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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
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017-07-24 to 2017-08-31
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017

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
Test material form:
liquid
Details on test material:
Appearance: clear liquid
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: DR74271215
- Expiration date of the lot/batch: 04-05-2017
- Purity: 98.2% (per Certificate of Analysis)

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature, protected from light
- Solubility and stability of the test substance in the solvent/vehicle: The test substance was soluble in water at a concentration of approximately 50 mg/mL, the maximum concentration tested for solubility.


TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing:
Test substance dilutions were prepared immediately before use and delivered to the test system at room temperature under filtered light.

Method

Species / strain
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
CELLS USED
- Source of cells: American Type Culture Collection, Manassas, VA
- The use of CHO cells has been demonstrated to be an effective method of detection of chemical clastogens
- In order to assure the karyotypic stability of the cell line, working cell stocks were not used beyond passage 15.

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: Exponentially growing CHO-K1 cells were seeded in complete medium (McCoy's 5A medium containing 10% fetal bovine serum, 1.5 mM L-glutamine, 100 units/mL penicillin, 100 μg/mL streptomycin and 2.5 μg/mL Amphotericin B) for each treatment condition at a target of 5 x 1E05 cells/culture.
- Properly maintained: yes, the cultures were incubated under standard conditions (37 ± 1°C in a humidified atmosphere of 5 ± 1% CO2 in air) for 16-24 hours.
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: In order to assure the karyotypic stability of the cell line, working cell stocks were not used beyond passage 15.
Additional strain / cell type characteristics:
not specified
Cytokinesis block (if used):
Colcemid at a final concentration of 0.1 µg/mL
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254-induced rat liver S9
Test concentrations with justification for top dose:
In the preliminary toxicity assay, the doses tested were 0.2, 0.6, 2, 6, 20, 60, 200, 600 and 2000 µg/mL. The top dose tested was the limit dose for this assay, based on OECD guidelines. In the chromosomal aberration assay, the doses tested were 100, 250, 500, 1000, 1500, and 2000 µg/mL with and without metabolic activation. The top dose tested was the limit dose for this assay, based on OECD guidelines.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle: Water was the vehicle of choice based on the solubility of the test substance, and compatibility with the target cells. In a solubility test conducted at BioReliance, the test substance was soluble in water at a concentration of approximately 50 mg/mL, the maximum concentration tested for solubility.
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: CHO cells were exposed to the test and control articles for 4 and 20 hours without S9 and for 4 hours with S9, and rinsed.
- Expression time (cells in growth medium): 20 hours (± 30 minutes), 1.5 normal cell cycles

STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: 1 in the preliminary toxicity assay; 2 in the chromosome aberration assay

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: To prepare slides, the cells were collected by centrifugation and the cells were resuspended in fresh fixative. The suspension of fixed cells was applied to glass microscope slides and air-dried. The slides were stained with Giemsa, permanently mounted.

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): 300 metaphase spreads containing 20 ± 2 centromeres from each dose (150 per duplicate treatment)

DETERMINATION OF CYTOTOXICITY- Method: RICC and mitotic index relative to solvent control

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: yes
- Cell density at seeding: 5 x 1E05 cells/culture
Evaluation criteria:
Evaluation of test results:
The test substance was considered to have induced a positive response if:
• at least one of the test concentrations exhibits a statistically significant increase when compared with the concurrent negative control (p ≤ 0.05), and
• the increase is concentration-related (p ≤ 0.05), and
• results are outside the 95% control limit of the historical negative control data.
The test substance was considered to have induced a clear negative response if none of the criteria for a positive response were met.

Criteria for Determination of a Valid Test
Vehicle Controls
The frequency of cells with structural chromosomal aberrations should ideally be within the 95% control limits of the distribution of the historical negative control database. If the concurrent negative control data fall outside the 95% control limits, they may be acceptable as long as these data are note extreme outliers (indicative of experimental or human error).
Positive Controls
The frequency of cells with structural chromosomal aberrations must be significantly greater than the concurrent vehicle control (p ≤ 0.05). In addition, the cytotoxicity response must not exceed the upper limit for the assay (60%).
Cell Proliferation
The average viable cell count in the vehicle control at harvest must be ≥ 1.5-fold the average viable cell baseline value.
Test Conditions
The test substance must be tested using a 4-hr treatment with and without S9, as well as a 20-hr treatment without S9. However, all three treatment conditions need not be evaluated in the case of a positive test substance response under any treatment condition.
Analyzable Concentrations
At least 300 metaphases must be analyzed from at least three appropriate test substance concentrations. The number of metaphases scored may be reduced when high numbers of cells with chromosomal aberrations (≥10% metaphases) are observed as with a positive test substance or the positive control substance.
Statistics:
Statistical analysis was performed using the Fisher's exact test (p ≤ 0.05) for a pairwise comparison of the frequency of aberrant cells in each treatment group with that of the vehicle control. The Cochran-Armitage trend test was used to assess dose-responsiveness.

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 nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: pH values were considered acceptable
- Effects of osmolality: Osmolality values were considered acceptable
- Water solubility: Soluble in water at a concentration of approximately 50 mg/mL, the maximum concentration tested for solubility.
- Precipitation: No visible precipitate observed
- Definition of acceptable cells for analysis: Metaphase spreads containing 20 ± 2 centromeres

RANGE-FINDING/SCREENING STUDIES:
The test substance was soluble in the treatment medium at all doses tested at the beginning and conclusion of the treatment period.
Cytotoxicity (≥ 50% reduction in cell growth index relative to the vehicle control) was not observed at any dose in any of the three exposure groups.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: Refer to main study report
- Negative (solvent/vehicle) historical control data: Refer to main study report

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: RICC and mitotic index relative to solvent control
- Other observations when applicable: Monolayer confluency

Applicant's summary and conclusion

Conclusions:
Under the conditions of the assay described in this report, the test substance was concluded to be negative for the induction of structural and numerical chromosome aberrations in the non-activated and S9-activated test systems in the in vitro mammalian chromosome aberration test using CHO cells.
Executive summary:

The test substance was tested to evaluate the potential to induce structural chromosomal aberrations using Chinese hamster ovary (CHO) cells in both the absence and presence of an exogenous metabolic activation system. CHO cells were treated for 4 hours in the absence and presence of S9, and for 20 hours in the absence of S9. Water was used as the vehicle.

In the preliminary toxicity assay, the doses tested ranged from 0.2 to 2000 µg/mL, which was the limit dose for this assay. Cytotoxicity ( 50% reduction in cell growth index relative to the vehicle control) was not observed at any dose in any of the three exposure groups.  Based upon these results, the doses chosen for the chromosome aberration assay ranged from 100 to 2000 µg/mL for all three exposure groups.

In the chromosome aberration assay, cytotoxicity ( 50% reduction in cell growth index relative to the vehicle control) was not observed at any dose in any of the three exposure groups. The doses selected for evaluation of chromosome aberrations were 500, 1000, and 2000 µg/mL for all three exposure groups.

In the non-activated 4 and 20-hour exposure groups, no significant or dose‑dependent increases in structural aberrations were observed at any dose (p > 0.05; Fisher’s Exact and Cochran-Armitage tests).

In the S9-activated 4-hour exposure group, statistically significant increases in structural aberrations (3.3%) were observed at doses 1000, and 2000 µg/mL (p  0.05; Fisher’s Exact). However, the Cochran-Armitage test was negative for a dose response (p > 0.05). In addition, the increase was within the historical 95% control limit of 0.00% to 3.88%. Since the significant increases in structural aberrations did not meet any criteria for a positive response, the statistical significance can be attributed to the 0.0% induction of structural aberrations in the vehicle control. Therefore, the statistically significant increase in structural chromosomal aberrations was considered biologically irrelevant.

No significant or dose‑dependent increases in numerical (polyploid or endoreduplicated cells) aberrations were observed at any dose in any of the treatment groups (p > 0.05; Fisher’s Exact and Cochran-Armitage tests). 

These results indicate that the test substance was negative for the induction of structural and numerical chromosome aberrations in the presence and absence of the exogenous metabolic activation system.