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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:
from 2008-11-27 to 2009-05-05
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report Date:
2009

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
adopted July 21, 1997
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
440/2008/EC
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian chromosome aberration test

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Chemical name: potassium cyanate
- Analytical purity of the test material: 98.2%

Method

Target gene:
none
Species / strain
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
not applicable.
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
Experiment A with 3/20 h treatment/sampling time
without and with S9 mix: 39.06, 78.12, 156.25, 312.5 and 625 µg/ml test item.
Experiment B with 20/28 h treatment/sampling time
without S9 mix: 39.06, 78.12, 156.25 and 234.37 µg/ml test item.
Experiment B with 3/28 h treatment/sampling time
with S9 mix: 39.06, 78.12, 156.25, 312.5 and 625 µg/ml test item.
Vehicle / solvent:
DME medium
Controlsopen allclose all
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
yes
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Positive controls:
yes
Positive control substance:
other: N-Nitrosodimethylamine
Details on test system and experimental conditions:
This in vitro test is a cytogenetic test, which detects structural chromosome aberrations in somatic and/or germ cells and plays an important role in the evaluation of genotoxicity of a given item or agent. Structural aberrations develop due to breaks in one or both DNA strands, resulting in chromosome fragments (breaks, deletions). Faulty reunion of chromosome fragments results in formation of exchanges. These aberrations can be detected and quantified by light microscope. Extensive chromosome breaks usually cause cell death; small changes (breaks, deletions, translocations, inversions etc.) are, however, not necessarily lethal and can be regarded as an indication of molecular events, which might lead to malignant transformation.

The purpose of this study was to establish the potential of the test item to induce structural chromosome aberrations in cultured Chinese hamster cells.

For treatment an asynchronous population of V79 cells in exponential growth was used. The expression time of around 20 hours after the start of treatment is appropriate, since the guidelines recommend expression times of about 1.5-fold of the normal cell cycle, which is 12-14 hours for the cell line used in this study. To ensure the test item does not cause extensive mitotic delay, expression is continued for a longer period of time (28 h) in a separate experiment.

At least three concentrations of the test item were used in each experiment. Where cytotoxicity occurs, these concentrations used cover the range from the maximum to little or no toxicity. At the time of harvesting, the highest concentration needs to show a significant reduction in the degree of confluence and cell count (at least 50 %). For relatively non-cytotoxic compounds the maximum concentration is 5 µl/ml, 5 mg/ml or 0.01 M, whichever is the lowest.

Additional time for expression is given in cases of strong toxic effects of the test item (delayed expression time of 28 hours), using the same concentration range, which induced reasonable cytotoxicity at shorter expression times.
18 and 26 h after the start of treatment, Colchicine is added to the cultures to arrest mitosis and 2 h later (20 and 28 h after start of treatment) metaphase spreads are prepared.

Chromosome aberrations are visualised under the microscope. Although the purpose of the assay is to detect structural chromosome aberrations, polyploidy and/or endoreduplication are reported when observed. To validate the assays, reference compounds are run concurrently to the test item.
Evaluation criteria:
At least 200 metaphase cells containing 2 N ± 2 centromeres were evaluated for structural aberrations from each experimental group. Chromatid and chromosome type aberrations (gaps, deletions and exchanges) were recorded separately. Additionally the number of polyploid and endoreduplicated cells were scored. The nomenclature and classification of chromosome aberrations were given based upon ISCN, 1985, and Savage, 1976, 1983.

Treatment of results
– The percentage of cells with structural chromosome aberration(s) was evaluated.
– Different types of structural chromosome aberrations are listed, with their numbers and frequencies for experimental and control cultures.
– Gaps were recorded separately and reported, but generally not included in the total aberration frequency.
– Concurrent measures of cytotoxicity for all treated and negative control cultures in the main aberration experiment (s) were recorded.
– Individual culture data were summarised in tabular form.
– Equivocal results were clarified by further testing preferably using modification of experimental conditions.

Interpretation of Results
The criteria for determining a positive result are:
– a concentration-related increase or a reproducible increase in the number of cells with aberrations.
– biological relevance of the results should be considered first, however, for the interpretation of the data both biological and statistical significance should be considered together.
– an increase in the number of polyploid cells may indicate that the test item has the potential to inhibit mitotic processes and to induce numerical chromosome aberrations.
– an increase in the number of cells with endoreduplicated chromosomes may indicate that the test item has the potential to inhibit cell cycle progression.
A test item for which the results do not meet the above criteria is considered non mutagenic in this system.
Statistics:
mean and standard deviation

Results and discussion

Test results
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Solubility and Dose Selection

Potassium cyanate was dissolved in DME medium. A clear solution was obtained up to a concentration of 25 mg/ml. There was no precipitation in the medium at any concentration tested.
The dose selection cytotoxicity assay was performed as part of this study to establish an appropriate concentration range for the Chromosome Aberration Assays, both in the absence and in the presence of a metabolic activation system (rodent S9-mix). Toxicity was determined by cell counting and results noted as cell survival in the treatment group (in %) in relation to the negative solvent control. These results were used to select concentrations of Potassium cyanate for the Chromosome Aberration Assays.
The following concentrations were selected ranging from little to maximum (< 50% survival) toxicity and evaluated in the main studies (Experiment A and B). All concentrations were run in duplicate (incl. negative and positive controls) and at least 200 well-spread metaphases were assessed:
Experiment A with 3/20 h treatment/sampling time
without and with S9 mix: 39.06, 78.12, 156.25, 312.5 and 625 µg/ml test item.
Experiment B with 20/28 h treatment/sampling time
without S9 mix: 39.06, 78.12, 156.25 and 234.37 µg/ml test item.
Experiment B with 3/28 h treatment/sampling time
with S9 mix: 39.06, 78.12, 156.25, 312.5 and 625 µg/ml test item.


Chromosome Aberration Assay

The cytotoxicity at the highest concentrations was adequate in the studies (experiment A and experiment B) as indicated by a reduction of % cell survival of at least 50 %.
In Experiment A, Potassium cyanate did not induce an increase in the number of cells with aberrations without gaps at any examined concentration, either in the absence or in the presence of metabolic activation, up to and including cytotoxic concentrations. There were no statistically significant differences between treatment and control groups and no dose-response relationship was noted.
In Experiment B, Potassium cyanate was examined (39.06, 78.12, 156.25 and 234.37 µg/ml) without S9 mix, over a long treatment period (20 hours). As well as in Experiment A, the frequency of the cells with structural chromosome aberrations without gaps did not show significant alterations compared to the concurrent controls. A three-hour treatment with KANTATE KC98 in the presence of S9 mix did not cause an increase in the number of cells with structural chromosome aberrations without gaps at 39.06, 78.12, 156.25, 312.5 and 625 µg/ml.

In Experiment A, the number of aberrant cells with gap after treatment of test item (each concentration without S9 mix and solvent control and each concentration with S9 mix) were slightly above (5-6 %) the upper range of the historical control data of LAB (2-4 %). The number of aberrant cells without gap after treatment of test item ( at concentrations of 78.12, 156.25 and 625 µg/ml and 625 µg/ml with S9 mix) were slightly above (2-3 %) the upper range of the historical control data (0-2- and 1-2 %). These slightly alterations were regarded as biologically irrelevant.
In Experiment B, the number of aberrant cells without gap at 156.25 and 234.37 µg/ml were a slightly above (3 %) the upper value of the historical control data of LAB (2 %). As well, the number of aberrant cells with gap after treatment of test item (solvent control, 78.12, 156.25 and 234.37 µg/ml without S9 mix and 78.12, 625 with S9 mix) were slightly above (5-6 %) the upper range of the historical control data of LAB (2-5%). These slightly alterations were regarded as biologically irrelevant.

As in Experiment A, in Experiment B no statistically significant differences between treatment and control groups and no dose-response relationships were noted. No increase in the rate of polyploid and endoreduplicated metaphases was found after treatment with the different concentrations of Potassium cyanate. In the control group the percentage of cells with structural aberration(s) without gap was equal or less than 5 %, proving the suitability of the cell line used. The positive controls Ethylmethane sulphonate (0.4 and 1.0 µl/ml) and N-Nitrosodimethylamine (1.0 µl/ml) caused the expected biologically relevant increases of cells with structural chromosome aberrations. The studies are, therefore, considered valid.

Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:
Interpretation of results:
negative

Potassium cyanate tested up to cytotoxic concentrations, both with and without metabolic activation, did not induce structural chromosome aberrations in this test in Chinese Hamster lung cells. Therefore, potassium cyanate is considered not clastogenic in this system.
Executive summary:

The test item, potassium cyanate was tested in a Chromosome Aberration Assay in V79 cells. The test item was dissolved in DME medium and the following concentrations were selected on the basis of cytotoxicity investigations made in a preliminary study (without and with metabolic activation).

In two independent experiments (both run in duplicate) at least 200 well-spread metaphase cells were analysed at concentrations and incubation/expression intervals given below, ranging from little to maximum (< 50 % survival) toxicity:

Experiment A with 3/20 h treatment/sampling time

without and with S9 mix: 39.06, 78.12, 156.25, 312.5 and 625 µg/ml test item.

Experiment B with 20/28 h treatment/sampling time

without S9 mix: 39.06, 78.12, 156.25 and 234.37 µg/ml test item.

Experiment B with 3/28 h treatment/sampling time

with S9 mix: 39.06, 78.12, 156.25, 312.5 and 625 µg/ml test item.

In Experiment A, there were no biologically significant increases in the number of cells showing structural chromosome aberrations, either in the absence or in the presence of metabolic activation, up to and including cytotoxic concentrations. There were no statistical differences between treatment and control groups and no dose-response relationships were noted. In Experiment B, the frequency of the cells with structural chromosome aberrations without gaps did not show significant alterations compared to the concurrent control, when potassium cyanate was examined up to cytotoxic concentrations (39.06, 78.12, 156.25 and 234.37 µg/ml) without S9 mix over a prolonged treatment period (20 hours). Further, a three-hour treatment with potassium cyanate up to cytotoxic concentrations (39.06, 78.12, 156.25, 312.5 and 625 µg/ml) in the presence of S9 mix did not cause an increase in the number of cells with structural chromosome aberrations without gaps.

In Experiment A and in Experiment B in some cases the number of aberrant cells with and without gap exceeded the historical control variation, however these biological alteration were considered of no biological relevance as there were no statistically significant differences between treatment and control groups and no dose-response relationships noted.

There were no biologically relevant increases in the rate of polyploid or endoreduplicated metaphases in either experiment in the presence or absence of metabolic activation.

The validity of the test was shown using Ethylmethane sulphonate (0.4 and 1.0 µl/ml) and N-Nitrosodimethylamine (1.0 µl/ml) as positive controls.

In conclusion, potassium cyanate tested up to cytotoxic concentrations, both with and without metabolic activation, did not induce structural chromosome aberrations in this test in Chinese Hamster lung cells. Therefore, potassium cyanate is considered not clastogenic in this system.