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Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2003-07-17 to 2004-01-07
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

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. et al.
Year:
2007
Bibliographic source:
The Toxicologist Vol. 96, No. 1

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)
Deviations:
yes
Remarks:
The top concentration used in the assay (3500 µg/mL) is slightly above the recommended testing limit (10 mM) for the assay.
GLP compliance:
yes
Type of assay:
mammalian cell gene mutation assay

Test material

Constituent 1
Reference substance name:
Reference substance 001
EC Number:
600-275-2
Cas Number:
10213-10-2
Specific details on test material used for the study:
TEST MATERIAL FORM
- solid: particulate/powder

DETAILS ON TEST MATERIAL
- Name of test material (as cited in study report): Sodium Tungstate Dihydrate
- Substance type: Active
- Physical state: White, crystalline powder
- Storage condition of test material: Room temperature
- Lot/batch No.: 91K3681

Method

Target gene:
Thymidine kinase locus
Species / strain
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
- Type and identity of media: The medium used for the study was RPMI 1640 supplemented with horse serum (10 % by volume), pluronic F68, L-glutamine, sodium pyruvate, penicillin and streptomycin. Treatment medium was Fischer's medium with the same medium supplements used in the culture medium except that the horse serum concentration was reduced to 5 % by volume. Cloning medium consisted of the RPMI 1640 culture medium with up to 20 % horse serum, without Pluronic F68 and with the addition of 0.24 % Noble agar to achieve a semi-solid state. Selection medium was cloning medium that contained 3 ug/mL of 5-trifluorothymidine.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes
Additional strain / cell type characteristics:
other: heterozygous at the TK locus
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254 induced rat liver S9
Test concentrations with justification for top dose:
Following concentrations have been tested in the assay: 62.5, 125, 250, 500, 1000, 1500, 2000, 2500, 3000 and 3500 µg/mL.
3500 µg/mL was the highest concentration and is slightly greater than the OECD Testing Guidelines of 10 mM (molecular weight= 329.86 g/mol; 10 mM=3299 µg/mL).
The test item remained soluble at all concentrations tested.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Water
Controlsopen allclose all
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
Non-activation assay Migrated to IUCLID6: 13 ug/mL
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: Methylcholanthrene-2 and 4 µg/mL
Remarks:
Activation assay
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium


DURATION
- Exposure duration: 4 hours
- Expression time (cells in growth medium): 2 days
- Selection time (if incubation with a selection agent): 14 days


SELECTION AGENT (mutation assays): 5-trifluorothymidine


NUMBER OF REPLICATIONS: 3


NUMBER OF CELLS EVALUATED: 3 x 10^6 cells


DETERMINATION OF CYTOTOXICITY
- Method: relative total growth


OTHER: Sizing Analysis- Both the small and large colonies were quantified for all cultures. A bimodal curve was generated and small and large colonies were quantitated by the areas under the curves. The large colonies presumably arose from point mutations and the small colonies from chromosome changes.

Evaluation criteria:
See below
Statistics:
no data

Results and discussion

Test results
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS:
- Precipitation: The test substance formed a transparent, colourless solution in water at 35 mg/mL, the highest concentration prepared for use in the assay. The test substance remained soluble at all concentrations tested.

RANGE-FINDING/SCREENING STUDIES: Cells were treated with the test substance for approximately 4 hours in the presence and absence of S9 activation at concentrations ranging from 6.90-3500 ug/mL. Test substance concentration for the main gene mutation assay were chosen to cover a toxicity range from 10 % to 20 % survival to no apparent effect on growth compared to the vehicle control. If little or no toxicity was observed and solubility was maintained, the mutation experiment was initiated with a maximum concentration of 5 mg/mL or 10 mM (whichever was lowest). If precipitation of the test substance occurred in the culture medium, the maximum applied dose was at least twice the solubility limit in culture medium. In this assay, the high dose was slightly higher than the OECD recommended guideline of 10 mM.
In the non-activation and activation assays, the test substance induced no cytotoxicity to weak cytotoxicity up to and including 1750 ug/mL and moderate cytotoxicity at 3500 ug/mL.

COMPARISON WITH HISTORICAL CONTROL DATA: One of the vehicle control cultures for the initial assay in the absence of metabolic activation (144.8 x 10^-6) was slightly above historical data range (36.4 to 135.7 x 10^-6) for mutant frequency. In this same assay one of the positive control cultures (522.0 x 10^-6) was also above the historical data range (227.0 to 487.4 x 10^-6) for mutant frequency. In addition, one vehicle control culture (139.1 x 10^-6) in the initial mutation assay with activation was slightly above the historical data range (34.0 to 123.9 x 10^-6) for mutant frequency.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
1. Initial Non-activation Mutation Assay-Concentrations at 62.5 and 125 ug/mL were discarded because a sufficient number of higher concentrations were available. The remaining eight treatments induced no cytotoxicity to moderate cytotoxicity (88.3 % (250 ug/mL) to 34.0 % (3000 ug/mL) relative growths).
2. Confirmatory Non-activation Mutation Assay- Concentrations at 62.5 and 125 ug/mL were terminated because there were sufficient higher concentrations available for analysis. The remaining eight concentrations induced no cytotoxicity to high cytotoxicity (120.3 % (250 ug/mL) to 13 % (3500 ug/mL) relative growth).
3. Initial Activation Mutation Assay- Concentrations at 62.5 and 125 ug/mL were terminated because there were sufficient higher concentrations available for analysis. The remaining eight concentrations induced no cytotoxicity (117.2 % to 81.8 % relative growths).
4. Confirmatory Activation Mutation Assay- Concentrations at 62.5 and 125 ug/mL were terminated because there were sufficient higher concentrations available for analysis. The remaining eight concentrations induced no cytotoxicity to moderate cytotoxicity (95.5 % (250 ug/mL) to 39.9 % (3500 ug/mL) relative growths).


OTHER: The average cloning efficiencies for the vehicle control were 91.4 % and 110.9 % without metabolic activation and 96.6 % and 109.5 % with metabolic activation, which demonstrated acceptable cloning conditions for the assays. The positive control cultures induced large increases in mutant frequencies that were greatly in excess of the minimum criteria.
Mutant colonies from all the cultures showed the expected bimodal distribution, and mutant colonies from the positive control cultures showed both small and large colonies.

Applicant's summary and conclusion

Conclusions:
Under the conditions of this assay, the test substance was reported as negative for inducing forward mutations at the TK locus in L5178Y mouse lymphoma cells (OECD 490) in the presence and absence of Aroclor 1254 induced rat liver S9.
Executive summary:

In a mammalian cell gene mutation assay (Mouse Lymphoma Forward Mutation Assay) cells cultured in vitro were exposed to Sodium tungstate dehydrate at concentrations of 62.5, 125, 250, 500, 1000, 1500, 2000, 2500, 3000 and 3500 µg/mL in the presence and absence of mammalian metabolic activation (S9). Sodium tungstate dihydrate was tested up to cytotoxicity. The positive controls did induce the appropriate response. There was no evidence of induced mutant colonies over background. This study is classified as acceptable. This study satisfies the requirement for Test Guideline OECD 490 for in vitro mutagenicity (mammalian forward gene mutation) data.