Potassium cyanate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

- Potassium cyanate is not mutagenic in Ames test without metabolic activation. Read across to sodium cyanate showed that it is also not mutagenic in an Ames test with metabolic activation.
- Potassium cyanate tested up to cytotoxic concentrations, both with and without metabolic activation, did not induce structural chromosome aberrations in Chinese Hamster lung cells. Therefore, potassium cyanate is considered not clastogenic in this system.
- Potassium cyanate tested both without and with metabolic activation (S9 mix), did not induce increases in mutant frequency in a HPRT-test. Therefore, potassium cyanate is considered not mutagenic in this in vitro mammalian cell gene mutation test performed with V79 (Chinese hamster lung) cells.

Therefore, the test substance was not mutagenic or clastogenic in the available in vitro studies.

Link to relevant study records

Reference

Reference 1

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

adopted July 21, 1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

440/2008/EC

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

none

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

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

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.

Conclusions:

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.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

from 2010-03-23 to 2010-05-31

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

adopted July 21, 1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

Commission Regulation (EC) No. 440/2008 of 30 May 2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Target gene:

Hypoxanthine-guanine phosphoribosyl transferase (hprt) locus

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

The cell line was purchased from ECACC (European Collection of Cells Cultures). The cell stocks were kept in a freezer at -80 ± 10 °C. Each batch of frozen cells was purged of HPRT mutants and was free of mycoplasma infections.

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 1, 3-hour treatment period (without and with S9 mix):
25.4, 50.7, 101.4, 202.8, 405.6, and 811.2 µg/mL test item

Experiment 2, 20-hour treatment period without S9 mix:
78.1, 117.2, 156.3, 234.4, 260.4, 286.5, and 312.5 µg/mL test item

Experiment 2, 3-hour treatment period with S9 mix:
25.4, 50.7, 101.4, 202.8, 405.6, and 811.2 µg/mL test item

Vehicle / solvent:

DME medium

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

Positive Control Item - without metabolic activation: 1.0 µL/mL (3h); 0.4 microL/mL (20 h)

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

Remarks:

Positive Control Item - with metabolic activation: 20 µg/mL

Details on test system and experimental conditions:

Study object: The mutation assay used in this study is based on the detection of mutations in the hprt locus located on the X chromosome. HPRT is a cellular enzyme that allows cells to salvage hypoxanthine and guanine from surrounding medium for use in DNA synthesis. If a toxic base analogue 6 thioguanine (6-TG) is present in the medium, then the analogue is phosphorylated via the HPRT pathway and incorporated into the nucleic acid. Thus, the cells die unless the enzyme is rendered inactive, by mutation.

Formulation: KANTATE KC98 was prepared in a concentration of 25 mg/mL with DME medium (stock solution) and diluted prior to treatment. The appropriate amount of these dosing formulations were diluted with Dulbecco’s Modified Eagle’s (DME) medium or DME + S9 mix to obtain the test concentrations. All dose formulations were prepared directly prior to the treatment of the cells.

Mutation Assay:

- Dose selection: In order to determine the treatment concentrations of test item in the gene mutation test a dose selection (cytotoxicity assay) was performed. Due to the low toxicity of test item the test concentrations of gene mutation test (3-hour treatment with and without S9 mix) were chosen on the basis of the maximum recommended concentration for soluble, non-cytotoxic substances; here: 0.01M. In case of prolonged treatment period (20-hour without S9 mix) the treatment concentrations were selected on the basis of cytotoxicity investigations made in a preliminary study.

- Main assay: Laboratory cultures of chinese hamster cells (V79 cell line) were grown in DME medium supplemented with 1 % of an antibiotic-antimycotic solution and heat-inactivated bovine serum (final conc. 10 %) (DME-10) at 37 °C in an incubator with a humidified atmosphere, set at 5 % CO2. During the 3 and 20 hours treatments with test item, the serum content was reduced to 5 % (DME-5). The selection medium for TG resistant mutants contained 10µM/mL of 6-thioguanine (6-TG) (DME-SEL). As a metabolic activation system the S9 fraction of phenobarbital and β-naphthoflavone induced rat liver was used. To validate the assays, reference compounds (solvent as negative control and as positive controls EMS and DMBA) were run concurrently to the test item. The pH and osmolality of the solvent control and test item solutions were determined.

- Plating for survival: Following adjustment of the cultures to 1 x 10E5 cells/mL, samples were diluted to 40 cells/mL. Five mL (200 cells/dish) of the final concentration of each culture was plated into 3 parallel 60-mm dishes. Cultures were incubated for 5 days for colony growing. The colonies were then fixed with methanol and counted after staining with Giemsa. Survivals were assessed by comparing the cloning efficiency of the treated groups to the solvent control.

- Expression of the mutant phenotype: During the phenotypic expression period the cultures were subcultured. Subcultures of approximately 5 x 10E5 cells were employed on days 1, 3, and 6, and selected on day 8.

- Selection of the mutant phenotype: At the end of the expression period the cultures from each of the dose levels were resided at 2 x 10E5 cells per 100-mm dish (five dishes) in selection medium.

- Plating for viability: 200 cells/60-mm dish in 5 mL of DME-10 medium were used for cloning efficiency determinations.

- Fixation and staining of colonies: After the selection period, the colonies were fixed, stained with Giemsa and counted for mutant selection and cloning efficiency determination.

Evaluation criteria:

Evaluation of experimental data/Treatment of results
- Mutation frequency: calculated by dividing the total number of mutant colonies by the number of cells selected (1 x 10E6 cells: 5 plates at 2 x 10E5 cells/plate), corrected for the cloning efficiency of cells prior to mutant selection (viability), and was expressed as 6-TG resistant mutants per 1 x 10E6 clonable cells.
- Relative Survival to Treatment: Relative Survival (%) = (average number of colonies per treated culture/average number of colonies per solvent control dish) x 100.
- Relative Population Growth (%): cumulative growth of the treated cells, relative to the control, over the expression period and prior to mutant selection: (Treated culture population increase over the expression period / solvent control culture population increase over the expression period) x 100.
- Colony forming ability of cells: measured by "Absolute Cloning Efficiency"(CE) at the time of mutant selection: CE (%) = (Average number of viable colonies per dish / 200) x100; 200 cells/60-mm dish in 5 mL of DME medium were used for CE determinations.
- pH and osmolality data were summarised in tabular form.

Assay acceptance criteria
The mutant frequency in the negative (solvent) control cultures is consistent with the historical control data.
The positive control chemicals induce a clear increase in mutant frequency.
The cloning efficiency of the negative controls is between the range of 60% to 140% on Day 0 and 70% to 130% on Day 7.

Evaluation of Results
The test item would have been considered to be mutagenic in this assay if all the following criteria were met:
- The assay is valid.
- The mutant frequency at one or more doses is significantly greater than that of the relevant control.
- Increase of the mutant frequency is reproducible.
- There is a clear dose-response relationship.
The test item would have been considered to have shown no mutagenic activity if no increases were observed which met the criteria listed above.

Statistics:

Mean and standard deviation, Wilcoxon-Mann-Whitney U-test was used to determine whether or not there are statistically significant increases in mutant frequency.

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

without metabolic activation (20 h treatment)

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

The test item, potassium cyanate was tested in a Mammalian Gene Mutation Test in V79 cells. Two independent main experiments (both run in duplicate) were performed at the concentrations given (cf. test concentrations).

In Experiment 1, there were slight increases in mutation frequency at the concentrations of 50.7, 101.4, 202.8, 405.6, and 811.2 µg/mL in the absence and at the concentrations of 101.4 and 405.6 µg/mL in the presence of metabolic activation compared to the concurrent control. These increases were not biologically or statistically significant and no dose-response relationships were noted.

In Experiment 2, the mutant frequency of the cells showed slight increases compared to the concurrent solvent control, but these alterations were not statistically significant when examined without S9 mix over a prolonged treatment period of 20 hours. In addition, a 3 hour treatment in the presence of S9 mix did not cause statistically significant increases in mutant frequency, further indicating that the findings in Experiment 1 were within the normal biological variation. As in Experiment 1, in Experiment 2 no statistical differences between treatment and solvent control groups and no dose response relationships were noted. With regard to pH value and osmolality, in experiments 1 and 2 no significant differences between treatment and control groups were observed.

The sensitivity of the tests and the efficacy of the S9 mix were demonstrated by large increases in mutation frequency in the positive control cultures. The mutation frequencies of the negative and positive control cultures were consistent with the historical control data from previous studies performed at the same laboratory.

Conclusions:

Potassium cyanate tested both without and with metabolic activation (S9 mix), did not induce increases in mutant frequency in this test. Therefore, potassium cyanate is considered not mutagenic in this in vitro mammalian cell gene mutation test performed with V79 (Chinese hamster lung) cells.

Executive summary:

The test item, potassium cyanate was tested in a Mammalian Gene Mutation Test in V79 cells. The test item (98.2 %) 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); due to the low toxicity of test item the test concentrations test were chosen on the basis of the maximum recommended concentration for soluble, non-cytotoxic substances; here: 0.01M.

 Two independent main experiments (both run in duplicate) were performed at the concentrations and treatment intervals given below:

Experiment 1, 3-hour treatment period without and with S9 mix:

25.4, 50.7, 101.4, 202.8, 405.6, and 811.2 µg/mL

Experiment 2, 20-hour treatment period without S9 mix:

78.1, 117.2, 156.3, 234.4, 260.4, 286.5, and 312.5 µg/mL

Experiment 2, 3-hour treatment period with S9 mix:

25.4, 50.7, 101.4, 202.8, 405.6, and 811.2 µg/mL

 In Experiment 1, there were slight increases in mutation frequency at the concentrations of 50.7, 101.4, 202.8, 405.6, and 811.2 µg/mL in the absence and at the concentrations of 101.4 and 405.6 µg/mL in the presence of metabolic activation compared to the concurrent control. These increases were not biologically or statistically significant and no dose-response relationships were noted.

 In Experiment 2, the mutant frequency of the cells showed slight increases compared to the concurrent solvent control, but these alterations were not statistically significant when examined without S9 mix over a prolonged treatment period of 20 hours. In addition, a 3‑hour treatment in the presence of S9 mix did not cause statistically significant increases in mutant frequency, further indicating that the findings in Experiment 1 were within the normal biological variation. As in Experiment 1 and Experiment 2 no statistical differences between treatment and solvent control groups and no dose‑response relationships were noted.

 The validity of the test was shown using Ethylmethane sulphonate (0.4 and 1.0 µL/mL) and 7,12-Dimethylbenz[a]anthracene (20 microgram/mL) as positive controls. The sensitivity of the tests and the efficacy of the S9 mix were demonstrated by large increases in mutation frequency in the positive control cultures, which did induce the appropriate response. The mutation frequencies of the negative and positive control cultures were consistent with the historical control data from previous studies performed at the same laboratory.

 In conclusion, potassium cyanate tested both without and with metabolic activation (S9 mix), did not induce increases in mutant frequency in this test in Chinese hamster lung cells. Therefore, potassium cyanate was not mutagenic in this in vitro mammalian cell gene mutation test performed with V79 cells.

 This study is classified as acceptable and satisfies the requirement for Test Guideline OECD 476 (“Genetic Toxicology: In vitro Mammalian Cell Gene Mutation Test”).

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1991

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1983

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

The purpose of the present investigation was to examine Natriumcyanat for mutagenic activity in five strains of Salmonella typhimurium both in the absence and in the presence of a metabolic activation system (S-9 mix), in compliance with OECD guideline 471 ("Salmonella typhimurium, Reverse Mutation Assay")
Target gene: HIS

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Species / strain / cell type:

S. typhimurium TA 1538

Metabolic activation:

with and without

Metabolic activation system:

S-9 cofactor mix (rat)

Test concentrations with justification for top dose:

0, 120, 370, 1110, 3333 and 10000 µg/mL

Vehicle / solvent:

water

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

water

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

only for TA 1535 and TA 100, in absence of the S-9 mix: 0.1 µg per plate

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

only for TA 1538 and TA 98, in absence of the S-9 mix: 2.0 µg per plate

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

only for TA 1537, in absence of the S-9 mix: 80 µg per plate

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

for all strains, in present of the S-9 mix

Details on test system and experimental conditions:

not indicated

Evaluation criteria:

A positive response in the assay system is taken to be a two-fold or greater increase in the mean number of revertant colonies appearing in the test plates over and above the background spontaneous reversion rate observed with the vehicle, together with evidence of a dose-response.

Statistics:

NA

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

At the highest level used, the test substance appeared to be slightly toxic for some strains, as was seen from the slightly less dense background lawn of bacterial growth.
The positive controls used in the present assays gave the expected strong increase in the number of his+ revertants, both in the absence and in the presence of the S-9 mix.

Conclusions:

The test substance is not genotoxic in the Ames test.

Executive summary:

In a reverse gene mutation assay in bacteria (Report No. V 91.048), strains TA 98, Ta 100, TA 1535, TA 1537 and TA 1538 of S. typhimurium were exposed to Sodium cyanate in water at concentrations of 0, 120, 370, 1110, 3333 and 10000 µg/mL in the presence and absence of mammalian metabolic activation S9 cofactor mix (rat).

Sodium cyanate was tested up to cytotoxic concentrations. At the highest level used, the test substance appeared to be slightly toxic for some strains, as was seen from the slightly less dense background lawn of bacterial growth. The positive controls used in the present assays gave the expected strong increase in the number of his+ revertants, both in the absence and in the presence of the S9 mix.

There was no evidence of induced mutant colonies over background.

This study is classified as acceptable. This study satisfies the requirements for Test Guideline EU B.13/14 and OECD 471 for in vitro mutagenicity (bacterial reverse gene mutation) data.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Key studies:

Ames test:

In a reverse gene mutation assay in bacteria, eight strains (unspecified) of S. typhimurium were exposed to small crystals of potassium cyanate in the absence of mammalian metabolic activation.

The positive controls induced did induce the appropriate responses in the corresponding strains. There was no evidence of induced mutant colonies over background.

This study is classified as acceptable. This study was conducted similar or equivalent to the requirement for Test Guideline EU B.14/14 and OECD 471 for in vitro mutagenicity (bacterial reverse gene mutation) data. However, no mammalian metabolic activation was used.

 

Thus, read across to sodium cyanate was made in addition of bacterial systems. In a reverse gene mutation assay in bacteria (Blijleven, 1991), strains TA 98, TA 100, TA 1535, TA 1537 and TA 1538 of S. typhimurium were exposed to sodium cyanate in water at concentrations of 0, 120, 370, 1110, 3333 and 10000 µg/mL in the presence and absence of mammalian metabolic activation S9 cofactor mix (rat).

Sodium cyanate was tested up to cytotoxic concentrations. At the highest level used, the test substance appeared to be slightly toxic for some strains, as was seen from the slightly less dense background lawn of bacterial growth. The positive controls used in the present assays gave the expected strong increase in the number of his+ revertants, both in the absence and in the presence of the S9 mix. There was no evidence of induced mutant colonies over background. This study satisfies the requirement for Test Guideline EU B.13/14 and OECD 471 for in vitro mutagenicity (bacterial reverse gene mutation) data.

 

Chromosome aberration test:

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.

 

HPRT test:

The test item, potassium cyanate was tested in a Mammalian Gene Mutation Test in V79 cells. The test item (98.2 %) 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); due to the low toxicity of test item the test concentrations were chosen on the basis of the maximum recommended concentration for soluble, non-cytotoxic substances; here: 0.01M.

 Two independent main experiments (both run in duplicate) were performed at the concentrations and treatment intervals given below:

Experiment 1, 3-hour treatment period without and with S9 mix:

25.4, 50.7, 101.4, 202.8, 405.6, and 811.2 µg/mL

Experiment 2, 20-hour treatment period without S9 mix:

78.1, 117.2, 156.3, 234.4, 260.4, 286.5, and 312.5 µg/mL

Experiment 2, 3-hour treatment period with S9 mix:

25.4, 50.7, 101.4, 202.8, 405.6, and 811.2 µg/mL

 In Experiment 1, there were slight increases in mutation frequency at the concentrations of 50.7, 101.4, 202.8, 405.6, and 811.2 µg/mL in the absence and at the concentrations of 101.4 and 405.6 µg/mL in the presence of metabolic activation compared to the concurrent control. These increases were not biologically or statistically significant and no dose-response relationships were noted.

 In Experiment 2, the mutant frequency of the cells showed slight increases compared to the concurrent solvent control, but these alterations were not statistically significant when examined without S9 mix over a prolonged treatment period of 20 hours. In addition, a 3‑hour treatment in the presence of S9 mix did not cause statistically significant increases in mutant frequency, further indicating that the findings in Experiment 1 were within the normal biological variation. As in Experiment 1 and Experiment 2 no statistical differences between treatment and solvent control groups and no dose‑response relationships were noted.

 The validity of the test was shown using Ethylmethane sulphonate (0.4 and 1.0 µL/mL) and 7,12-Dimethylbenz[a]anthracene (20 microgram/mL) as positive controls. The sensitivity of the tests and the efficacy of the S9 mix were demonstrated by large increases in mutation frequency in the positive control cultures, which did induce the appropriate response. The mutation frequencies of the negative and positive control cultures were consistent with the historical control data from previous studies performed at the same laboratory.

 In conclusion, potassium cyanate tested both without and with metabolic activation (S9 mix), did not induce increases in mutant frequency in this test in Chinese hamster lung cells. Therefore, potassium cyanate was not mutagenic in this in vitro mammalian cell gene mutation test performed with V79 cells.

 This study is classified as acceptable and satisfies the requirement for Test Guideline OECD 476 (“Genetic Toxicology: In vitro Mammalian Cell Gene Mutation Test”).

 

Supporting studies:

Point mutation test in V79 cells:

In a mammalian cell gene mutation assay (Na+/K+-ATPase locus), V79 cells cultured in vitro were exposed to potassium cyanate in water at concentrations of 0.01, 0.003, 0.1, 0.3 and 1 mM in the absence of mammalian metabolic activation.

Potassium cyanate was tested up to cytotoxic concentrations. The positive controls did induce the appropriate response. There was no evidence of induced mutant colonies over background.

This study is classified as acceptable. This study satisfies the requirement as supplementary study. No mammalian metabolic activation was used.

 

Gene mutation (TK-locus and OUA-locus) and micronucleous study:

In a mammalian cell gene mutation assay (TK-locus and OUA-locus) and a micronucleous assay, CHO cells cultured in vitro were exposed to potassium cyanate at concentration range from 4-2500 µg/mL in the presence of mammalian metabolic activation (S9 homogenate fraction).

Potassium cyanate was tested up to cytotoxic concentrations. The positive controls did induce the appropriate response (MNU, tested in parallel). In all the tests, no mutagenic and clastogenic effects were observed with potassium cyanate at doses of 4-60 µg/mL for 3 exposure durations (1, 2 or 17 h). No effects with regard to mutagenicity were noted at any concentrations. At a concentration of 1000 µg/mL and 2500 µg/mL, potassium cyanate caused an increase in the number of cells with micronuclei and fragmented nuclei indicating clastogenic effects. The results for the 2 highest concentrations are not regarded as biologically relevant, because severe cytotoxic effects were observed (fragmented nuclei). The concentrations clearly exceeded the maximum test concentrations according to OECD test guidelines and represent thus most likely rather artificial than real biologically relevant effects.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

 

Based on the results obtained in in vitro studies potassium cyanate is not considered to be genotoxic/mutagenic or clastogenic and thus has not to be classified for genetic toxicity according to Regulation (EC) No 1272/2008 (CLP), as amended for the seventeenth time in Regulation (EU) 2021/849.