Registration Dossier

Administrative data

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:
weight of evidence
Study period:
2003-07-16 to 2004-01-06
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
The reliability of this study for the substance tested is a K1, but in application of read-across to a different substance ECHA’s guidance specifies that the score can be a maximum of K2. Due to similar physical-chemical properties, similar or lower transformation/dissolution results and similar or lower in vitro bioaccessibility in synthetic body fluids for tungsten dioxide (the target substance) than the source substances, the resulting toxicity potential would also be expected to be similar or lower, so read-across is appropriate. Therefore, the dose descriptors are expected to be sufficiently similar or higher for the target substance, and read-across to the source chemical is adequately protective. 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 Dioxide
3. CATEGORY APPROACH JUSTIFICATION: See Annex 1 in CSR
4. DATA MATRIX: See Annex 1 in CSR
Cross-reference
Reason / purpose:
read-across: supporting information

Data source

Referenceopen allclose all

Reference Type:
study report
Title:
Unnamed
Year:
2004
Report Date:
2004
Reference Type:
publication
Title:
Genotoxicity Evaluation of Sodium Tungstate Dihydrate and Tungsten Powder.
Author:
Reddy G, McCain WC, Leach GJ.
Year:
2007
Bibliographic source:
The Toxicologist, Vol.96, No. 1, March 2007

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
- Name of test material (as cited in study report): Sodium Tungstate Dihydrate (CAS 13472-45-2)
- Substance type: Active
- Physical state: White, crystalline powder

Test animals

Species:
mouse
Strain:
other: Crl:CD-1 (ICR)BR
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Portage MI
- Age at study initiation: 9 weeks at time of dosing
- Weight at study initiation: 30.3 to 37.9 grams at the time of dosing
- Assigned to test groups randomly: yes, by a computer program
- Housing: The animals were housed in sanitary polycarbonate cages containing Sani-Chips Hardwood Chip Laboratory bedding. The animals were housed, separated by gender, up to five animals per cage during acclimation, and by full dose group/harvest timepoint after randomization.
- Diet (e.g. ad libitum): PMI Feeds, Inc. Certified Rodent Diet #5002 ad libitum
- Water (e.g. ad libitum): tap water ad libitum
- Acclimation period: at least 5 days


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


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

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: corn oil
- Concentration of test material in vehicle: 25, 50, and 100 mg/ml for initial test and 75 mg/ml for repeat test
- Amount of vehicle (if gavage or dermal): 10 ml/kg
- Lot/batch no. (if required): 12-406
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: Prior to dosing, each concentration of the test substance was prepared by adding the appropriate volume of the vehicle to a pre-weighed quantitiy of the test substance and mixing, forming homogeneous suspensions. The formulations were held at room temperature prior to dosing and stirred during the dosing procedure.

Duration of treatment / exposure:
Animals received a single oral gavage dose of the test substance.
Frequency of treatment:
Animals received a single oral gavage dose of the test substance.
Post exposure period:
24 hours (all dose groups) and 48 hours (vehicle control, positive control, 750 mg/kg and 1000 mg/kg groups only)
Doses / concentrationsopen allclose all
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

Examinations

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.

Results and discussion

Test results
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.

Any other information on results incl. tables

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.

Applicant's summary and conclusion

Conclusions:
The test substance was reported as negative in the mouse bone marrow micronucleus assay, under the conditions of this study.
In a mouse bone marrow micronucleus assay, 6 male rats/dose were treated orally with Sodium tungstate at doses of 0, 250, 500 and 1000 mg/kg bw. Bone marrow cells were harvested at 24h post-treatment. The vehicle was corn oil.
Toxic signs were observed at the 1000 mg/kg dose level including soft feces, hypoactivity, rough hair coat 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 hair coats, irregular respiration, and/or recumbence. In addition, one animal died in the 750 mg/kg group. The positive control induced the appropriate response. There was not a significant increase in the frequency of micronucleated polychromatic erythrocytes in bone marrow after any treatment time.
This study is classified as acceptable. This study satisfies the requirement for Test Guideline OECD 474 for in vivo cytogenetic mutagenicity data.
Executive summary:

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