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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:
07 August 2014 to 07 November 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report Date:
2015

Materials and methods

Test guidelineopen allclose all
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Qualifier:
according to
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Qualifier:
according to
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test

Test material

Reference
Name:
Unnamed

Method

Target gene:
NA
Species / strain
Species / strain / cell type:
lymphocytes:
Details on mammalian cell type (if applicable):
Cells were grown in Eagle's minimal essential medium with HEPES
buffer (MEM), supplemented "in-house" with L-glutamine, penicillin/ streptomycin, amphotericin B and 15% foetal calf serum
Cytokinesis block (if used):
yes, democolcine
Metabolic activation:
with and without
Metabolic activation system:
phenobarbitone/β-naphthoflavone induced rat liver S9
Test concentrations with justification for top dose:
Experiment 1 (μg/ml) without S9: 0, 5.63, 11.25, 22.5, 45, 90, 180 0, 5.63, Experiment 1 (μg/ml) with S9: 11.25, 22.5, 45, 90, 180 μg/ml
Experiment 2 (μg/ml) without S9: 0, 5.63, 11.25, 22.5, 45, 90, 180, 360
Experiment 2 (μg/ml) with S9: 0, 5.63, 11.25, 22.5, 45, 90, 180, 360

Vehicle / solvent:
- Vehicle(s)/solvent(s) used: acetone
- Justification for choice of solvent/vehicle: The test item was not sufficiently soluble in water or DMSO.
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
acetone
Positive controls:
yes
Positive control substance:
not specified
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium; preincubation; in suspension; as impregnation on paper disk
With Metabolic Activation: Cultures were established approximately 48 hours prior to treatment. Cultures were incubated at 37°C for 4 hours in the presence of the test material prior to washing.

Without Metabolic Activation: Cultures were established approximately 48 hours prior to treatment. In Experiment 1, cultures were incubated at 37°C for 4 hours in the presence of the test material prior to washing. In Experiment 2, the cultures were incubated in the presence of the substance at 37°c for 24 hours

DURATION
- Preincubation period: 48
- Exposure duration: 4 or 24 hours
- Expression time (cells in growth medium): 20 or 0 hours
- Selection time (if incubation with a selection agent): N/A
- Fixation time (start of exposure up to fixation or harvest of cells): Mitosis was arrested by addition of democolcine two hours prior to the required harvest time and the cells were harvested and fixed

SELECTION AGENT (mutation assays): N/A
SPINDLE INHIBITOR (cytogenetic assays): yes
STAIN (for cytogenetic assays): 5% Gurrs Giemsa
NUMBER OF REPLICATIONS: Treatments performed in duplicate.
NUMBER OF CELLS EVALUATED: A total of 2000 lymphocyte cell nuclei were counted and the number of cells in metaphase recorded and expressed as the mitotic index and as a percentage of the vehicle control value. Where possible the first 100 consecutive well-spread metaphases from each culture were counted.

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

OTHER EXAMINATIONS:
- Determination of polyploidy: Yes in comparison to controls
- Determination of endoreplication: If the chromosomes are arranged in closely apposed pairs, ie. 4 chromatids instead of 2, the cell is scored as endoreduplicated
Evaluation criteria:
A positive response was recorded for a particular treatment if the % cells with aberrations, excluding gaps, markedly exceeded that seen in the concurrent control, either with or without a clear dose relationship.
For modest increases in aberration frequency a dose response relationship is generally required and appropriate statistical tests may be applied in order to record a positive response.
Statistics:
The frequency of cells with aberrations excluding gaps and the frequency of polyploid cells was compared, where necessary, with the concurrent vehicle control value using Fisher's Exact test.

Results and discussion

Test results
Species / strain:
lymphocytes: primary culture
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
concentrations limited by the presence of precipitate, see text
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Preliminary Toxicity Test
The dose range for the Preliminary Toxicity Test was 0, 11.11, 22.23, 44.45, 88.91, 177.81, 355.63, 711.25, 1422.5, 2845 μg/ml.
The maximum dose was the 10 mM concentration. A precipitate of the test item was observed at and above 44.45 μg/ml in both non-S9 exposure groups and at and above 88.91 ug/ml in the with-S9 exposure group.
Microscopic assessment of the slides prepared from the exposed cultures showed that metaphase cells were present at up to 2845 ug/mL in both of the 4(20)-hour exposure groups. In the 24-hour exposure group metaphase cells were present up to 2845 ug/mL. The selection of the
maximum dose level was based on toxicity and also on the onset of the formation of a greasy/oily precipitate, where it was considered that the maximum exposure to the test material was occurring.
Experiment 1
No inhibition of mitotic index was observed in the absence of 2% S9 or in the presence of S9. All of the vehicle control cultures had frequencies of cells with chromosome aberrations within the expected range. The positive control items induced statistically significant increases in the frequency of cells with aberrations indicating that the sensitivity of the assay and the efficacy of the S9-mix were validated. The test item did not induce any statistically significant increases in the frequency of cells with aberrations, either in the absence or presence of metabolic activation. There was no significant increase in the incidence of polyploidy.

Experiment 2
A dose-related inhibition of mitotic index was observed in both exposure groups. In the absence of S9, 25% mitotic inhibition was observed at 180 ug/mL. In the presence of 1% S9, a maximum of 28% mitotic inhibition was achieved at 90 ug/mL. The maximum dose level selected for metaphase analysis was based on toxicity and precipitate in both exposure groups.

Results
All vehicle (acetone) controls had frequencies of cells with aberrations within the range expected for normal human lymphocytes. All the positive control items induced statistically significant increases in the frequency of cells with aberrations indicating that the sensitivity of the assay and the efficacy of theS9-mix were validated. The test item was slightly toxic and exposure was limited by the solubility of the test substance. The test substance did not induce any statistically significant increases in the frequency of cells with aberrations, in either of two separate experiments.

Applicant's summary and conclusion

Conclusions:
The test item was considered to be non-clastogenic to human lymphocytes in vitro.