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Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In an in vitro bacterial gene mutation assay conducted according to OECD 471, two in vitro chromosome aberration assays conducted according to OECD 473, and an in vitro micronucleus assay, tungsten carbide (WC) was deemed to be negative for mutagenicity. No in vitro L5178Y TK +/- mouse lymphoma forward mutation assay data are available for WC (target substance). However, in vitro L5178Y TK +/- mouse lymphoma forward mutation assay data are available for tungsten (source substance), which are used for read-across. 

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Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Reliability assigned by the secondary source. Only the secondary source reviewed.
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A pKM 101
Metabolic activation:
with and without
Metabolic activation system:
Aroclor induced rat liver S9 mix
Test concentrations with justification for top dose:
5, 15, 50, 150, 500, 1500, 5000 µg/plate
Untreated negative controls:
yes
Positive controls:
yes
Details on test system and experimental conditions:
Type: Ames test
METHOD OF APPLICATION: 2 independent assays were performed: range finding (standard plate incorporation) and repeat (including a pre-incubation stage)

Statistics:
no data
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
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
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
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
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A pKM 101
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Positive controls validity:
valid
Additional information on results:
No signs of toxicity were noted toward any of the tested strains at any dose. No substantial increase in revertant colony numbers over control counts were obtained with any of the tester strains at any tungsten carbide concentration in either the presence or the absence of S9 mix. The positive controls were functional.
Conclusions:
Interpretation of results (migrated information):
negative
Endpoint:
in vitro cytogenicity / micronucleus study
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
data from handbook or collection of data
Remarks:
Well documented scientifically sound study with sufficient information provided on methods and results to evaluate data. Reliability assigned by secondary source. Primary and secondary source reviewed.
Qualifier:
equivalent or similar to guideline
Guideline:
other: OECD TG 487 In vitro Micronucleus assay
Deviations:
yes
Remarks:
: percentage of binucleated cells in control group was 38% vs. recommended 50%. Treatment time was only 15 minutes vs. the recommended 3-6 hours.
Principles of method if other than guideline:
The assay was performed according to the method of Fenech and Morley, 1985 which with some minor exceptions is comparable to draft method OECD 487.
GLP compliance:
not specified
Type of assay:
in vitro mammalian cell micronucleus test
Species / strain / cell type:
lymphocytes: human peripheral blood
Additional strain / cell type characteristics:
not specified
Metabolic activation:
without
Test concentrations with justification for top dose:
10, 50, 100 ug/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Sterile deionized water
Untreated negative controls:
yes
Remarks:
no further data provided
Negative solvent / vehicle controls:
not specified
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
other: MMC
Details on test system and experimental conditions:
METHOD: Blood samples were drawn by venipuncture from two donors (one male and one female; age < 28 years) into heparinized Vacutainer tubes. The blood was diluted 1:1 with PBS and layered on top of Ficolt paque. After 40 min centrifugation at 400 g, the ring of PBMC was recovered and the cells were washed with phosphate buffered saline (PBS). The cells were cultured in Hann's F10 medium supplemented with 15% foetal calf serum and 2% purified phytohemaglobin HA (PHA) - (duplicate 1 mL aliquots kept at 37 °C).
24 hours after the onset of PHA stimulation, 10 uL of test substance wass added to the cells. After 15 minutes of exposure (37 °C), the cultures were centrifuged at 400 g (10 minutes), after which the supernatant was removed and the cells were resuspended in PBS or fresh medium while keeping the cultures on a magnetic source to remove the majority of the particles. The cells were further incubated.
Cytochalasin B was added 44 hours after the start of the culture to block cytokinesis, and after a total of 72 hours the cells were cytospinned onto a normal microscope slide, fixed with 100% methanol and stained with 5% Giemsa in Sorensen buffer.Slides from duplicate cultures were analysed for each treatment point. A sample of 1000 cytokinesis-blocked lymphocytes were examined per culture for the presence of micronuclei. In addition, the percentages of bionucleated cells, polynucleated cells, mitotic figures and mononucleated cells with micronuclei were recorded. As a measure for cell cycle delay and/or cytotoxicity, the relative division index was calculated based on 1000 cells/culture.

STAIN (for cytogenetic assays): %5 Giemsa in Sorensen buffer
Statistics:
The statistical analysis of the dose-effect relationship was performed for each donor separately. An effect was considered positive when either a statistically significant trend was obtained or when in the absence of this trend, at least two of three tested doses were statistically significantly different from the concurrent control. Since both analyses were performed in parallel, the Bosferoni correction of the significance level was applied. Differences between treated and control samples were determined by Student's t-test. The dose dependency was evaluated by a trend test. Comparison of the chromosome/genome mutation frequencies in the micronucleus test and DNA migration in the Comet assay were performed by Pearson's correlation analysis. All analyses were performed using SPSS and Statview software packages.
Species / strain:
lymphocytes: human peripheral blood
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
not specified
Untreated negative controls validity:
valid
Positive controls validity:
valid
Conclusions:
The test substance did not result in an increased frequency in the percentage of micronuclei in 1000 cytokinesis blocked binucleated lymphocytes.
Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
data from handbook or collection of data
Remarks:
Reliability assigned by secondary source. Only secondary source reviewed.
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test
Species / strain / cell type:
lymphocytes: human peripheral blood
Metabolic activation:
with and without
Metabolic activation system:
rat-liver S-9 mix
Test concentrations with justification for top dose:
2.44, 4.88, 9.77, 19.53, 39.06, 78.13, 156.25 and 312.5 ug/mL
Tungsten carbide was suspended in culture medium. As the suspension showed precipitates at 312.5 ug/mL, this was selected as the highest dose.
True negative controls:
yes
Positive controls:
yes
Details on test system and experimental conditions:
Lymphocytes were incubated with tungsten carbide for 3 hours with and without rat-liver S9-mix (first test), then washed and further incubated for 17 hours for recovery. In the second test incubation was continuously for 20 hours without S9 mix or for 3 hours with rat-liver S9-mix like in the first test.

No cytotoxicity was found at the highest dose tested. Mitotic indices were determined for 1000 cells; metaphase analysis was performed for 100 cells. Polyploidy was analysed for 500 cells each of untreated control and highest tungsten carbide concentration tested.
Statistics:
no data
Species / strain:
lymphocytes: human peripheral blood
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
other: high values (mean = 3% excluding gaps)
Positive controls validity:
valid
Additional information on results:
Two separate tests were performed. In both tests no cytoxicity of tungsten carbide was observed towards human lymphocytes. There was no increase in the number of polyploid metaphase cells compared to the solvent control.

First test:
- Without S9 mix: no biologically relevant increases of chromosomal aberrations at any tungsten carbide concentration.
- With S9 mix: statistically significant increase in number of chromosomal aberrations at 78.13 and 156.25 µg/ml were identified when compared to solvent control. No reproducible values were observed between replicate cultures at 78.13 µg/ml. However, cultures exposed to tungsten carbide contained cells with a frequency of aberrations (excluding gaps) that exceeded the upper 99 % limit of the historical negative control range at dose levels of 78.13 µg/ml and above.

Second test:
- Without S9 mix: no biologically relevant increases of chromosomal aberrations at any tungsten carbide concentration.
- With S9 mix: no statistically singnificant increase in number of chromosomal aberrations at any tungsten carbide concentration. However, the statistical significance was decreased due to high solvent control values (mean = 3 % excluding gaps) and cultures exposed to tungsten carbide at the two highest concentrations contained cells with a frequency of aberrations (excluding gaps) that exceeded the upper 99 % limit of the historical negative control range. The positive controls were functional.
Remarks on result:
other: other: Human lymphocytes
Remarks:
Migrated from field 'Test system'.
Conclusions:
Under the conditions of this assay the test substance exhibited no evidence of clastogenic activity in the absence of S9 mix and equivocal evidence of clastogenic activity in the presence of S9 mix.
Endpoint:
in vitro cytogenicity / micronucleus study
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Remarks:
Well documented scientifically sound study with sufficient methods and results provided to evaluate data.
Reason / purpose for cross-reference:
reference to same study
Qualifier:
equivalent or similar to guideline
Guideline:
other: draft OECD 487: In Vitro Micronucleus Test
Deviations:
yes
Remarks:
(1) No exogenous metabolic activation. (2) The highest test concentration did not achieve 50% cytotoxicity. (3) treatment time was 15 minutes vs. recommended 3-6 hours.
GLP compliance:
not specified
Type of assay:
in vitro mammalian cell micronucleus test
Species / strain / cell type:
lymphocytes: human
Details on mammalian cell type (if applicable):
- Human peripheral blood samples were drawn from the same health volunteer (age ≤ 30 years) by venipuncture in heparinized tubes.
- Leukocytes (lymphocytes + monocytes) were isolated on a Ficoll-Paque gradient, washed with PBS and resuspended in fresh medium containing 15% fetal calf serum (FCS).
- Lymphocytes were stimulated to divide by 2% phytohemagglutinin (PHA).
- Cultures were set up at a concentration of 0.5e-6 cells/mL in glass tubes and incubated at 37 degrees C.
Metabolic activation:
without
Test concentrations with justification for top dose:
0, 10, 50, 75 and 100 ug/mL
Vehicle / solvent:
- Water a.d.
Negative solvent / vehicle controls:
yes
Remarks:
Water a.d.
Positive controls:
yes
Remarks:
Mitomycin C (MMC) at 0.15 ug/mL
Details on test system and experimental conditions:
- After 24 hours of Phytohemaglobin stimulation, tungsten carbide particles were dispersed in 1 mL cultures.
- Water a.d. (10 uL) was used as metal vehicle.
- Lymphocytes were incubated at 37 °C for 15 minutes, and then further incubated for a total of 72 hours.
- Cytochalasin B (6 ug/mL) was added after 44 hours to block cytokinesis.
- After the total 72 hours, the cells were placed on slides using a cytospin.
- All slides were fixed in 100% methanol for 20 minutes and stained with 5% Giemsa in Sörensen buffer (pH 6.8).
- Cell processing was performed as described by Singh et al (1988), with some modifications.
Evaluation criteria:
- Duplicate cultures were analysed for each dose tested: 1000 cytokinesis-blocked (binucleated) lymphocytes (CBs) were examined per culture for the presence of one, two, or three micronuclei.
- The percentages of binucleated cells with micronuclei were recorded.
- As a measures for cell cycle delay and/or cytotoxicity, the relative division index (RDI) was used: RDI = [(CB + 2polyN)/n treated sample]/[(CB + 2polyN)/n control sample] x 100.
- All slides were coded and analysed at 1250 X magnification.
Statistics:
- Statistical differences between controls and treated samples were determined with the chi-square test.
Species / strain:
lymphocytes: human
Metabolic activation:
without
Genotoxicity:
ambiguous
Remarks:
Positive only at low doses, no dose dependent increase was identified.
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
At highest concentration tested
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
- The relative division index was 95, 73, 88 and 68 for the 10, 50, 75 and 100 ug/mL groups, respectively.
- A statistically significant, but non-dose-dependent increase of multi-nucleated cytokinesis blocked cells (MNCB) was observed after tungsten carbide (WC) treatment at concentrations > 10 ug/mL (p < 0.01). The test substance was only able to induce a statistically significant increase at low doses. No further dose dependent increases were noted.
- A decrease of the percentage of Cytokinesis blocked cells was observed at the 100 µg/mL concentration only.
- Data obtained from the regression analysis comparing mean tail length and frequencies of MNCB determined after exposure of leukocytes from the same donor for 15 minutes to WC (r = 0.95) were consistent with a causal relationship between DNA breakage and micronucleus induction (not obvious for WC).

Metal particles were removed using a magnetic source for some compounds evaluated in conjunction with this assay. It is unclear if this procedure was used for the assay with tungsten carbide particles and whether the magnetic source was used for the negative control as well. This is significant, as magnetic fields alone have been reported to be able to induce chromosomal damage in lymphocytes.

Conclusions:
A non-dose-dependent, statistically significant increase in the percentage of binucleated cells containing micronuclei was observed in human leukocytes exposed to tungsten carbide. Increases were only noted at low doses. Based on the lack of dose dependency, the results were deemed equivocal.
Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2007-02-27 to 2008-01-23
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
The study was performed according to OECD guideline 473 with minor deviations that did not impact the validity of the study. GLP study.
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
yes
Remarks:
however none that would affect the validity of the study.
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test
Species / strain / cell type:
other: Chinese hamster lung (CHL) cells
Details on mammalian cell type (if applicable):
- Type and identity of media: McCoy's 5A medium supplemented with 10 % heat inactivated foetal calf serum and 100 ug/mL gentamycin
Metabolic activation:
with and without
Metabolic activation system:
Aroclor induced rat liver S9
Test concentrations with justification for top dose:
Experiment 1
- 3-hour treatment without S9: 125.0, 250.0, 375.0, 500.0, 625.0, 750.0, 875.0, 1000, 1500, 2000, 2500, 3000, 3750, 4500 and 4997 ug/mL
- 3-hour treatment with S9: 125.0, 250.0, 500.0, 750.0, 1000, 1250, 1500, 1750, 2000, 2250, 2500 and 3000 ug/mL

Experiment 2, Trial 1
- 20-hour treatment in the absence of S9: 50.0, 100.0, 200.0, 300.0, 400.0, 500.0, 600.0, 750.0, 900.0, 1100, 1250, 1500 and 2000 ug/mL
- 3-hour treatment in the presence of S9: 250.0, 500.0, 1000, 1250, 1500, 1750, 2000, 2250, 2500, 2750 and 3000 ug/mL

Experiment 2, Trial 2
- 3 hour treatment in the presence of S9: 250.0, 500.0, 750.0, 900.0, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1800, 2000 and 2500 ug/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: McCoy's 5A culture medium.
- Justification for choice of solvent/vehicle: Preliminary solubility data indicated that the test substance formed a homogeneous suspension in the medium which was considered suitable for dosing, at concentrations up to approximately 5.26 mg/mL.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
without metabolic activation Migrated to IUCLID6: 0.25 and 0.30 ug/mL
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
without metabolic activation Migrated to IUCLID6: 12.50 and 25.00 ug/mL
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
with metabolic activation Migrated to IUCLID6: 6.25 and 12.50 ug/mL
Details on test system and experimental conditions:
METHOD OF APPLICATION: In medium

DURATION
- Exposure duration and Expression time (cells in growth medium): Experiment 1 was comprised of a 3 hour treatment + and -S9 followed by a 17 hour recovery period. Experiment 2 was comprised of a 3 hour treatment +S9 followed by a 17 hour recovery period, together with a continuous 20 hour treatment in the absence of S9. Approximately 1.5 hours prior to harvest (20 hours), colchicine was added to give a final concentration of approximately 1 ug/mL to arrest dividing cells in metaphase.
- Fixation time (start of exposure up to fixation or harvest of cells): Cells were harvested 20 hours after the beginning of treatment.

SPINDLE INHIBITOR (cytogenetic assays): Colchicine
STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: Quadruplicate cultures were treated with the vehicle and duplicate cultures were treated with the test substance and positive controls.

NUMBER OF CELLS EVALUATED: 100 metaphase spreads per slide where appropriate.

DETERMINATION OF CYTOTOXICITY
- Method: Population doubling relative to controls.

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

SLIDE PREPARATION: Cells were kept in fixative at 1-10 degrees C before slides were made, but slides were not made on the day of harvest to ensure that cells were adequately fixed. Cells were centrifuged and resuspended in a minimal amount of fresh fixative (if required) to give a milky suspension.Several drops of 45 % (v/v) aqueous acetic acid were added to each suspension to enhance chromosome spreading, and several drops of suspension were transferred to clean microscope slides labeled with the appropriate study details. After the slides had dried, the cells were stained for 5 minutes in filtered 4 % (v/v) Giemsa in pH 6.8 buffer. The slides were rinsed, dried and mounted with coverslips.

SLIDE ANALYSIS: Slides from the positive controls were checked to ensure that the system was operating satisfactorily. Where appropriate, one hundred metaphases from each slide were analyzed for chromosome aberrations. Where 10 cells with structural aberrations (excluding gaps) were noted on a slide, analysis was terminated. Only cells with 23 to 27 (modal number +/- 2) chromosomes were considered acceptable for analysis. Any cells with more than 27 chromosome (polyploid, hyperdiploid or endoreduplicated cells) observed during the evaluation were recorded separately.
After completion of scoring and decoding of slides, the numbers of aberrant cells in each culture were categorized as follows:
1. Cells with structural aberrations including gaps.
2. Cells with structural aberrations excluding gaps.
3. Polyploid, endoreduplicated or hyperdiploid cells.
The totals for category 2 in the vehicle control cultures were compared with the current laboratory historical negative control (normal) ranges to determine whether the assay was acceptable or not. The totals for category 2 in the test substance treated cultures were also compared with normal ranges.
Evaluation criteria:
Evaluation Criteria- For a valid test, the test substance was considered to induce clastogenic events if:
1. A proportion of cells with structural aberrations at one or more concentrations that exceeded the normal range were observed in both replicate cultures.
2. A statistically significant increase in the proportion of cells with structural aberrations (excluding gaps) was observed (p<= 0.05).
3. There was a concentration-related trend in the proportion of cells with structural aberrations (excluding gaps).
- The test substance was considered positive in the assay if all the above criteria were met.
- The test substance was considered negative in the assay if none of the above criteria were met.
- Results which only partially satisfied the above criteria were dealt with on a case-by-case basis. Evidence of a concentrated-related effect was considered useful but not essential in the evaluation of a positive result. Biological relevance was taken into account, for example consistency of response within and between concentrations and (where appropriate) between experiments, or effects occurring only at high or very toxic concentrations, and the types and distribution of aberrations.
Statistics:
The statistical significance of increases in the percentage of cells with structural aberrations for any data set was only taken into consideration if the frequency of aberrant cells in both replicate cultures at one or more concentrations exceeded the normal range. The statistical method used was the Fisher's exact test. Probability values of p <= 0.058 were accepted as significant. The proportions of cells in categories 1 and 3 were examined in relation to normal ranges and may have been analyzed by Fisher's exact test.
Species / strain:
other: Chinese hamster lung (CHL) 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: Range-finding assay - Precipitation was observed in the 3 hour treatment assays in the absence and presence of S9 at concentrations of 388.6 ug/mL and above. In the 20 hour exposure assay in the absence of S9, precipitation was observed at concentrations of 647.6 ug/mL and above.
Chromosome aberration assay - Precipitation was observed at all concentrations in the 3 and 20 hour treatment in the presence or absence of metabolic activation, except at the 50 ug/mL dose level in the 20 hour treatment assay in the absence of metabolic activation.

RANGE-FINDING/SCREENING STUDIES: The test substance was evaluated for toxicity (population doubling relative to controls) in a range-finding experiment at concentrations ranging from 18.13-4997 ug/mL in the presence and absence of metabolic activation. Duplicate cultures were treated with the vehicle and single cultures were treated with the test substance. Positive controls were not included. The experiment was comprised of a 3 hour treatment + and -S9 followed by a 17 hour recovery period, together with a continuous 20 hour treatment in the absence of S9.
1. 3 hour treatment in the absence of S9- Cytotoxicity ranged from 0-3 % at concentrations ranging from 18.13-647.6 ug/mL, and from 71-90 % at concentrations of 1079 ug/mL and above.
2. 3 hour treatment in the presence of S9-Cytotoxicity ranged from 0-4 % at concentrations ranging from 18.13-1079 ug/mL, and from 44-100 % at concentrations of 1799 ug/mL and above.
3. 20 hour treatment in the absence of S9-Cytotoxicity ranged from 0-1 % at concentrations ranging from 18.13-647.6 ug/mL, and from 23-99 % at concentrations of 1079 ug/mL and above.

COMPARISON WITH HISTORICAL CONTROL DATA: The proportion of cells with structural aberrations (excluding gaps) in the vehicle control cultures fell within the normal historical range. Treatment of cultures with the test substance in the absence and presence of S9 resulted in frequencies of cells with structural aberrations (excluding gaps) which were similar to those in observed concurrent vehicle control cultures in all assays. The numbers of aberrant cells (excluding gaps) in all test substance treated cultures fell within normal ranges.
No increases in the frequency of cells with numerical aberrations, which exceeded the concurrent controls and the normal range, were observed in cultures treated with the test substance in the absence and presence of S9.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Experiment 1
- 3-hour treatment in the absence of S9: Cytotoxicity levels ranged from 20-100 % at the concentrations originally tested (125-4997 ug/mL). The 125, 500, and 625 ug/mL cultures were chosen for chromosomal aberration analysis. Cytotoxicity levels at 125, 500 and 625 ug/mL were 20, 42, and 53 %, respectively.
- 3-hour treatment in the presence of S9: Cytotoxicity levels ranged from 10-72 % at the concentrations originally tested (125-3000 ug/mL). The 500, 1500 and 2500 ug/mL cultures were chosen for chromosomal aberration analysis. Cytotoxicity levels at 500, 1500 and 2500 ug/mL were 16, 30, and 56 %, respectively.

Experiment 2, Trial 1
- 20-hour treatment in the absence of S9: Cytotoxicity levels ranged from 12-100 % at the concentrations originally tested (50-2000 ug/mL). The 50, 200 and 300 ug/mL cultures were chosen for chromosomal aberration analysis. Cytotoxicity levels at 50, 200 and 300 ug/mL were 12, 43 and 52 %, respectively.
- 3-hour treatment in the presence of S9: Cytotoxicity levels ranged from 5-100 % at the concentrations originally tested (250-3000 ug/mL). Based on the steep concentrated related toxicity a second trial was run under this treatment condition, using a narrower concentration range with more closely spaced concentrations. This trial was not included in chromosomal aberration analysis.

Experiment 2, Trial 2
- 3 hour treatment in the presence of S9: Cytotoxicity levels ranged from 8-69 % at the concentrations orginally tested (250-2500 ug/mL). The 500, 1000, 1500 and 2500 ug/mL cultures were chosen for chromosomal aberration analysis. Cytotoxicity levels at 500, 1000, 1500 and 2500 ug/mL were 26, 14, 13 and 69 %, respectively.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Conclusions:
The test substance did not induce chromosome aberrations in cultured Chinese hamster lung (CHL) cells when tested to the limit of cytotoxicity in both the absence and presence of rat liver metabolic activation system (S9).
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:
2004-03-18 to 2004-07-26
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
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: Tungsten Metal
Target: Tungsten Carbide
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:
no
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 culture medium used in 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 semisolid state. Selection medium was cloning medium that contained Trifluorothymidine (TFT).
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: 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:
Initital non-activated and activated assays- 9.85, 19.7, 39.3, 78.5, 157, 313, 625, 1250, 2500 and 5000 ug/mL
Confirmatory non-activated and activated assays- 19.7, 39.3, 78.5, 157, 313, 625, 1250, 2500 and 5000 ug/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: 10 % saline
- Justification for choice of solvent/vehicle: The test substance formed an opaque, black, homogeneous suspension in the vehicle at 50 mg/mL, the highest concentration prepared. Upon further dilution, the test substance became a transparent, light-grey solution at concentrations as high as 3.13 mg/mL and remained soluble at all lower concentratoins.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
Non-activation test system 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:
Activated test system
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 hours
- Expression time (cells in growth medium): 2days
- Selection time (if incubation with a selection agent): 13 days


SELECTION AGENT (mutation assays): 5-trifluorothymidine


NUMBER OF REPLICATIONS: 3


NUMBER OF CELLS EVALUATED: 3E-6 cells


DETERMINATION OF CYTOTOXICITY
- Method: relative total growth


Evaluation criteria:
see other information on materials and methods
Statistics:
no data
Species / strain:
mouse lymphoma L5178Y cells
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
- Precipitation: The test substance was observed to precipitate from treatment medium by the termination of treatment at concentrations as low as 313 ug/mL.
- Other confounding effects: The test substance formed an opaque, black, homogeneous suspension in the vehicle at 50 mg/mL, the highest concentration prepared. Upon further dilution, the test substance became a transparent, light-grey solution at concentrations as high as 3.13 mg/mL and remained soluble at all lower concentrations.


RANGE-FINDING/SCREENING STUDIES: Cells were treated with the test substance for approximately 4 hours in the presence and absence of metabolic activation at concentrations ranging from 9.85-5000 ug/mL. The test substance induced no cytotoxicity to weak cytotoxicity. Based on these results, 5000 ug/mL was chosen as the top concentration for the definitive assay.


COMPARISON WITH HISTORICAL CONTROL DATA: All vehicle and positive control values were within historical data ranges for mutant frequencies except Methylcholanthrene (MCA) (activation assay) at 4 ug/mL. In the initial activation assay MCA had a mutant frequency 608.1E-6 and in the confirmatory assay MCA had a value of 607.5E-6. The historical range for mutant frequencies is 200.2 to 524.0E-6.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Initial non-activation and activation assays- Concentrations of 9.85 and 19.7 ug/mL were discarded because a sufficient number of higher concentrations were available.

Confirmatory non-activation and activation assays- The concentration of 19.7 ug/mL was terminated because there were sufficient higher concentrations available for analysis.

Control Values- The average cloning efficiencies for the vehicle controls were 99.3 and 130.6 % without metabolic activation and 104.9 and 116.2 % 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.

Sizing analysis- The L5178Y TK +/- mutation assay produces a bimodal distribution of large and small mutant colonies. The origin of the bimodal of mutant colony sizes is considered to reflect the types of genetic damage, with the large colonies derived from cells with intragenic mutations that affect only the TK gene and the small colonies the result of larger mutations that affect cell growth as well as the TK gene. Mutant colonies from all the cultures showed the expected bimodal distribution, and mutant colonies from the positive control treated cultures showed both small and large colonies.

Conclusions:
The test substance was did not induce forward mutations at the TK locus in L5178Y mouse lymphoma cells, under the activation and non-activation conditions of this study.
Executive summary:

No mammalian cell mutation data of sufficient quality are available for tungsten carbide (target substance). However, mammalian cell mutation data are available for tungsten metal (source substance), which are used for read-across. Due to similar water solubility and lower toxicity for the target substance compared to the source substance, the resulting read-across from the source substance to the target substance is appropriate. In addition, read-across is appropriate because the classification and labelling is similar 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 on Annex 3 in the CSR.

Endpoint:
in vitro DNA damage and/or repair study
Remarks:
Type of genotoxicity: DNA damage and/or repair
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Remarks:
Well documented scientifically sound study with sufficient methods and results provided to evaluate data.
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to other study
Qualifier:
no guideline available
Principles of method if other than guideline:
Similar to Singh et al, 1988, which is the standard for in vitro single-cell gel comet assay testing, with no major deviations. The study is an alkaline single cell gel electrophoresis (comet) assay in human leukocytes (lymphocytes + monocytes) conducted to quantify DNA breakage. The DNA damaging activity of tungsten carbide was determined.
GLP compliance:
not specified
Type of assay:
comet assay
Species / strain / cell type:
lymphocytes: From a single healthy human volunteer
Details on mammalian cell type (if applicable):
- Human peripheral blood samples were drawn from the same healthy volunteer (age ≤ 30 years) by venipuncture in heparinized tubes.
- Leukocytes (lymphocytes + monocytes) were isolated on a Ficoll-Paque gradient, washed with PBS and resuspended in fresh medium containing 15% fetal calf serum (FCS).
- Lymphocytes were stimulated to divide by 2% phytohemagglutinin (PHA)
- Cultures were set up at a concentration of 0.5e-6 cells/mL in glass tubes and incubated at 37 deg C
Metabolic activation:
without
Test concentrations with justification for top dose:
0, 10, 50, 75, 100 ug/mL WC
Vehicle / solvent:
- Water a.d. (10 uL)
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
Water a.d.
Positive controls:
yes
Remarks:
Ethyl methane sulfonate (EMS) at 2 mM
Details on test system and experimental conditions:
- Cells were processed after 15 minutes of exposure to tungsten carbide
- EMS was used as the positive control (1-hour exposure)
- Cell processing was performed as described by Singh et al (1988), with some minor modifications
- Fully frosted slides were covered with 1% NMP agarose and a coverslip
- The agarose was allowed to solidify at room temperature and removed by scraping with a coverslip, then the slides were covered with 300 µL NMP agarose (0.5%) and a coverslip, and placed on ice for 10 min to let agarose solidify
- After removal of the coverslip, 5000 - 50,000 cells (in 10 µL incubation solution) were mixed with 90 µL of 0.6% LMP agarose and carefully layered on top, covered with a coverslip and put on ice to solidify
- The coverslips were removed, and the slides put in cold, freshly made lysing solution (2.5 M NaCl, 10 mM Tris, 100 mM EDTA disodium salt and 1% w/v N-lauroylsarcosine, pH 10, supplemented with 10% v/v DMSO and 1% v/v Triton X-100 before use) for at least 1-hour at 4 °C
- For electrophoresis the slides were placed in a horizontal electrophoresis box filled with freshly made alkaline electrophoresis buffer (300 mM NaOH, 1 mM EDTA, pH > 12) for 40 min at 18 °C, to allow the DNA to unwind
- Electrophoresis (300 mA, 0.7 V/cm) was performed in the same buffer for 20 min at 18 °C
- Slides were removed from the buffer, the excess alkali was neutralized with 0.4 mM Tris (pH 7.5) and the slides stored with coverslip in a moist chamber at 4 °C until analysis.
- Ethidium bromide (20 µg/mL) stained nuclei were analysed by a computer-guided image analysis system
- Tail length (TL) of the comet was measured by defining manually the centre and the leading edge of the nucleus, and the end of the tail.
- Tail moment (TM = TL X fraction of DNA content in the tail) and the percentage of DNA in the tail (% DNAtail) were assessed.
Statistics:
- Statistical differences between controls and treated samples were determined with the non-parametric Mann-Whitney U-test.
Species / strain:
lymphocytes: From a single healthy human volunteer
Metabolic activation:
without
Genotoxicity:
ambiguous
Cytotoxicity / choice of top concentrations:
not specified
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
- WC induced a significant increase in DNA migration as compared to controls, although the increase was not dose-dependent.
- The Tail Length was significantly different from the control at all tested concentrations, p < 0.01 for 10 and 50 ug/mL and p < 0.0001 at 75, and 100 ug/mL.
- Tail moment was also significantly increased at all tested concentrations, p < 0.05 at 10 ug/mL, p < 0.01 at 75 and 100 ug/mL and p < 0.0001 at 50 and 242 ug/mL.

Although tungsten carbide was able to induce some significant DNA migration, no dose-dependent increase was identified. This suggests that the test substance allowed some uncoiling of the chromatin loops or induced the formation of slowly migrating DNA fragments. By uncoiling the chromatin, tungsten carbide might amplify the clastogenic effects when found in a mixture.

Conclusions:
The test substances showed a slight but statistically significant increase in the tail length and tail moment at all concentrations tested. This effect showed no dose dependency.
Endpoint:
in vitro DNA damage and/or repair study
Remarks:
Type of genotoxicity: DNA damage and/or repair
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
data from handbook or collection of data
Remarks:
ell documented scientifically sound study with sufficient information provided on methods and results to evaluate data. Reliability assigned by secondary source. Primary and secondary source reviewed.
Reason / purpose for cross-reference:
reference to same study
Qualifier:
no guideline available
Principles of method if other than guideline:
Performed according to the method of Singh et al. 1988.
GLP compliance:
not specified
Type of assay:
comet assay
Species / strain / cell type:
lymphocytes: human peripheral blood (two donors:(1 male and 1 female <28 years old)
Additional strain / cell type characteristics:
not specified
Metabolic activation:
without
Test concentrations with justification for top dose:
10, 50, 100 ug/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Sterile deionized water.
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
not specified
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
other: ethyl methane sulfonate
Details on test system and experimental conditions:
METHOD OF APPLICATION: Blood samples were drawn by venipuncture from two donors (one male and one female; age < 28 years) into heparinized Vacutainer tubes. The blood was diluted 1:1 with phosphate buffered saline (PBS) and layered on top of Ficolt paque. After 40 minutes of centrifugation at 400 g, the ring of Peripheral blood mononucleated cells was recovered and washed with PBS (10 minutes of centrifugation at 400 g). The cells were cultured in Hann's F10 medium supplemented with 15% foetal calf serum and 2% purified phytohemoglobin HA (PHA).
24 hours after the onset of PHA stimulation, 10 uL of the test substance suspension was added to the cells. After 15 minutes of exposure (37 °C), the cultures were centrifuged at 400 g (10 minutes), after which the supernatant was removed and the cells were resuspended in PBS or fresh medium while keeping the cultures on a magnetic source to remove the majority of the particles. The cells were processed immediately.

SELECTION AGENT (mutation assays): N/A
STAIN (for cytogenetic assays): Ethidium bromide

NUMBER OF REPLICATIONS: Duplicates

NUMBER OF CELLS EVALUATED: 100 cells/treatment points

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: The comet assay was performed according to the method developed by Singh et al (1988). An aliquot of 10 uL of PBMC was mixed with 300 uL of low melting point agarose (0.8% w/v in PBS) and loaded onto a normal pre-coated microscopic slide (normal melting point agarose 1% w/v in water). Briefly, after lysis at 4 degrees C (2.5 M NaCl, 100 mM Na2EDTA, 10 mM Tris, supplemented with 1% Triton X-100 and 10% MDSO just before use), unwinding of the DNA was performed in a buffer containing 300 mM NaOH. 1 mM Na2EDTA over 40 minutes. Alkaline electrophoresis was performed in the same buffer for 20 minutes at 0.7 V/cm. The slides were than neutralized three times for 5 minutes each with 0.4 M Tris and dehydrated in 100% ice-cold ethanol. To visualize and analyse the DNA damage, the slides were rehydrated with deionized water and stained with ethididium bromide. Fifty randomly chosen, non-overlapping comets per coded slide coming from duplicate cultures were captured using a Leitz fluorescence microscope coupled to a CCD camera and an image analysis system. Percentage of DNA in the tail and tail length were recorded as DNA damage parameters.
Statistics:
The statistical analysis of the dose-effect relationship was performed for each donor separately. An effect was considered positive when either a statistically significant trend was obtained or when in the absence of this trend, at least two of three tested doses were statistically significantly different from the concurrent control. Since both analyses were performed in parallel, the Bosferoni correction of the significance level was applied. Differences between treated and control samples were determined by the Mann-Whitney U-test. The dose dependency was evaluated by a trend test. Comparison of the chromosome/genome mutation frequencies in the micronucleus test and DNA migration in the Comet assay were performed by Pearson's correlation analysis. All analyses were performed using SPSS and Statview software packages.
Species / strain:
lymphocytes: human peripheral blood
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
not specified
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Tungsten Carbide did not induce significant DNA migration in human lymphocytes from the two donors.
Remarks on result:
other: other: Human lymphocytes (peripheral blood mononucleated cells)
Remarks:
Migrated from field 'Test system'.
Conclusions:
Tungsten Carbide did not induce significant DNA migration in human lymphocytes from the two donors.
Endpoint:
in vitro DNA damage and/or repair study
Remarks:
Type of genotoxicity: DNA damage and/or repair
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Remarks:
Well documented scientifically sound study with sufficient information provided on methods and results to evaluate the data.
Reason / purpose for cross-reference:
reference to same study
Qualifier:
no guideline available
Principles of method if other than guideline:
The DNA-breakage potency of the test substance was assessed by quantifying the radioactivity recovered with the eluted fraction.
GLP compliance:
not specified
Type of assay:
other: alkaline elution
Species / strain / cell type:
lymphocytes: human
Details on mammalian cell type (if applicable):
- Type and identity of media: Ham F10 medium.
- Collection and culturing of cells: Blood samples were collected from healthy individuals by venipuncture in heparinized tubes and the lymphocytes were isolated on a Ficoll-Paque gradient. The cells were washed with phosphate buffered saline and suspended in Ham F10 medium containing 15 % fetal calf serum and 8 ug/ ml phytohaemagglutinin HA 16. After 72 hours of culture, the cells were labelled with 1 uCi/ml of [3H]Thymidine triphosphate (TTP).
Metabolic activation:
without
Test concentrations with justification for top dose:
25, 50, 100 and 250 ug/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Water
Untreated negative controls:
yes
Remarks:
No further data provided
Negative solvent / vehicle controls:
not specified
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
Migrated to IUCLID6: 100 mM
Details on test system and experimental conditions:
METHOD OF APPLICATION: In medium


DURATION
- Exposure duration: 10, 15, or 20 minutes
- Exposure temperature: 37C

ALKALINE ELUTION TEST: 5-6e-4 TTP treated lymphocytes were dispersed in each well of a filtration plate and exposed to the test substance suspended in complete Ham F10 medium. After exposure, sodium formate was added at a final concentration of 1 M and the cells were lysed in a solution adjusted at pH 9 containing 0.04 M Na4EDTA, 2 M NaCl, 0.2 % N-lauroylsarcosine, 0.5 mg/ml proteinase K as well as 1 M sodium formate to protect the DNA from the active oxygen species which could have been further produced during the lysis phase. The DNA remaining on the filters was washed with a solution containing 0.02 mM Na4EDTA. Elution buffer (0.1 M tetrapropylammonium hydroxide, 0.02 M EDTA, pH 12.1) was added in a volume of 280 ul and eluted by vacuum (~16 kPa) in a single fraction. The DNA-breakage potency of the test substance was assessed by quantifying the radioactivity recovered with the eluted fraction.
Statistics:
Statistics were performed with the Tukey-Kramer multiple comparisons tests.
Species / strain:
lymphocytes: human
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
The test substance did not produce more single strand DNA breaks than in controls up to a dose of 250 ug/mL.
Conclusions:
The test substance did not produce more single strand DNA breaks than controls up to a dose of 250 ug/mL, using the Alkaline Elution Assay.
Endpoint:
in vitro DNA damage and/or repair study
Remarks:
Type of genotoxicity: DNA damage and/or repair
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Remarks:
Well documented scientifically sound study with sufficient information provided on methods and results to evaluate the data.
Reason / purpose for cross-reference:
reference to same study
Qualifier:
no guideline available
Principles of method if other than guideline:
Determination of the test substances ability to produced single strand break in the DNA of human lymphocytes.
GLP compliance:
not specified
Type of assay:
comet assay
Species / strain / cell type:
primary culture, other: human lymphocytes
Details on mammalian cell type (if applicable):
- Type and identity of media: Ham F10 medium containing 15 % fetal calf serum.
- Cell preparation: Blood samples were collected from healthy individuals by venipuncture in heparinized tubes and the lymphocytes were isolated on a Ficoll Paque gradient. The cells were washed with phosphate buffered saline and resuspended in Ham F10 medium containing 15 % fetal calf serum and 8 ug/ml phytohaemagglutinin HA 16 (PHA). Twenty-four hours after PHA stimulation the lymphocytes were used for the assay.
Metabolic activation:
without
Test concentrations with justification for top dose:
10, 20, 75 and 100 ug/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Water
Untreated negative controls:
yes
Remarks:
No further data provided
Negative solvent / vehicle controls:
not specified
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
Migrated to IUCLID6: 2 mM
Details on test system and experimental conditions:
METHOD OF APPLICATION: In medium

DURATION
- Exposure duration: The test substance was dispersed in 1 ml of culture medium and incubated with 5e-6 lymphocytes during 15 minutes at 37 °C.

COMET ANALYSIS: Fully frosted slides were covered with 1 % normal melting point agarose and a coverslip. Agarose was left to solidify at room temperature and removed by scraping with a coverslip. When the slides were completely dry they were covered with 300 ul NMP-agarose (0.5 %) and a coverslip and further placed on ice for 10 minutes to let agarose solidify. Coverslips were removed and 5-50 x 10e-3 lymphocytes in 5 ul of incubation solution were mixed with 100 ul of low melting point agarose and carefully layered on top, covered again with a coverslip and put on ice to solidify. Coverslips were removed and a final top-layered of 100 ul LMP-agarose (0.6 %) and a coverslip were added. The slides were then put on ice for 10 minutes, the coverslips removed and the cells lysed overnight at 4 °C in a solution consisting of NaCl (2.5 M), Tris (10mM), EDTA disodium salt (100 mM) and N-lauroylsarcosine (1 % w/v) (pH 10) supplemented with 10 % (v/v) DMSO and 1 % (v/v) Triton X-100. The slides were then placed in a horizontal electrophoresis box filled with freshly made electrophoresis buffer (300 mM NaOH, 1 mM EDTA, pH > 13) for 40 minutes at 19 degrees C to allow DNA to unwind. Electrophoresis was performed in the same buffer for 20 minutes at 18 °C (25 V, 300mA). The excess alkali was neutralized with three subsequent rinsings in 0.4 M Tris (pH 7.5). The cells were stained with 100 ul ethidium bromide (20 ug/ml water) for 10 minutes, rinsed with distilled water and kept in a moist chamber at 4°C. For analysis, images from a Zeiss fluorescence microscope (magnification X 300) were captured with an air cooled camera type Coolview on a frame grabber type DT 2855 (Photonic Science). Images were selected randomly and only overlapping comets (nucleus + tail) were omitted for analysis. Fifty cells per concentration were captured and measure. The DNA content of the image was calculated from the sum of light intensities of all pixels in that area; for each separate image, the light intensity of neighboring control area was subtracted. The comet length was measured by defining manually on the screen, sequentially the centre of the nucleus, the leading edge of the nucleus and the end of the tail. All the data were transferred to and analyzed with a Macintosh Quadra 650. The parameters extracted from the measurements were essentially the ‘DNA content’ defined as the total fluorescence (above background) associated with an image and ‘tail moment’ defined as the fraction of DNA in tail (DNA content in tail/DNA content in nucleus + tail) multiplied by the tail length.
Statistics:
Statistics were performed with the Mann-Whitney U-test.
Species / strain:
primary culture, other: human lymphocytes
Metabolic activation:
without
Genotoxicity:
ambiguous
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
not applicable
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
The mean tail length and moment of the comets induced by the positive control clearly differed from the negative control. Exposure of lymphocytes to the test substance up to 100 ug/mL did not induce any DNA breakage above the level measured in negative control.

When correlations between DNA content and the length of the tail were analyzed, the positive control induced comets with a percentage of DNA in the tail higher than the control comets. Nonetheless, after treatment with 75 and 100 ug/mL of the test substance, although no dose-dependent increased tail length or tail moment was observed, the test substance treated cells presented a relatively higher DNA content in the tail than in negative control comets.
Conclusions:
After treatment with 75 and 100 ug/mL of the test substance, although no dose-dependent increased tail length or tail moment was observed, the test substance treated cells presented a relatively higher DNA content in the tail than in control comets. These results may suggest that the test substance either allows some uncoiling of the chromatin loops or induces the formation of slowly migrating fragments.
The results suggest that tungsten carbide may modify the structure of the chromatin leading to an increased sensitivity to clastogenic effects.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

There is no in vivo mutagenicity studies on tungsten carbide.  An in vivo micronucleus assay conducted according to OECD 474 is available on sodium tungstate is available and was used for read-across. Sodium tungstate was not mutagenic in the in vivo micronucleus.

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:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
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: Tungsten Carbide
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: 64-79 degrees F
- Humidity: 30-70%
- Air changes: at least 10 per hour
- Photoperiod: 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 quantity 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, eg toxic signs, death, or depression of the ratio of PCEs to normochromatic erythrocytes (NCEs).


TREATMENT AND SAMPLING TIMES: 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 genetic toxicity in vivo of sufficient quality are available for tungsten carbide (target substance). However, genetic toxicity in vivo data are available for sodium tungstate (source substance), which are used for read-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 on Annex 3 in the CSR.

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

Additional information

Due to similar water solubility, in vitro bioaccessibility in synthetic alveolar, lysosomal, and interstitial fluids simulating inhalation exposure, and available toxicity data for tungsten carbide (target substance) and tungsten metal (source substance), 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 labelling 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.

Due to lower water solubility and lower toxicity for the tungsten carbide (target substance) compared to the sodium tungstate (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 attached description of the read-across category approach in Annex 3.

Justification for classification or non-classification

The in vitro bacterial reverse gene mutation assay, in vitro chromosome aberration assays, and in vitro micronucleus assay of sufficient quality on tungsten carbide were negative for mutagenicity. In addition, an in vitro L5178Y TK +/- mouse lymphoma forward mutation assay conducted on tungsten and an in vivo micronucleus assay conducted on sodium tungstate dihydrate, used for read-across to tungsten carbide, were negative for mutagenicity. Therefore, based on the weight-of-evidence (WoE) from the available data, tungsten carbide does not warrant classification for mutagenicity.