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Genetic toxicity in vitro

Description of key information

Based on the lack of mutagenicity reported in all three in vitro assays, an in vivo assay is not required for tungsten metal (target substance). However, an in vivo micronucleus assay conducted according to OECD 474 was available for sodium tungstate (source substance), which was used for read-across. Due to lower water solubility and lower toxicity for the target substance (tungsten metal) 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, lower for the source substance. For more details, refer to the attached description of the read-across approach.

Tungsten metal was negative for mutagenicity in an in vitro bacterial reverse mutation assay conducted according to OECD 471, an in vitro chromosome aberration assay conducted according to OECD 473, and an in vitro L5178Y TK +/- Mouse Lymphoma Forward Mutation Assay conducted according to OECD 476.

Link to relevant study records

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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:
2004-04-08 to 2004-04-08
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
Only an unsigned draft report available for review., however the study was well documented study conducted according to OECD guideline 473 and GLP.
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 cells were grown in McCoy's 5a culture medium supplemented with ~10 % heat-inactivated fetal bovine serum (FBS), L-glutamine (2mM), penicillin G (100 units/mL), and streptomycin (100 ug/mL).
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
Additional strain / cell type characteristics:
other: The CHO-WBL subclone is a permanent cell line with an average cycle time of 12 to 14 hours and a modal chromosome number of 21.
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254 induced rat liver S9
Test concentrations with justification for top dose:
Initial assay with and without metabolic activation (3-hour treatment)- 13.6, 19.4, 27.7, 39.5, 56.5, 80.7, 115, 165, 235, 336, 480, 686, 980, 1400 and 2000 ug/mL
Confirmatory assay without metabolic activation (20 hour treatment)- 3.13, 6.25, 12.5, 25.0, 50.0, 100, 200, 300, 400 and 500 ug/mL
Confirmatory assay with metabolic activation (3-hour treatment)- 50.0, 100, 200, 300, 400 and 500 ug/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: 0.9 % NaCl USP Saline
- Justification for the choice of solvent/vehicle: In saline, the test substance formed a homogeneous, opaque, dark grey suspension with the test substance settling down slowly at a concentration of ~20.0 mg/mL. At a dose concentration of 2000 ug/mL (dosed in the absence of cells using a 10 % dosing volume in McCoy's 5a culture medium), the test substance formed a homogenous, slightly translucent, light red suspension with a precipitate settling down slowly in the dilution tube, and pH was 8.5 (pH of the culture medium was 8.5).
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
Non-activation assay Migrated to IUCLID6: 0.750 and 1.50 ug/mL, for the 3-hour treatment; 0.200 and 0.400, for the 20-hour treatment
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
Activation assay Migrated to IUCLID6: 7.50 and 12.5 ug/mL
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: Initial assay with and without S9- 3 hours; Confirmatory assay- 20 hours without S9, 3 hours with S9
- Expression time (cells in growth medium): Initial assay with and without S9- approximately 15 hours; Confirmatory assay- approximately 15 hours for cultures treated in the presence of S9 and no expression period for cultures treated in the absence of S9
- Fixation time (start of exposure up to fixation or harvest of cells): approximately 20 hours, with Colcemid present during the last 2 +/-0.5 hours


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


NUMBER OF REPLICATIONS: 2


NUMBER OF CELLS EVALUATED: 100 cells, if possible, from each replicate culture were analyzed for the different types of chromosomal aberrations. At least 25 cells were analyzed from those cultures that had greater than 25 % of cells with one or more aberrations.


DETERMINATION OF CYTOTOXICITY
- Method: mitotic index, visual assessment of the percent confluence of the cell monolayer as compared to the vehicle control.


OTHER EXAMINATIONS:
- Determination of polyploidy: Yes, by evaluating at least 100 metaphases, if available.
- Determination of endoreplication: Yes, by evaluating at least 100 metaphases, if available.
Evaluation criteria:
The following factors were taken into account in evaluation of the data:
- The number and percentages of aberrant cells excluding and including gaps
- Evidence of a dose-response relationship.
Evaluation of a Positive Response- The test substance was considered positive for inducing chromosomal aberrations if a significant increase (the difference was considered significant when p<= 0.01) in the number of cells with chromosomal aberrations was observed at one or more concentrations. The linear trend test evaluated the dose responsiveness. A dose-response should be observed if a significant increase was seen at one or more concentrations.
Evaluation of a Negative Response-The test substance was considered negative for inducing chromosomal aberration if no significant increase was observed in the number of cells with chromosomal aberrations at any of the concentrations.
Equivocal Evaluation- Most assays give clearly positive or negative results, but in rare cases the data would preclude making a definitive judgment about the activity of the test substance. Results might remain equivocal or questionable regardless of the number of times the assay was repeated.
-In certain circumstances the Study Director might use additional considerations to obtain a final evaluation of the test substance based upon the Study Director's scientific judgment.
Statistics:
Statistical analysis employed a Cochran-Armitage test for linear trend and Fisher's Exact Test to compare the percentage of cells with aberrations in treated cells to the results obtained for the vehicle controls.
Statistical analysis was also performed for cells exhibiting polyploidy and/or endoreduplication in order to indicate significant (p<= 0.01) increases in these events as indicators of possible induction of numerical aberrations; however, the groups were evaluated only for structural aberrations and not for numerical aberrations by this protocol.
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
Precipitation and pH: In the initial assay without metabolic activation with a 3-hour treatment, a precipitate was observed after dosing and prior to washing of the cultures treated with >/=980 ug/mL. A precipitate was observed prior to harvest of the cultures treated with >/= 336 ug/mL. In the initial assay with metabolic activation with a 3 hour treatment, a precipitate was observed after dosing and prior to wash of the cultures treated with >/= 480 ug/mL. A precipitate was observed prior to harvest of the cultures treated with >/= 336 ug/mL. In the confirmatory assay without metabolic activation with a 20-hour treatment, a precipitate was observed after dosing and prior to harvest of the cultures treated with >/=300 ug/mL. In the confirmatory assay with metabolic activation with a 3-hour treatment, a precipitate was observed after dosing and prior to harvest of the cultures treated with >/=300 ug/mL.


COMPARISON WITH HISTORICAL CONTROL DATA: Vehicle control and positive control values for % of cells with chromosome aberrations, % of cells with chromosome aberrations + % of cells with gaps, % of polyploidy cells and % endoreduplication cells were all within historical data ranges, except for the % of cells with chromosome aberrations + % of cells with gaps in the vehicle control group of the initial assay in the absence of metabolic activation. The average value for this group was 4.5 % which was slightly above the historical data range of 0-4 %.


ADDITIONAL INFORMATION ON CYTOTOXICITY:
1. Initial assay (3-hour treatment): In the assay without metabolic activation, the monolayer confluence as compared to vehicle control was 86 % at 2000 ug/mL; at all other concentrations it was 100 %. Reductions of 0, 0, 0, 21, 0 and 4 % were observed in the mitotic indices of the cultures treated with 115, 165, 235, 336, 686 and 2000 ug/mL, respectively, as compared with the vehicle control cultures. In the assay with metabolic activation, the monolayer confluence as compared to the vehicle control was 86 % at 1400 and 2000 ug/mL; at all other concentrations it was 100 %. Reductions of 0, 2, 0, 0, 0, 0, and 0 % were observed in the mitotic indices of the cultures treated with 115, 165, 235, 336, 686, 1400 and 2000 ug/mL, respectively, as compared with the vehicle control cultures. Since there was no toxicity exhibited at any of the doses tested, the lowest dose with a precipitate at the time of harvest was selected as the highest dose selected for chromosome aberration analysis as recommended by OECD Testing Guidelines. Chromosome aberrations were analyzed from the cultures treated with 115, 165, 235 and 336 ug/mL.
2. Confirmatory assay: In the assay without metabolic activation, reductions of 3, 3, 3 and 17 % were observed in the mitotic indices of the cultures treated with 50, 200, 300 and 500 ug/mL, respectively, as compared with the vehicle control cultures. In the assay with metabolic activation, reductions of 0, 0, and 1 % were observed in the mitotic indices of the cultures treated with 200, 300 and 500 ug/mL, respectively, as compared with the vehicle control cultures. Since there was no toxicity exhibited at any of the doses tested in the absence or presence of metabolic activation, the lowest dose with a precipitate at the time of harvest was selected as the highest dose selected for analysis as recommended by the OECD Testing Guidelines. In the absence of S9, chromosomal aberrations were analyzed from the cultures treated with 25, 50, 200 and 300 ug/mL, and in the presence of S9, chromosomal aberrations were analyzed from the cultures treated with 50, 100, 200 and 300 ug/mL

The sensitivity of the cell cultures for induction of chromosomal aberrations was shown by the increase frequency of aberrations in the cells treated with both positive controls.

Conclusions:
The test substance was considered negative for inducing structural chromosomal aberrations in CHO cells with and without metabolic activation under conditions of this assay.
Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2004-03-18 to 2004-07-26
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Well documented study performed according to OECD guideline 476 and GLP.
Qualifier:
according to
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
Key result
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.

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.
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:
2004-03-29 to 2004-05-05
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Well document study according to OECD guideline 471 and GLP.
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
Salmonella tyhimurium: Histidine locus and E.coli: tryptophan locus
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
other: See below in additional information on materials and methods.
Species / strain / cell type:
E. coli WP2 uvr A
Additional strain / cell type characteristics:
other: See below in additional information on materials and methods.
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254 induced rat liver S9
Test concentrations with justification for top dose:
33.3, 100, 333, 1000, 3330 and 5000 ug/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Saline (0.9 % NaCl)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
Migrated to IUCLID6: Used in the presence of S9 for strain TA98 at 2.5 ug/plate
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
2-nitrofluorene
Remarks:
Migrated to IUCLID6: Used in the absence of S9 for strain TA98 at 1.0 ug/plate
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene used in the presence of S9 for strains TA100, TA1535, TA1537 and WP2uvrA at 2.5, 2.5, 2.5 and 25.0 ug/plate, respectively.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
Migrated to IUCLID6: Used in the absence of S9 for strains TA100 and TA1535 at 2.0 ug/plate
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: ICR-191-used in the absence of S9 for strain TA1537 at 2.0 ug/plate
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
Migrated to IUCLID6: Used in the absence of S9 for strain WP2uvrA at 1.0 ug/plate
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

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

SELECTION AGENT (mutation assays): histidine (Salmonella) and tryptophan (E. coli)

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: decrease in the number of revertant colonies per plate and/or thinning or disappearance of the bacterial background lawn

CONFIRMATION OF TESTER STRAIN GENOTYPE-Tester strain cultures were checked for the following genetic markers on the day of their use in the mutagenicity assay:
1. rfa Wall Mutation - exhibited by crystal violet sensitivity
2. pKM101 Plasmid - exhibited by resistance to ampicillin
3. Characteristic Number of Spontaneous Revertants: TA98 (8-60), TA100 (60-240), TA1535 (4-45), TA1537 (2-25), WP2uvrA (5-40)

OTHER: The most concentrated test substance dilution and the S9 mix were checked for sterility.

Evaluation criteria:
Tester Strains TA98, TA100 and WP2uvrA- The test substance was considered positive, if it produce at least a 2-fold increase in the mean revertants per plate in at least one of the tester strains over the mean revertants per plate of the appropriate vehicle control. This increase in the mean number of revertants per plate had to be accompanied by a dose response to increasing concentrations of the test substance.
Tester Strains TA1535 and tA1537- The test substance was considered positive, if it produce at least a 3-fold increase in the mean revertant per plate in at least one of the tester strains over the mean revertants per plate of the appropriate vehicle control. This increase in the mean number of revertants per plate had to be accompanied by a dose response to increasing concentrations of the test substance.
Statistics:
no data
Key result
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
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 and Mutagenicity assay- Test substance precipitate was observed on the plates at >/=3330 ug/plate.
- Other confounding effects: The test substance was observed to form an opaque, dark-grey, non-viscous suspension at 50 mg/mL, which was the most concentrated stock dilution prepared for the mutagenicity assay. The test substance was observed to dilute to a solution at 3.33 mg/mL and all succeeding lower dilutions prepared.


RANGE-FINDING/SCREENING STUDIES: The test substance was evaluated for toxicity in a range-finding assay using tester strains TA100 and UP2uvrA in the presence and absence of S9 activation at concentrations ranging from 6.67-5000 ug/plate.
No toxicity was observed in either strain at any concentration level in the presence and absence of S9 as evidenced by non dose-related decreases in the number of revertants per plate and normal bacterial background lawns. Therefore, 5000 ug/plate was selected as the maximum dose level for the mutagenicity test.


COMPARISON WITH HISTORICAL CONTROL DATA: The vehicle and positive control values for revertants/plate fell in historical control data range.


Conclusions:
The test substance was evaluated for mutagenic potential in Bacterial Reverse Mutation Assay. Under the conditions of the study, the test substance did not cause a positive increase in the mean number of revertants per plate with any of the tester strains in the presence or absence of S9 activation.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Sodium tungstate (read across substance) 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:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
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 for the target substance (tungsten metal) 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, lower for the source 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 metal
3. CATEGORY APPROACH JUSTIFICATION: See Annex 1 in CSR
4. DATA MATRIX: See Annex 1 in CSR
Reason / purpose:
read-across: supporting information
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: PMI Feeds, Inc. Certified Rodent Diet #5002 ad libitum
- Water: tap water ad libitum
- Acclimation period: at least 5 days

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

IN-LIFE DATES: From: 2003-08-25 and 2003-08-26
Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: corn oil
- Concentration of test material in vehicle: 25, 50, and 100 mg/ml for initial test and 75 mg/ml for repeat test
- Amount of vehicle (if gavage or dermal): 10 ml/kg
- Lot/batch no. (if required): 12-406
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: Prior to dosing, each concentration of the test substance was prepared by adding the appropriate volume of the vehicle to a pre-weighed 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, 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.
Executive summary:

No in vivo mutagenicity data of sufficient quality are available for tungsten metal (target substance). However, in vivo 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)

Additional information

Justification for classification or non-classification

Tungsten metal was negative for mutagenicity in an in vitro bacterial reverse mutation assay conducted according to OECD 471, an in vitro chromosome aberration assay conducted according to OECD 473, and an in vitro L5178Y TK +/- Mouse Lymphoma Forward Mutation Assay conducted according to OECD 476. In addition, sodium tungstate was negative for mutagenicity in an in vivo micronucleus assay conducted according to OECD 474, which was used for read across. Therefore, based on the weight of evidence from the available data, tungsten metal does not warrant classification.