4. DATA MATRIX: See Annex 3 in CSR

Reason / purpose for cross-reference:

read-across: supporting information

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

version adopted May 26, 1983.

Deviations:

yes

Remarks:

A S. typhimurium strain TA102 or an E.coli strain was not included as per the current guideline.

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

histidine locus

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Additional strain / cell type characteristics:

other: See "any other information on materials and methods" section

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254 induced rat liver S9

Test concentrations with justification for top dose:

10, 100, 333.3, 1000 and 5000 ug/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Water bidest
- Justification for choice of solvent/vehicle: The solvent was chosen to its solubility properties and its relative non-toxicity for the bacteria.

Untreated negative controls:

yes

Remarks:

untreated

Negative solvent / vehicle controls:

yes

Remarks:

Water bidest (vehicle)

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

Without metabolic activation Migrated to IUCLID6: for strains TA1535 and TA100

Untreated negative controls:

yes

Remarks:

untreated

Negative solvent / vehicle controls:

yes

Remarks:

Water bidest (vehicle)

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 4-nitro-o-phenylene-diamine for strains TA1537 and TA98

Remarks:

Without metabolic activation

Untreated negative controls:

yes

Remarks:

untreated

Negative solvent / vehicle controls:

yes

Remarks:

Water bidest (vehicle)

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene for all strains tested.

Remarks:

With metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 72 hours
- Selection time (if incubation with a selection agent): 72 hours (simultaneous with exposure)


SELECTION AGENT (mutation assays): L-histidine


NUMBER OF REPLICATIONS: 3 plates per dose level and strain.


DETERMINATION OF CYTOTOXICITY
- Method: Cytotoxicity was evaluated by a reduction in the number of spontaneous revertants and/or clearing of bacterial background lawn.

OTHER: 2 independent experiments were performed.

Evaluation criteria:

The generally accepted conditions for the evaluation of the results were:
-corresponding background growth on both negative control and test plates
-normal range of spontaneous reversion rates.
Range of spontaneous reversion frequencies were as follows:
TA1535: 3-37 reversions/plate
TA1537: 4-31 reversions/plate
TA98: 15-60 reversions/plate
TA100: 75-200 reversions/plate
The test substance was considered as positive if either a significant dose-related increase in the number of revertants or a significant and reproducible increase of at least one test concentration was induced.
A test substance producing neither a significant dose-related increase in the number of revertants nor a significant and reproducible positive response at any one of the test points was considered non-mutagenic.
A significant response was described as follows:
The test substance was considered a mutagen if in strain TA100 the number of reversions was at least twice as high and in strains TA1535, TA1537 and TA98 it was at least three times higher as compared to the spontaneous reversion rates.
Also, a dose-dependent increase in the number of revertants was regarded as an indication of possible existing mutagenic potential of the test substance regardless whether the highest dose induced the above described enhancement factors or not.

Statistics:

Due to international guidelines a statistical evaluation of the results was recommended. However, no evaluated statistical procedures could be recommended for analysis of data from the bacterial assay at the time of study.

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98 and 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:

valid

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES: To evaluate the toxicity of the test substance a pre-study was performed with strains TA98 and TA100 in the absence and presence of metabolic activation. 8 concentrations (1-5000 ug/plate) were tested for toxicity and mutation induction with each 3 plates. The experimental conditions in the pre-study were the same as the main study. Toxicity of the test substance was evidenced by a reduction in the number of spontaneous revertants, a clearing of the bacterial background lawn, or by degree of survival of treated cultures. The plates with the test substance showed normal background growth up to 5000 ug/plate in both strains.

ADDITIONAL INFORMATION ON CYTOTOXICITY: No toxic effects, evidenced by a reduction in the number of spontaneous revertants, occurred in the test substance groups with and without metabolic activation up to 5000 ug/plate. In addition, the test substance plates showed normal background growth up to 5000 ug/plate with and without metabolic activation in all strains used.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Up to the highest investigated dose, no significant and reproducible dose-dependent increases in revertant colony numbers were obtained in any of the strains used. The presence of metabolic activation did not influence these findings.

A weak increase in the number of revertants in the strains TA1535 (exp. 1, without S9 mix at 100, 1000, and 5000 ug/plate and exp 2, without S9 mix at 1000 ug/plate and with S9 mix at 10 ug/plate); TA1537 (exp. 1, with S9 mix at 10, 100 and 5000 ug/plate); and TA98 (exp. 1, with S9 mix at 10 ug/plate) was identifid, however they were considered not to be relevant as no dose-dependency was obtained. The positive controls showed a significant increase in induced revertant colonies.

Conclusions:

Sodium metatungstate was assessed for its potential to induce gene mutations according to the plate incorporation test using Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100. Based on the results, it was concluded that the test substance did not induce point mutations by base pair changes or frameshifts in the genome of the strains used in the presence or absence of metabolic activation.

Executive summary:

No in vitro genotoxicity data of sufficient quality were available specifically on ammonium metatungstate (target substance). However, in vitro genotoxicity data are available on sodium metatungstate (source substance), which are used for read-across. Due to similar water solubility for the target substance compared to the source substance, the resulting read-across from the source substance to the target substance is appropriate to estimate of potential toxicity for this endpoint. 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.

Reference 4

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

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

Adequacy of study:

key study

Study period:

2003-07-16 to 2004-01-02

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

The study was conducted according to OECD guideline 471 and GLP. The reliability of this study for this 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 a K2.

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 metatungstate
3. CATEGORY APPROACH JUSTIFICATION: See Annex 3 in CSR
4. DATA MATRIX: See Annex 3 in CSR

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 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:

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:

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.

Executive summary:

No in vitro mutagenicity data of sufficient quality were available specifically on ammonium metatungstate (target substance). However, in vitro mutagencity data are available on sodium tungstate (source substance), which are used for read-across. Due to similar water solubility and toxicity for the target substance compared to the source substance, the resulting read-across from the source substance to the target substance is appropriate for this endpoint. In addition, read-across is appropriate because the classification and labelling is similar for the source substance and 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 read-across category approach description in the Category section of this IUCLID submission or Annex 3 of the CSR.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

No in vivo mutagenicity data of sufficient quality are available on ammonium metatungstate (target substance). However, in vivo mutagencity data are available on sodium metatungstate and sodium tungstate (source substances), which were used for read-across. Sodium metatungstate was negative for mutagenicity in an in vivo micronucleus assay conducted according to OECD 474. In addition, sodium tungstate was negative for mutagenicity in an OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test) . Based on the lack of in vivo mutagenicity of the source substances reported, ammonium metatungstate is not considered a mutagen.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

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

Adequacy of study:

supporting study

Study period:

1989-03-06 to 1989-06-16

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

Well documented, scientifically sound study conducted according to OECD Guideline 474 and GLP. While the reliability of this study for the substance tested is a K1, in application of read-across to a different substance ECHA’s guidance specifies that the score can be a maximum of K2.

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 Metatungstate
Target: Ammonium metatungstate
3. CATEGORY APPROACH JUSTIFICATION: See Annex 3 in CSR
4. DATA MATRIX: See Annex 3 in CSR

Reason / purpose for cross-reference:

read-across: supporting information

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

yes

Remarks:

Limited details were provided on test substance (i.e. purity was missing), relative humidity was not regulated.

GLP compliance:

yes

Type of assay:

micronucleus assay

Species:

mouse

Strain:

NMRI

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Wiga GmbH
- Age at study initiation: 10 weeks (at start of acclimatization)
- Weight at study initiation: approximately 30 g
- Fasting period before study: Yes; Approximately 18 hours before treatment the animals received no food but water ad libitum.
- Housing: Animals were housed singly in Makrolon Type I cages with wire mesh tops. Granulated soft wood bedding was provided.
- Diet: Pelleted standard diet was provided. No information was provided if it was available ad libitum.
- Water: Tap water was provided ad lbiitum.
- Acclimation period: minimum of 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 +/- 3 degrees C
- Humidity (%): Relative humidity was not regulated.
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: aqua dest.
- Justification for choice of solvent/vehicle: The vehicle was chosen due to its non-toxicity for the animals.
- Amount of vehicle (if gavage or dermal): 10 mL/kg
- Other: A vehicle control group was included for each sampling time.

Details on exposure:

At the beginning of the treatment the animals were weighed and the individual volume (10 mL/kg) to be administered was adjusted to the animal's body weight.

Frequency of treatment:

Animals were treated once by oral gavage.

Post exposure period:

24, 48 and 72 hours

Remarks:

Doses / Concentrations:
2000 mg/kg bw
Basis:
actual ingested

No. of animals per sex per dose:

6 per sex per sampling time.

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide
- Route of administration: oral gavage
- Doses / concentrations: 30 mg/kg
- The positive control animals were sampled at 24 hours.

Tissues and cell types examined:

bone marrow

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: The dose was chosen based on results from a pre-experiment where the maximum tolerated dose was determined. 4 animals per dose level received orally a single dose of 1000, 1500, 2000, 2500, or 3000 mg/kg bw, respectively, Sodium metatungstate dissolved in aqua dest.. The volume administered was 10 mL/kg bw..

On the basis of the pre-experiment toxicity, 2000 mg/kg bw was estimated to be the maximum tolerated dose.

TREATMENT AND SAMPLING TIMES: At the beginning of the treatment the animals were weighed and the individual volume to be administered was adjusted to the animal's body weight. The animals received the test article once. Sampling of the bone marrow was done at 24, 48 and 72 hours after treatment.

DETAILS OF SLIDE PREPARATION: The animals were sacrificed by cervical dislocation. The femora were removed, the epiphyses were cut off and the marrow was flushed out with 2.0 ml fetal calf serum, using a 5 ml syringe, into 1 ml fetal calf serum. The cell suspension was centrifuged at 1000 rpm for 5 minutes and the supernatant was discarded. A small drop of the resuspended cell pellet was spread on a slide. The smear was air-dried and then stained with May-Grunswald/Giemsa. Cover slips were mounted with EUKITT. At least one slide was made from each bone marrow sample.

METHOD OF ANALYSIS: Evaluation of the slides was performed using NIKON microscopes with 100x oil immersion objectives. 1000 PCEs were analyzed per animal for micronuclei. To describe a cytotoxic effect the ratio between polychromatic and normochromatic erythrocytes was determined in the same sample and expressed in normochromatic erythrocytes per 1000 PCEs. The analysis was performed with coded slides.

Five animals per sex and group were evaluated as described. The remaining animal of each test group was evaluated in case an animal had died in its test group spontaneously or due to gavage error.

Evaluation criteria:

The test substance was classified as mutagenic if it induced either a statistically significant dose-related increase in the number of micronucleated polychromatic erythrocytes or a reproducible statistically significant positive response for at least one of the test points. If the test substance produced neither a statistically significant dose-related increase in the number of micronucleated polychromatic erythrocytes nor a statistically significant and reproducible positive response at any one of the test points it was considered non-mutagenic in the system. Biological significance was considered along with the statistical significance.

Statistics:

Statistical significance was determined by means of the nonparametric Mann-Whitney test. (p<0.05)

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

apathy and death

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: 1000-3000 mg/kg bw
- Solubility: The test substance was soluble in aqua dest..
- Clinical signs of toxicity in test animals: 1000 mg/kg bw group-One male and one female expressed reduction of spontaneous activity followed by apathy. None of the animals died. 1500 mg/kg bw group-All animals expressed reduction of spontaneous activity followed by apathy. One male died shortly after administration. This lethal effect was considered not be induced by the test substance and was regarded to be due to gavage error. 2000 mg/kg bw group-All animals expressed reduction of spontaneous activity followed by apathy within a period of at least 6 hours after treatment. None of the animals died. 2500 mg/kg bw-All animals expressed reduction of spontaneous activity followed by apathy. Additionally, two males and one female expressed abdominal position and eyelid closure. One male died within 72 hours after treatment. 3000 mg/kg bw group- All animals expressed reduction of spontaneous activity, abdominal position, eyelid closure and apathy. Two males and 1 female died within 24 hours after treatment.
- Other: A preliminary study on acute toxicity was performed with the same strain of animals and under identical conditions as in the mutagenicity study. 4 animals (2 males and 2 females) per dose level received orally a single dose of the test substance in a volume of 10 ml/kg bw. On the basis of the results, 2000 mg/kg bw was estimated to be the maximum tolerated dose.

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): In comparison with the corresponding vehicle controls there was no substantial enhancement in the frequency of the detected micronuclei at preparation intervals 24 hours and 72 hour after application of the test substance. The mean values of micronuclei observed after treatment were in the same range as compared to the vehicle control groups. However, biometric analysis of the results demonstrated a significant increase of the micronucleus frequency after administration of the test substance at preparation interval 48 hours (p<0.05).
This statistical significant effect was considered to be of minor importance:
1. The corresponding actual vehicle control rate in the study was unusually low (0.01 % PCEs with micronuclei).
2. The micronucleus frequency of 0.07 % PCEs with micronuclei after treatment with 2000 mg/kg bw was still in the range of the historical control data presented.
3. The 48 hour value of 0.07 % did not substantially deviate from the data obtained at preparation interval 24 hours (0.10 %) and 72 hours (0.17%) after treatment with the test substance.
Therefore, the statistical significant response was not considered to be an indication of an induced mutagenic effect due to the test substance.
The positive control showed a distinct increase of induced micronucleus frequency.

- Ratio of PCE/NCE (for Micronucleus assay): The number of normochromatic erythrocytes was not increased after treatment with the test substance as compared to the mean values of NCEs of the corresponding vehicle controls, indicating that the test substance had no cytotoxic properties.

All animals treated with 2000 mg/kg bw expressed apathy within a period of at least 6 hours after treatment. One male and one female died at preparation interval 24 hours and one male and one female died at preparation interval 48 hours.

Conclusions:

Sodium metatungstate was assessed in the micronucleus assay for its potential to induce micronuclei in polychromatic erythrocytes in the bone marrow of the mouse at a dose level of 2000 mg/kg bw. Based on the results, it was determined that the test substance did not induce micronuclei as determined by the micronucleus test at preparation intervals of 24, 48 and 72 hours.

Executive summary:

No in vivo genotoxicity data of sufficient quality were available specifically on ammonium metatungstate (target substance). However, in vivo genotoxicity data are available on sodium metatungstate (source substance), which are used for read-across. Due to similar water solubility for the target substance compared to the source substance, the resulting read-across from the source substance to the target substance is appropriate to estimate of potential toxicity for this endpoint. 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.

Reference 2

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

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 2004-01-06

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

Study was conducted according to OECD guideline 474 and GLP. The reliability of this study for this 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 a K2.

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 metatungstate
3. CATEGORY APPROACH JUSTIFICATION: See Annex 3 in CSR
4. DATA MATRIX: See Annex 3 in CSR

Reason / purpose for cross-reference:

read-across: supporting information

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

micronucleus assay

Species:

mouse

Strain:

other: Crl:CD-1 (ICR)BR

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Portage MI
- Age at study initiation: 9 weeks at time of dosing
- Weight at study initiation: 30.3 to 37.9 grams at the time of dosing
- Assigned to test groups randomly: yes, by a computer program
- Housing: The animals were housed in sanitary polycarbonate cages containing Sani-Chips Hardwood Chip Laboratory bedding. The animals were housed, separated by gender, up to five animals per cage during acclimation, and by full dose group/harvest timepoint after randomization.
- Diet: PMI Feeds, Inc. Certified Rodent Diet #5002 ad libitum
- Water: tap water ad libitum
- Acclimation period: at least 5 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 64-79 degrees F
- Humidity (%): 30-70
- Air changes (per hr): at least 10 per hour
- Photoperiod (hrs dark / hrs light): 12 hours light/12 hours dark


IN-LIFE DATES: From: 2003-08-25 and 2003-08-26

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: corn oil
- Concentration of test material in vehicle: 25, 50, and 100 mg/ml for initial test and 75 mg/ml for repeat test
- Amount of vehicle (if gavage or dermal): 10 ml/kg
- Lot/batch no. (if required): 12-406

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: Prior to dosing, each concentration of the test substance was prepared by adding the appropriate volume of the vehicle to a pre-weighed quantitiy of the test substance and mixing, forming homogeneous suspensions. The formulations were held at room temperature prior to dosing and stirred during the dosing procedure.

Duration of treatment / exposure:

Animals received a single oral gavage dose of the test substance.

Frequency of treatment:

Animals received a single oral gavage dose of the test substance.

Post exposure period:

24 hours (all dose groups) and 48 hours (vehicle control, positive control, 750 mg/kg and 1000 mg/kg groups only)

Remarks:

Doses / Concentrations:
250, 500, and 1000 mg/kg
Basis:
actual ingested
initial assay

Remarks:

Doses / Concentrations:
750 mg/kg
Basis:
actual ingested
repeat assay

No. of animals per sex per dose:

6 male animals/dose/time point (only 5 animals/dose/time point were used for the actual analysis)

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide
- Route of administration: oral gavage
- Doses / concentrations: 80 mg/kg

Tissues and cell types examined:

erythrocytes (bone marrow)

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: The high dose in the micronucleus assay was the maximum tolerated dose determined by the range-finding study. This dose should have produced some indication of toxicity, e.g., toxic signs, death, or depression of the ratio of PCEs to normochromatic erythrocytes (NCEs).

TREATMENT AND SAMPLING TIMES (in addition to information in specific fields): 24 and 48 hours (vehicle and high dose group only

DETAILS OF SLIDE PREPARATION: At the appropriate harvest timepoint, the animals were euthanized by CO2 inhalation followed by incision of the diaphragm. The hind limb bones (tibias) were removed from marrow extraction from five surviving animals in each treatment and control group. For each animal, the marrow flushed from the bones was combined in an individual centrifuge tube containing 3 to 5 ml fetal bovine serum.
Following centrifugation to pellet the tissue, the supernatant was removed by aspiration and portions of the pellet were spread on slides and air-dried. The slides were fixed in methanol, stained in May-Grunswald solution followed by Giemsa, and protected by permanently mounted coverslips.

METHOD OF ANALYSIS: The slides were scored for micronuclei and the PCE to NCE cell ratio. The micronucleus frequency (expressed as percent micronucleated cells) was determined by analyzing the number of micronucleated PCEs from at least 2000 PCEs per animal. The PCE:NCE ratio was determined by scoring the number of PCEs and NCEs observed while scoring at least the first 500 erythrocytes per animal.
Micronuclei were darkly stained and generally round, although almond and ring shaped micronuclei occasionally occurred. Micronuclei were sharp bordered and generally between one-twentieth and one-fifth the size of the PCEs. The unit of scoring was the micronucleated cell, not the micronucleus; thus, the occasional cells with more than one micronucleus was counted as one micronucleated PCE, not two (or more) micronuclei.
The staining procedure permitted the differentiation by color of PCEs and NCEs (bluish-gray and red, respectively).
The historical background frequency of micronucleated cells was expressed as the percentage of micronucleated cells based on the number of PCEs analyzed.

Evaluation criteria:

The criteria for a positive response were the detection of a statistically significant increase in micronucleated PCEs for at least one dose level, and a statistically significant dose-related response. If both of these were not present, than the result was negative. Statistical significance was not the only determinate of a positive response; the Study Director also considered the biological relevance of the results in the final evaluation.

Statistics:

Assay data analysis was performed using an analysis of variance on untransformed proportions of cells with micronuclei per animal and on untransformed PCE:NCE ratios when the variances were homogenous. Ranked proportions were used for heterogeneous variances. If the analysis of variance was statistically significant (p<=0.05), a Dunnett's t-test was used to determine which dose groups, if any, were statistically significantly different from the vehicle control. Analyses were performed separately for each sampling time.

Sex:

male

Genotoxicity:

negative

Toxicity:

yes

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 mouse bone marrow micronucleus assay, under the conditions of this study.

Executive summary:

No in vivo mutagenicity data of sufficient quality were available specifically on ammonium metatungstate (target substance). However, in vivo mutagencity data are available on sodium tungstate (source substance), which are used for read-across. Due to similar water solubility and toxicity for the target substance compared to the source substance, the resulting read-across from the source substance to the target substance is appropriate for this endpoint. In addition, read-across is appropriate because the classification and labelling is similar for the source substance and 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 read-across category approach description in the Category section of this IUCLID submission or Annex 3 of the CSR.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

No in vitro or in vivo mutagenicity data are available for ammonium metatungstate (target substance). However, in vitro and in vivo mutagenicity data are available for sodium metatungstate and sodium tungstate (source substances), which were used for read-across. 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.

Justification for classification or non-classification

No mutagenicity data are available on ammonium metatungstate (target substance); however, data are available on sodium metatungstate and sodium tungstate (source substances). Sodium metatungstate was negative for mutagenicity in an in vitro bacterial reverse mutation assay and an in vivo micronucleus assay of sufficient quality. In addition, sodium tungstate was negative for mutagenicity in an in vitro chromosome aberration assay and an in vitro L5178Y TK+/- mouse lymphoma forward mutation assay, and in an in vivo Mammalian Erythrocyte Micronucleus Test of sufficient quality. Therefore, based on the weight-of-evidence from the available data of source substances, ammonium metatungstate does not warrant classification.