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Well documented, scientifically sound GLP study conducted according to OECD Guideline 473 (In vitro Mammalian Chromosome Aberration Test) with no deviations.
O3W
trioxotungsten
- Name of test material (as cited in study report): tungsten yellow oxide; 1314-35-8
- Substance type: active
- Physical state: powder
- Stability under test conditions:
- Storage condition of test material: room temperature
- Type and identity of media: The CHO cultures used for treatment were initiated in McCoy's 5A medium supplemented with approximately 10 % Fetal Bovine Serum (FBS), penicillin G (100 units/mL), streptomycin (100 ug/mL) and L-glutamine (at least 2 mM). This medium was referred to as complete McCoy's 5A culture medium. The cultures were incubated at 37 +/- 2 degrees C, in a humidified atmosphere of 5 +/- 1.5 % CO2. On the day following cell seeding, the culture medium was replaced with fresh serum free medium for the 3 hour exposures with and without metabolic activation and with fresh complete medium for the 24 hour exposure without metabolic activation. After the 3 hour exposure, cultures were rinsed and fresh complete medium was added.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes/no
- Periodically "cleansed" against high spontaneous background: yes/no
- Other: CHO cells exposed to test substances in vitro are routinely used to detect clastogenicity. The cell line used in this study, obtained from Merck Research Laboratories (West Point, PA), was sub-cloned at IITRI, aliquoted into freezing vials and stored in liquid nitrogen. Stock cultures are maintained for up to 12 weeks after thawing a vial from frozen stock.
Additional strain / cell type characteristics:
Aroclor 1254 induced rat liver S9
Experiment 1/cytotoxicity test with and without metabolic activaiton-0, 0.01, 0.02, 0.039, 0.078, 0.156, 0.313, 0.625, 1.25, 2.5 and 5 mg/mL
Experiment 2 with metabolic activation-0, 0.50, 0.75, 1.0, 1.5, 2.0, 2.5 and 3.0 mg/mL
Experiment 2 without metabolic activation-0, 0.10, 0.20, 0.25, 0.50, 0.75, 1.0, 1.5, 2.0, 2.5 and 3.0 mg/mL
The maximum target concentration of test substance, 5.0 mg/mL, in the chromosome aberration assay was based upon the upper limit for testing in OECD Guideline 473.
Concentration confirmation analysis of at least the lowest and highest test substance formulations used in the study were performed by the Analytical Chemistry Division at IITRI.
- Vehicle(s)/solvent(s) used: serum-free McCoy's 5A cell culture media
- Justification for choice of solvent/vehicle: no data
Controlsopen allclose all
serum-free McCoy's 5A cell culture media
mitomycin C
without metabolic activation Migrated to IUCLID6: 0.5 ug/mL
with metabolic activation Migrated to IUCLID6: 10 ug/mL
METHOD OF APPLICATION: in medium
- Exposure duration: 3 hours (Experiment 1 with and without metabolic activation and Experiment 2 with metabolic activation), 24 hours (Experiment 2 without metabolic activation)
- Expression time (cells in growth medium): ~19 hours (3 hour treatment) and 0 hours (24 hour treatment)
- Fixation time (start of exposure up to fixation or harvest of cells): Cultures were harvested 24 hours after initiation of treatment. The cultures were trypsinized and a measured aliquot of cells were counted (using a hemacytometer and trypan blue). After centrifuging the cell suspension, the pelleted cells were re-suspended in at least 10 mL of 0.075M KCl hypotonic solution and incubated. This treatment helps to swell the cells and disperse the chromosomes on a 'plate' such that each chromosome is visible. After the treatment with hypotonic buffer, a 0.5 mL aliquot of Carnoy's fixative (3 parts methanol: 1 part glacial acetic acid) was added to each tube and the cells were centrifuged and the supernatant discarded. The cell pellets were re-suspended in Carnoy's fixative and centrifuged twice, and then they were stored refrigerated in fresh Carnoy's fixative until they were dropped onto glass slides.
SPINDLE INHIBITOR (cytogenetic assays): Colcemid
STAIN (for cytogenetic assays): 5 % Giemsa
NUMBER OF REPLICATIONS:
NUMBER OF CELLS EVALUATED:
100 cells per replicate were evaluated for all groups except the positive control in which 25-50 cells per replicate were evaluated.
- Method: At least 1000 cells per slide were counted to establish a mitotic index for each slide selected for analysis based on cytotoxicity. After the mitotic index was determined for each culture, dose levels were selected for chromosome analysis. Cytotoxicity was measured as the percent cell number reduction relative to the vehicle control cultures.
OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: yes
- Other: Chromatid, and isochromatid gaps and uncoiled chromosomes were noted but were not added into the totals for structural aberration assessment since they are not considered to represent true breaks. In addition Gaps were recorded separately and reported but were not included in the total aberration frequency. The cultures were assessed for test substance precipitation at the time of dosing and after three hours in the 3 hour exposure cultures and at harvest in the 24 hour exposure. The osmolality and pH of at least the highest dosing solution concentration in culture medium were measured. If the resulting measurements record an upward shift in the osmolality by 50 mOsm or more (relative to the vehicle) or a shift in pH of one pH unit or more (up or down), relative to the vehicle The Sponsor would by notified.
Once the assay acceptance criteria had been met, the following factors were taken into account in evaluation of the test substance data:
-Percentage of cells with structural aberrations
-Percentage of cells with more than one structural aberration
-Evidence for increasing amounts of damage with increasing test substance concentration
Since the experimental unit was the cell, the percentage of cells with structural aberrations was the basis of evaluation for clastogenicity. The test substance was considered positive for inducing chromosomal aberrations if a significant increase (ANOVA), p
The test substance was considered negative for inducing chromosomal aberration if no statistically significant increase was observed in the percentage of cells with chromosomal aberrations at any of the concentrations.
Statistical analysis consisted of a one-way analysis of variance (ANOVA) to compare the percentages of cells with aberrations in treated cells with the percentage of cells with aberrations from the vehicle controls. No statistical analysis was used to assess polyploidy and/or endoreduplication. Increases in polyploidy and endoreduplication may indicate possible induction of numerical chromosome changes via spindle apparatus disruption of one kind or another.
- Effects of pH:
- Effects of osmolality:
- Precipitation: The test substance formed a yellow precipitate at most of the concentrations tested and was formulated as a suspension.
A cytotoxicity experiment was performed. Since acceptable cytotoxicity criteria were met by at least three of the dose levels of the test substance within an exposure time group, dose levels were selected for analysis of chromosome aberrations. This cytotoxicity experiment was reported as experiment 1 described above.
COMPARISON WITH HISTORICAL CONTROL DATA:
1. Experiment 1 with metabolic activation-The lowest concentration that approximated at 50% reduction in cell number relative to the vehicle control (i.e., 2.5 mg/mL) and did not have an obscuring level of precipitation was selected as the highest dose level for the chromosome aberration analysis. The cytotoxicity at the 2.5 mg/mL concentration was approximately 58 %. The remaining concentrations selected for analysis were 0.078 and 1.25 mg/mL. Percent reductions in cell numbers of approximately 0 % and 51 %, (relative to the vehicle control) were observed at these dose levels, respectively.
2. Experiment 1 without metabolic activation-The lowest concentration that approximated a 50 % reduction in cell number relative to the vehicle control (i.e., 2.5 mg/mL) and did not have an obscuring level of precipitation was selected as the highest dose level for the chromosome aberration analysis. The cytotoxicity at the 2.5 mg/mL concentration was approximately 45 % The emaining concentrations selected for analysis were 0.156 and 1.25 mg/mL. Percent reductions in cell numbers of approximately 5 % and 25 %, (relative to the vehicle control) were observed at these dose levels, respectively.
3. Experiment 2 with metabolic activation-The lowest concentration that approximated a 50 % reduction in cell number relative to the vehicle control (i.e., 1.5 mg/mL) and did not have an obscuring level of precipitation was selected as the highest dose level for the chromosome aberration analysis. The cytotoxicity at the 1.5 mg/mL concentration was approximately 39 %. The remaining concentrations selected for analysis were 0.75 and 1.0 mg/mL. Percent reductions in cell numbers of approximately 39 % and 18 %, (relative to the vehicle control) were observed at these dose levels, respectively.
4. Experiment 2 without metabolic activation-Chromosome aberrations were evaluated from the cultures treated with concentrations of 1.0, 1.5 and 2.0 mg/mL. The reduction in cell numbers at these dose levels, relative to the vehicle control were 38 %, 40 % and 70 %, respectively.
Dosing Formulation Analysis:
Analysis of the dose formulations showed that the target concentrations for the test substance were not verified. This was most likely due to the nature of the suspension of the test substance in culture media when samples for analytical chemistry were collected. Since the target concentrations could not be verified other factors were used to select dosing levels for analysis (cytotoxicity and test substance precipitation).
Chromosome Aberration Induction:
1. Experiment 1 with metabolic activation-No significant increase in cells with structural chromosomal aberrations was observed in the cultures treated with the test substance relative to the vehicle control. The percentage of polyploid and endoreduplicated cells was slightly elevated relative to the vehicle control in the presence of metabolic activation but these elevations were not statistically significant. The positive control chromosome aberration response was elevated relative to the vehicle and the response was statistically significant (t-test, p</=0.01).
2. Experiment 1 without metabolic activation- No significant increase in cells with structural chromosomal aberrations was observed in the cultures treated with the test substance relative to the vehicle control. The percent of polyploid and endoreduplicated cells was not significantly elevated over the vehicle control vales at any of the doses tested in the absence of metabolic activation. The positive control chromosome aberration response was elevated relative to the vehicle and the response was statistically significant (t-test, p</=0.01).
3. Experiment 2 with metabolic activation-No significant increases in cells with structural chromosomal aberrations were observed in the test substance treated cultures relative to the vehicle control. The percentage of polyploid and endoreduplicated cells was not elevated relative to the vehicle control in the absence of metabolic activation. The positive control chromosome aberration response was elevated relative to the vehicle and the response was statistically significant (t-test, p</=0.01).
4. Experiment 2 without metabolic activation- No significant increases in cells with structural chromosomal aberrations were observed in the cultures treated with the test substance relative to the vehicle control. The percentage of polyploid and endoreduplicated cells was not elevated relative to the vehicle control in the absence of metabolic activation. The positive control chromosome aberration response was elevated relative to the vehicle and the response was statistically significant (t-test, p</=0.01).
negative with and without metabolic activation
The test substance was evaluated for potential clastogenic effects using the structural chromosome aberration assay in Chinese hamster ovary cells, with and without metabolic activation. The test substance failed to induce any clastogenic responses in the CHO cells when tested in the presence and absence of metabolic activation up to dose levels that resulted in cytotoxicity and/or precipitation.