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Toxicological information

Genetic toxicity: in vitro

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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:
2007-02-27 to 2008-01-23
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
The study was performed according to OECD guideline 473 with minor deviations that did not impact the validity of the study. GLP study.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2008
Report Date:
2008

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
yes
Remarks:
however none that would affect the validity of the study.
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Tungsten Carbide
- Substance type: Active
- Physical state: Grey powder
- Analytical purity: 99.98 %
- Stability under test conditions: Information supplied by the Sponsor stated that the test substance would remain stable for a minimum of 1 year if stored in a closed container at ambient temperature.
- Storage condition of test material: Room temperature in the dark.

Method

Species / strain
Species / strain / cell type:
other: Chinese hamster lung (CHL) cells
Details on mammalian cell type (if applicable):
- Type and identity of media: McCoy's 5A medium supplemented with 10 % heat inactivated foetal calf serum and 100 ug/mL gentamycin
Metabolic activation:
with and without
Metabolic activation system:
Aroclor induced rat liver S9
Test concentrations with justification for top dose:
Experiment 1
- 3-hour treatment without S9: 125.0, 250.0, 375.0, 500.0, 625.0, 750.0, 875.0, 1000, 1500, 2000, 2500, 3000, 3750, 4500 and 4997 ug/mL
- 3-hour treatment with S9: 125.0, 250.0, 500.0, 750.0, 1000, 1250, 1500, 1750, 2000, 2250, 2500 and 3000 ug/mL

Experiment 2, Trial 1
- 20-hour treatment in the absence of S9: 50.0, 100.0, 200.0, 300.0, 400.0, 500.0, 600.0, 750.0, 900.0, 1100, 1250, 1500 and 2000 ug/mL
- 3-hour treatment in the presence of S9: 250.0, 500.0, 1000, 1250, 1500, 1750, 2000, 2250, 2500, 2750 and 3000 ug/mL

Experiment 2, Trial 2
- 3 hour treatment in the presence of S9: 250.0, 500.0, 750.0, 900.0, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1800, 2000 and 2500 ug/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: McCoy's 5A culture medium.
- Justification for choice of solvent/vehicle: Preliminary solubility data indicated that the test substance formed a homogeneous suspension in the medium which was considered suitable for dosing, at concentrations up to approximately 5.26 mg/mL.
Controlsopen allclose all
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
without metabolic activation Migrated to IUCLID6: 0.25 and 0.30 ug/mL
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
without metabolic activation Migrated to IUCLID6: 12.50 and 25.00 ug/mL
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
with metabolic activation Migrated to IUCLID6: 6.25 and 12.50 ug/mL
Details on test system and experimental conditions:
METHOD OF APPLICATION: In medium

DURATION
- Exposure duration and Expression time (cells in growth medium): Experiment 1 was comprised of a 3 hour treatment + and -S9 followed by a 17 hour recovery period. Experiment 2 was comprised of a 3 hour treatment +S9 followed by a 17 hour recovery period, together with a continuous 20 hour treatment in the absence of S9. Approximately 1.5 hours prior to harvest (20 hours), colchicine was added to give a final concentration of approximately 1 ug/mL to arrest dividing cells in metaphase.
- Fixation time (start of exposure up to fixation or harvest of cells): Cells were harvested 20 hours after the beginning of treatment.

SPINDLE INHIBITOR (cytogenetic assays): Colchicine
STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: Quadruplicate cultures were treated with the vehicle and duplicate cultures were treated with the test substance and positive controls.

NUMBER OF CELLS EVALUATED: 100 metaphase spreads per slide where appropriate.

DETERMINATION OF CYTOTOXICITY
- Method: Population doubling relative to controls.

OTHER EXAMINATIONS:
- Determination of polyploidy: Yes
- Determination of endoreplication: Yes

SLIDE PREPARATION: Cells were kept in fixative at 1-10 degrees C before slides were made, but slides were not made on the day of harvest to ensure that cells were adequately fixed. Cells were centrifuged and resuspended in a minimal amount of fresh fixative (if required) to give a milky suspension.Several drops of 45 % (v/v) aqueous acetic acid were added to each suspension to enhance chromosome spreading, and several drops of suspension were transferred to clean microscope slides labeled with the appropriate study details. After the slides had dried, the cells were stained for 5 minutes in filtered 4 % (v/v) Giemsa in pH 6.8 buffer. The slides were rinsed, dried and mounted with coverslips.

SLIDE ANALYSIS: Slides from the positive controls were checked to ensure that the system was operating satisfactorily. Where appropriate, one hundred metaphases from each slide were analyzed for chromosome aberrations. Where 10 cells with structural aberrations (excluding gaps) were noted on a slide, analysis was terminated. Only cells with 23 to 27 (modal number +/- 2) chromosomes were considered acceptable for analysis. Any cells with more than 27 chromosome (polyploid, hyperdiploid or endoreduplicated cells) observed during the evaluation were recorded separately.
After completion of scoring and decoding of slides, the numbers of aberrant cells in each culture were categorized as follows:
1. Cells with structural aberrations including gaps.
2. Cells with structural aberrations excluding gaps.
3. Polyploid, endoreduplicated or hyperdiploid cells.
The totals for category 2 in the vehicle control cultures were compared with the current laboratory historical negative control (normal) ranges to determine whether the assay was acceptable or not. The totals for category 2 in the test substance treated cultures were also compared with normal ranges.
Evaluation criteria:
Evaluation Criteria- For a valid test, the test substance was considered to induce clastogenic events if:
1. A proportion of cells with structural aberrations at one or more concentrations that exceeded the normal range were observed in both replicate cultures.
2. A statistically significant increase in the proportion of cells with structural aberrations (excluding gaps) was observed (p<= 0.05).
3. There was a concentration-related trend in the proportion of cells with structural aberrations (excluding gaps).
- The test substance was considered positive in the assay if all the above criteria were met.
- The test substance was considered negative in the assay if none of the above criteria were met.
- Results which only partially satisfied the above criteria were dealt with on a case-by-case basis. Evidence of a concentrated-related effect was considered useful but not essential in the evaluation of a positive result. Biological relevance was taken into account, for example consistency of response within and between concentrations and (where appropriate) between experiments, or effects occurring only at high or very toxic concentrations, and the types and distribution of aberrations.
Statistics:
The statistical significance of increases in the percentage of cells with structural aberrations for any data set was only taken into consideration if the frequency of aberrant cells in both replicate cultures at one or more concentrations exceeded the normal range. The statistical method used was the Fisher's exact test. Probability values of p <= 0.058 were accepted as significant. The proportions of cells in categories 1 and 3 were examined in relation to normal ranges and may have been analyzed by Fisher's exact test.

Results and discussion

Test results
Species / strain:
other: Chinese hamster lung (CHL) 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: Range-finding assay - Precipitation was observed in the 3 hour treatment assays in the absence and presence of S9 at concentrations of 388.6 ug/mL and above. In the 20 hour exposure assay in the absence of S9, precipitation was observed at concentrations of 647.6 ug/mL and above.
Chromosome aberration assay - Precipitation was observed at all concentrations in the 3 and 20 hour treatment in the presence or absence of metabolic activation, except at the 50 ug/mL dose level in the 20 hour treatment assay in the absence of metabolic activation.

RANGE-FINDING/SCREENING STUDIES: The test substance was evaluated for toxicity (population doubling relative to controls) in a range-finding experiment at concentrations ranging from 18.13-4997 ug/mL in the presence and absence of metabolic activation. Duplicate cultures were treated with the vehicle and single cultures were treated with the test substance. Positive controls were not included. The experiment was comprised of a 3 hour treatment + and -S9 followed by a 17 hour recovery period, together with a continuous 20 hour treatment in the absence of S9.
1. 3 hour treatment in the absence of S9- Cytotoxicity ranged from 0-3 % at concentrations ranging from 18.13-647.6 ug/mL, and from 71-90 % at concentrations of 1079 ug/mL and above.
2. 3 hour treatment in the presence of S9-Cytotoxicity ranged from 0-4 % at concentrations ranging from 18.13-1079 ug/mL, and from 44-100 % at concentrations of 1799 ug/mL and above.
3. 20 hour treatment in the absence of S9-Cytotoxicity ranged from 0-1 % at concentrations ranging from 18.13-647.6 ug/mL, and from 23-99 % at concentrations of 1079 ug/mL and above.

COMPARISON WITH HISTORICAL CONTROL DATA: The proportion of cells with structural aberrations (excluding gaps) in the vehicle control cultures fell within the normal historical range. Treatment of cultures with the test substance in the absence and presence of S9 resulted in frequencies of cells with structural aberrations (excluding gaps) which were similar to those in observed concurrent vehicle control cultures in all assays. The numbers of aberrant cells (excluding gaps) in all test substance treated cultures fell within normal ranges.
No increases in the frequency of cells with numerical aberrations, which exceeded the concurrent controls and the normal range, were observed in cultures treated with the test substance in the absence and presence of S9.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Experiment 1
- 3-hour treatment in the absence of S9: Cytotoxicity levels ranged from 20-100 % at the concentrations originally tested (125-4997 ug/mL). The 125, 500, and 625 ug/mL cultures were chosen for chromosomal aberration analysis. Cytotoxicity levels at 125, 500 and 625 ug/mL were 20, 42, and 53 %, respectively.
- 3-hour treatment in the presence of S9: Cytotoxicity levels ranged from 10-72 % at the concentrations originally tested (125-3000 ug/mL). The 500, 1500 and 2500 ug/mL cultures were chosen for chromosomal aberration analysis. Cytotoxicity levels at 500, 1500 and 2500 ug/mL were 16, 30, and 56 %, respectively.

Experiment 2, Trial 1
- 20-hour treatment in the absence of S9: Cytotoxicity levels ranged from 12-100 % at the concentrations originally tested (50-2000 ug/mL). The 50, 200 and 300 ug/mL cultures were chosen for chromosomal aberration analysis. Cytotoxicity levels at 50, 200 and 300 ug/mL were 12, 43 and 52 %, respectively.
- 3-hour treatment in the presence of S9: Cytotoxicity levels ranged from 5-100 % at the concentrations originally tested (250-3000 ug/mL). Based on the steep concentrated related toxicity a second trial was run under this treatment condition, using a narrower concentration range with more closely spaced concentrations. This trial was not included in chromosomal aberration analysis.

Experiment 2, Trial 2
- 3 hour treatment in the presence of S9: Cytotoxicity levels ranged from 8-69 % at the concentrations orginally tested (250-2500 ug/mL). The 500, 1000, 1500 and 2500 ug/mL cultures were chosen for chromosomal aberration analysis. Cytotoxicity levels at 500, 1000, 1500 and 2500 ug/mL were 26, 14, 13 and 69 %, respectively.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:
The test substance did not induce chromosome aberrations in cultured Chinese hamster lung (CHL) cells when tested to the limit of cytotoxicity in both the absence and presence of rat liver metabolic activation system (S9).