Registration Dossier

Toxicological information

Genetic toxicity: in vitro

Currently viewing:

Administrative data

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.

Data source

Referenceopen allclose all

Reference Type:
study report
Title:
Unnamed
Year:
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 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Tungsten powder, APS 1-5 micron
- Molecular weight (if other than submission substance): 184
- Substance type: Active
- Physical state: Dark grey powder
- Storage condition of test material: Room temperature

Method

Species / strain
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).
Controlsopen allclose all
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.

Results and discussion

Test results
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

Any other information on results incl. tables

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.

Applicant's summary and conclusion

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.