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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In an in vitro bacterial reverse mutation assays conducted according to OECD 471, tungsten blue oxide was negative for mutagenicity. No relevant mammalian, chromosomal aberration, and in vivo mutagencity studies are avialble on tungsten blue oxide. Therfeore, read across to tungsten trioxide and sodium tungstate (read across source substances) was conducted. In an in vitro chromosome aberration assay conducted according to OECD 473, tungsten trioxide was negative for mutagenicity. An in vitro L5178Y TK +/- Mouse Lymphoma Forward Mutation Assay data are available for sodium tungstate dihydrate and was negative for mutagenicity.

Link to relevant study records

Referenceopen allclose all

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:
2003-06-06 to 2003-06-29
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Well documented, scientifically sound study conducted according to GLP and OECD guideline 471 (Bacterial Reverse Mutation Assay).
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
Histidine locus
Species / strain / cell type:
other: TA97a, TA98, TA100, TA102 and TA1535
Additional strain / cell type characteristics:
other: See additional information on methods below
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254 induced rat liver S9
Test concentrations with justification for top dose:
- 62, 185, 556, 1667 and 5000 ug/plate
-The test substance solution of the highest concentration group was prepared by suspending 500 mg of the test substance in 10 mL of 0.5 % carboxymethyl cellulose. The suspensions for the lower concentrations were prepared by subsequent dilution of one volume of the higher concentrated solution with two volumes of 0.5 % carboxymethyl cellulose. The test substance was suspended and diluted immediately before the start of the Ames test. The time between the preparation and the administration to the last petri dish was less than 3 hours.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: 0.5 % carboxymethyl cellulose
Untreated negative controls:
yes
Remarks:
Vehicle control
Negative solvent / vehicle controls:
yes
Remarks:
0.5 % carboxymethyl cellulose
True negative controls:
no
Remarks:
N/A
Positive controls:
yes
Remarks:
N/A
Positive control substance:
other: 2-Aminoanthracene- used for tester strains TA98 (1 ug/plate), TA100 (2 ug/plate) and TA1535 ( 2 ug/plate)
Remarks:
with metabolic activation
Untreated negative controls:
yes
Remarks:
Vehicle control
Negative solvent / vehicle controls:
yes
Remarks:
0.5 % carboxymethyl cellulose
True negative controls:
no
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
Remarks:
with metabolic activation Migrated to IUCLID6: used for tester strain TA97a (10 ug/plate)
Untreated negative controls:
yes
Remarks:
Vehicle control
Negative solvent / vehicle controls:
yes
Remarks:
0.5 % carboxymethyl cellulose
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 1,8-Dihydroxy-anthraquinone- used for tester strain TA102 (50 ug/plate)
Remarks:
with metabolic activation
Untreated negative controls:
yes
Remarks:
Vehicle control
Negative solvent / vehicle controls:
yes
Remarks:
0.5 % carboxymethyl cellulose
True negative controls:
no
Positive controls:
yes
Positive control substance:
2-nitrofluorene
Remarks:
without metabolic activation Migrated to IUCLID6: used for tester strain TA98 (2 ug/plate)
Untreated negative controls:
yes
Remarks:
Vehicle control
Negative solvent / vehicle controls:
yes
Remarks:
0.5 % carboxymethyl cellulose
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
without metabolic activation Migrated to IUCLID6: used for tester strains TA100 (2 ug/plate) and TA1535 (1 ug/plate)
Untreated negative controls:
yes
Remarks:
Vehicle control
Negative solvent / vehicle controls:
yes
Remarks:
0.5 % carboxymethyl cellulose
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 4-Nitro-o-phenylenediamine- used for tester strain TA97a (10 ug/plate)
Remarks:
without metabolic activation
Untreated negative controls:
yes
Remarks:
Vehicle control
Negative solvent / vehicle controls:
yes
Remarks:
0.5 % carboxymethyl cellulose
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: t-Butyl-hydroperoxide- used for tester strain TA102 (50 ug/plate)
Remarks:
without metabolic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION
-In agar (plate incorporation).

DURATION
- Exposure duration: 2 days
- Selection time (if incubation with a selection agent): 2 days

SELECTION AGENT (mutation assays)
-Histidine

NUMBER OF REPLICATIONS
-3 replications were used for the test substance and positive control, and 6 replications were used for the solvent control.

NUMBER OF CELLS EVALUATED
-The number of viable cells in the overnight-cultures was in the range of 2 x E8 cells/mL.

DETERMINATION OF CYTOTOXICITY
- Method: Reduction in bacterial background lawn.


OTHER: The actual batch of the strains was tested for ampicillin resistance (TA102: ampicillin/tetracycline resistance), UV-sensitivity and sensitivity against crystal violet, for spontaneous mutation frequencies and for sensitivities against the positive control substances in September 2000. The bacteria were store frozen since that time.
Evaluation criteria:
Means and standard deviations were calculated for the number of mutants in every concentration group.
The criteria for a positive result were:
A reproducible increase of the number of revertants to more than the following threshold values for at least one of the concentrations:
-For the strains with a low spontaneous revertant rate (TA98 and TA1535): 2 1/2 fold of the amount of the spontaneous revertants.
-For the strains with a high spontaneous revertant rate (TA97a, TA100 and TA1020: 1 2/3 fold of the amount of the spontaneous revertants.
Key result
Species / strain:
other: TA97a, TA98, TA100, TA102 and TA1535
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: 118 mg/L at 20 degrees C
- Precipitation: Although the test substance was not soluble, the particles were so fine that they were not visible in any of the concentration groups.

RANGE-FINDING/SCREENING STUDIES
The test substance was evaluated in a preliminary toxicity test using the Plate Incorporation Assay method. The test substance was tested at concentrations ranging from 21-5000 ug/plate in tester strain TA100 in the absence of metabolic activation. The test substance was not toxic up to 5000 ug/plate. It was therefore decided to use 5000 ug/plate as the highest concentration for the Ames Assay, which was the limit concentration according to the guidelines. Each of the other 4 concentrations were 1/3 of the preceding one.

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

OTHER
All positive control substances increased the mutation frequency to more than the threshold values stated above. As 2-aminoanthracene, 1,8-dihydroxy-anthraquinone and 7,12-dimethyl-benz[a]anthracene require metabolic activation for mutagenicity, the result of these substances demonstrated the efficiency of the metabolizing system.
There were no substantial increases in the number of mutants in any of the tested bacterial strains at any of the tested concentrations of the test substance. The addition of S9 metabolic activation did not change these results.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

The results were verified by a second independent experiment.

Conclusions:
Interpretation of results (migrated information):
negative

Under the conditions of this assay, Tungsten Oxide Blue was non-mutagenic in the Ames test with Salmonella typhimurium strains TA97a, TA98, TA100, TA102 and TA1535 in the presence and absence of metabolic activation up to 5000 ug/plate, which was the limit concentration for this substance.
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:
2009-09-15 to 2010-06-07
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. Due to similar water solubility, in vitro bioaccessibility in synthetic alveolar, lysosomal, interstitial, sweat, and gastric fluids, and available toxicity data for the target (TBO) and source (WO3) substances, the resulting toxicity potential would also be expected to be similar so read across is appropriate between these substances. In addition, read across is appropriate for this endpoint because the classification and labeling is the same for the source and target substances, the PBT/vPvB profile is the same, and the dose descriptors are, or are expected to be, sufficiently similar or more conservative for the target substance. For more details refer to the attached description of the read across approach.
Justification for type of information:
REPORTING FORMAT FOR THE CATEGORY APPROACH
1. HYPOTHESIS FOR THE ANALOGUE 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: Tungsten trioxide
Target: Tungsten blue oxide
3. CATEGORY APPROACH JUSTIFICATION: See Annex 1 in CSR
4. DATA MATRIX: See Annex 1 in CSR
Qualifier:
according to
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 cultures used for treatment were initiated in McCoy's 5A medium supplemented with approximately 10 % Fetal Bovine Serum (FBS), penicillin G (100 units/mL), streptomycin (100 ug/mL) and L-glutamine (at least 2 mM). This medium was referred to as complete McCoy's 5A culture medium. The cultures were incubated at 37 +/- 2 degrees C, in a humidified atmosphere of 5 +/- 1.5 % CO2. On the day following cell seeding, the culture medium was replaced with fresh serum free medium for the 3 hour exposures with and without metabolic activation and with fresh complete medium for the 24 hour exposure without metabolic activation. After the 3 hour exposure, cultures were rinsed and fresh complete medium was added.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes/no
- Periodically "cleansed" against high spontaneous background: yes/no
- Other: CHO cells exposed to test substances in vitro were routinely used to detect clastogenicity. The cell line used in this study, obtained from Merck Research Laboratories (West Point, PA), was sub-cloned at IITRI, aliquoted into freezing vials and stored in liquid nitrogen. Stock cultures were maintained for up to 12 weeks after thawing a vial from frozen stock.
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254 induced rat liver S9
Test concentrations with justification for top dose:
Experiment 1/cytotoxicity test with and without metabolic activation - 0, 0.01, 0.02, 0.039, 0.078, 0.156, 0.313, 0.625, 1.25, 2.5 and 5 mg/mL
Experiment 2 with metabolic activation-0, 0.50, 0.75, 1.0, 1.5, 2.0, 2.5 and 3.0 mg/mL
Experiment 2 without metabolic activation-0, 0.10, 0.20, 0.25, 0.50, 0.75, 1.0, 1.5, 2.0, 2.5 and 3.0 mg/mL
The maximum target concentration of test substance, 5.0 mg/mL, was based upon the upper limit for testing in OECD Guideline 473.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: serum-free McCoy's 5A cell culture media
- Justification for choice of solvent/vehicle: no data
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
serum-free McCoy's 5A cell culture media
True negative controls:
no
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
without metabolic activation Migrated to IUCLID6: 0.5 ug/mL
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
serum-free McCoy's 5A cell culture media
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
with metabolic activation Migrated to IUCLID6: 10 ug/mL
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium


DURATION
- Exposure duration: 3 hours (Experiment 1 with and without metabolic activation and Experiment 2 with metabolic activation), 24 hours (Experiment 2 without metabolic activation)
- Expression time (cells in growth medium): ~19 hours (3 hour treatment) and 0 hours (24 hour treatment)
- Fixation time (start of exposure up to fixation or harvest of cells): Cultures were harvested 24 hours after initiation of treatment. The cultures were trypsinized and a measured aliquot of cells were counted (using a hemacytometer and trypan blue). After centrifuging the cell suspension, the pelleted cells were re-suspended in at least 10 mL of 0.075M KCl hypotonic solution and incubated. This treatment helps to swell the cells and disperse the chromosomes on a 'plate' such that each chromosome is visible. After the treatment with hypotonic buffer, a 0.5 mL aliquot of Carnoy's fixative (3 parts methanol: 1 part glacial acetic acid) was added to each tube and the cells were centrifuged and the supernatant discarded. The cell pellets were re-suspended in Carnoy's fixative and centrifuged twice, and then they were stored refrigerated in fresh Carnoy's fixative until they were dropped onto glass slides.


SPINDLE INHIBITOR (cytogenetic assays): Colcemid
STAIN (for cytogenetic assays): 5 % Giemsa


NUMBER OF REPLICATIONS:
2

NUMBER OF CELLS EVALUATED:
100 cells per replicate were evaluated for all groups except the positive control in which 25-50 cells per replicate were evaluated.

DETERMINATION OF CYTOTOXICITY
- Method: At least 1000 cells per slide were counted to establish a mitotic index for each slide selected for analysis based on cytotoxicity. After the mitotic index was determined for each culture, dose levels were selected for chromosome analysis. Cytotoxicity was measured as the percent cell number reduction relative to the vehicle control cultures.


OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: yes
- Other: Chromatid, and isochromatid gaps and uncoiled chromosomes were noted but were not added into the totals for structural aberration assessment since they are not considered to represent true breaks. In addition Gaps were recorded separately and reported but were not included in the total aberration frequency. The cultures were assessed for test substance precipitation at the time of dosing and after three hours in the 3 hour exposure cultures and at harvest in the 24 hour exposure. The osmolality and pH of at least the highest dosing solution concentration in culture medium were measured. If the resulting measurements record an upward shift in the osmolality by 50 mOsm or more (relative to the vehicle) or a shift in pH of one pH unit or more (up or down), relative to the vehicle The Sponsor would by notified.

Evaluation criteria:
Once the assay acceptance criteria had been met, the following factors were taken into account in evaluation of the test substance data:
-Percentage of cells with structural aberrations
-Percentage of cells with more than one structural aberration
-Evidence for increasing amounts of damage with increasing test substance concentration
Since the experimental unit was the cell, the percentage of cells with structural aberrations was the basis of evaluation for clastogenicity. The test substance was considered positive for inducing chromosomal aberrations if a significant increase (ANOVA), pThe test substance was considered negative for inducing chromosomal aberration if no statistically significant increase was observed in the percentage of cells with chromosomal aberrations at any of the concentrations.
Statistics:
Statistical analysis consisted of a one-way analysis of variance (ANOVA) to compare the percentages of cells with aberrations in treated cells with the percentage of cells with aberrations from the vehicle controls. No statistical analysis was used to assess polyploidy and/or endoreduplication.
Key result
Species / strain:
Chinese hamster Ovary (CHO)
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
- Effects of pH:
- Effects of osmolality:
- Evaporation from medium:
- Precipitation: The test substance formed a yellow precipitate at most of the concentrations tested and was formulated as a suspension.

RANGE-FINDING/SCREENING STUDIES:
A cytotoxicity experiment was performed. Since acceptable cytotoxicity criteria were met by at least three of the dose levels of the test substance within an exposure time group, dose levels were selected for analysis of chromosome aberrations. This cytotoxicity experiment was reported as experiment 1 described above.

COMPARISON WITH HISTORICAL CONTROL DATA:
no data

ADDITIONAL INFORMATION ON CYTOTOXICITY:
1. Experiment 1 with metabolic activation-The lowest concentration that approximated at 50% reduction in cell number relative to the vehicle control (i.e., 2.5 mg/mL) that did not have an obscuring level of precipitation was selected as the highest dose level for the chromosome aberration analysis. The cytotoxicity at the 2.5 mg/mL concentration was approximately 58 %. The remaining concentrations selected for analysis were 0.078 and 1.25 mg/mL. Percent reductions in cell numbers of approximately 0 % and 51 %, (relative to the vehicle control) were observed at these dose levels, respectively.
2. Experiment 1 without metabolic activation-The lowest concentration that approximated a 50 % reduction in cell number relative to the vehicle control (i.e., 2.5 mg/mL) and did not have an obscuring level of precipitation was selected as the highest dose level for the chromosome aberration analysis. The cytotoxicity at the 2.5 mg/mL concentration was approximately 45 % The emaining concentrations selected for analysis were 0.156 and 1.25 mg/mL. Percent reductions in cell numbers of approximately 5 % and 25 %, (relative to the vehicle control) were observed at these dose levels, respectively.
3. Experiment 2 with metabolic activation-The lowest concentration that approximated a 50 % reduction in cell number relative to the vehicle control (i.e., 1.5 mg/mL) and did not have an obscuring level of precipitation was selected as the highest dose level for the chromosome aberration analysis. The cytotoxicity at the 1.5 mg/mL concentration was approximately 39 %. The remaining concentrations selected for analysis were 0.75 and 1.0 mg/mL. Percent reductions in cell numbers of approximately 39 % and 18 %, (relative to the vehicle control) were observed at these dose levels, respectively.
4. Experiment 2 without metabolic activation-Chromosome aberrations were evaluated from the cultures treated with concentrations of 1.0, 1.5 and 2.0 mg/mL. The reduction in cell numbers at these dose levels, relative to the vehicle control were 38 %, 40 % and 70 %, respectively.
Remarks on result:
other: all strains/cell types tested

Data Acceptability Criteria: The vehicle control cultures (with and without activation) were within the acceptance criteria of less than approximately 5 % cells with aberrations. The sensitivity of the cell cultures for the induction of chromosomal aberrations was shown by an increased frequency of aberrations in the cells exposed to the positive controls. In trials utilizing metabolic activation, a significant positive response to the positive control treatment also demonstrated the integrity of the activation system. Toxicity was seen as the percent of cell number reduction of the treated groups relative to the vehicle controls. At least three concentrations were analyzed for aberrations for each condition tested.

Dosing Formulation Analysis:

Analysis of the dose formulations showed that the target concentrations for the test substance were not verified. This was most likely due to the nature of the suspension of the test substance in culture media when samples for analytical chemistry were collected. Since the target concentrations could not be verified other factors were used to select dosing levels for analysis (cytotoxicity and test substance precipitation).

Chromosome Aberration Induction:

1. Experiment 1 with metabolic activation-No significant increase in cells with structural chromosomal aberrations was observed in the cultures treated with the test substance relative to the vehicle control. The percentage of polyploid and endoreduplicated cells was slightly elevated relative to the vehicle control in the presence of metabolic activation but these elevations were not statistically significant. The positive control chromosome aberration response was elevated relative to the vehicle and the response was statistically significant (t-test, p</=0.01).

2. Experiment 1 without metabolic activation- No significant increase in cells with structural chromosomal aberrations was observed in the cultures treated with the test substance relative to the vehicle control. The percent of polyploid and endoreduplicated cells was not significantly elevated over the vehicle control vales at any of the doses tested in the absence of metabolic activation. The positive control chromosome aberration response was elevated relative to the vehicle and the response was statistically significant (t-test, p</=0.01).

3. Experiment 2 with metabolic activation-No significant increases in cells with structural chromosomal aberrations were observed in the test substance treated cultures relative to the vehicle control. The percentage of polyploid and endoreduplicated cells was not elevated relative to the vehicle control in the absence of metabolic activation. The positive control chromosome aberration response was elevated relative to the vehicle and the response was statistically significant (t-test, p</=0.01).

4. Experiment 2 without metabolic activation- No significant increases in cells with structural chromosomal aberrations were observed in the cultures treated with the test substance relative to the vehicle control. The percentage of polyploid and endoreduplicated cells was not elevated relative to the vehicle control in the absence of metabolic activation. The positive control chromosome aberration response was elevated relative to the vehicle and the response was statistically significant (t-test, p</=0.01).

Conclusions:
Interpretation of results: negative with and without metabolic activation

The test substance was evaluated for potential clastogenic effects using the structural chromosome aberration assay in Chinese hamster ovary cells, with and without metabolic activation. The test substance failed to induce any clastogenic responses in the CHO cells when tested in the presence and absence of metabolic activation up to dose levels that resulted in cytotoxicity and/or precipitation.


Executive summary:

No in vitro chromosomal aberration data of sufficient quality are available for tungsten oxide (target substance). However, in vitro chromosomal aberration data are available for sodium tungstate (source substance), which will be used for reading across. Due to lower water solubility and lower toxicity for the target substance compared to the source substance, the resulting read across from the source substance to the target substance is appropriate as a conservative estimate of potential toxicity for this endpoint. In addition, read across is appropriate because the classification and labelling is more protective for the source substance than the target substance, the PBT/vPvB profile is the same, and the dose descriptors are, or are expected to be, lower for the source substance. For more details, refer to the read-across category approach included in the Category section of this IUCLID submission and/or as an Annex in the CSR.

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. Due to lower water solubility and lower toxicity values for the target substance (TBO) 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:
REPORTING FORMAT FOR THE CATEGORY APPROACH
1. HYPOTHESIS FOR THE ANALOGUE 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: Tungsten blue oxide
3. CATEGORY APPROACH JUSTIFICATION: See Annex 1 in CSR
4. DATA MATRIX: See Annex 1 in CSR
Qualifier:
according to
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 E6 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 in Any other information on materials and methods Section
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 E-6) was slightly above historical data range (36.4 to 135.7 x E-6) for mutant frequency. In this same assay one of the positive control cultures (522.0 x E-6) was also above the historical data range (227.0 to 487.4 x E-6) for mutant frequency. In addition, one vehicle control culture (139.1 x E-6) in the initial mutation assay with activation was slightly above the historical data range (34.0 to 123.9 x E-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:
Interpretation of results:negative

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 under conditions of this study.
Executive summary:

No in vitro mammalian mutagenicity data of sufficient quality are available for tungsten oxide (target substance). However, in vitro mammalian mutagenicity data are available for sodium tungstate (source substance), which will be used for reading across. Due to lower water solubility and lower toxicity for the target substance compared to the source substance, the resulting read across from the source substance to the target substance is appropriate as a conservative estimate of potential toxicity for this endpoint. In addition, read across is appropriate because the classification and labelling is more protective for the source substance than the target substance, the PBT/vPvB profile is the same, and the dose descriptors are, or are expected to be, lower for the source substance. For more details, refer to the read-across category approach included in the Category section of this IUCLID submission and/or as an Annex in the CSR.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

No in vivo mutagencity study on tungsten blue oxide is available. Therefore, the endpint was read across to sodium tungstate. Sodium tungstate was negative for mutagenicity in an in vivo micronucleus assay conducted according to OECD 474.

Link to relevant study records
Reference
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:
study well documented, meets generally accepted scientific principles, acceptable for assessment
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 lower water solubility and lower toxicity values for the target substance (TBO) 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:
REPORTING FORMAT FOR THE CATEGORY APPROACH
1. HYPOTHESIS FOR THE ANALOGUE 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: Tungsten blue oxide
3. CATEGORY APPROACH JUSTIFICATION: See Annex 1 in CSR
4. DATA MATRIX: See Annex 1 in CSR
Qualifier:
according to
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 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 (e.g. ad libitum): PMI Feeds, Inc. Certified Rodent Diet #5002 ad libitum
- Water (e.g. ad libitum): 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.
Key result
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.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

No in vitro L5178Y TK +/- Mouse Lymphoma Forward Mutation Assay data are available for tungsten trioxide (target substance). However, in vitro L5178Y TK +/- Mouse Lymphoma Forward Mutation Assay data are available for sodium tungstate (source substance), which will be used for read-across. Due to lower water solubility and lower toxicity values for the target substance (tungsten trioxide) 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 labelling is more protective for the source substance than the target substance, the PBT/vPvB profile is the same, and the dose descriptors are, or are expected to be, conservative for the target substance. For more details, refer to the attached description of the read-across approach. In addition, although not required under REACH because of the lack of mutagenicity observed in the available in vitro mutagenicity assays, an in vivo micronucleus assay on sodium tungstate was also used for read across to support the weight of evidence that tungsten trioxide is not a mutagen.

Justification for classification or non-classification

One in vitro reverse gene mutation assay on tungsten blue oxide and an in vitro chromosome aberration assay of sufficient quality on tungsten trioxide were negative for mutagenicity. In addition, an in vitro L5178Y TK +/- Mouse Lymphoma Forward Mutation Assay and an in vivo micronucleus assay conducted according to OECD 474 on sodium tungstate dihydrate, used for read-across to tungsten blue oxide, were negative for mutagenicity. Therefore, based on the weight-of-evidence from the available data, tungsten blue oxide does not warrant classification for mutagenicity.