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Genetic toxicity: in vitro

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in vitro gene mutation study in mammalian cells
Type of genotoxicity: gene mutation
Type of information:
migrated information: read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Study period:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study, performed under GLP. Recognised as valid by the World Health Organisation (WHO)

Data source

Referenceopen allclose all

Reference Type:
study report
Report date:
Reference Type:
review article or handbook

Materials and methods

Test guideline
equivalent or similar to guideline
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
GLP compliance:
Type of assay:
mammalian cell gene mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Potassium cyanide
EC Number:
EC Name:
Potassium cyanide
Cas Number:
Molecular formula:
potassium cyanide
Details on test material:
99% pure


Target gene:
Species / strain
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Metabolic activation system:
Arochlor 1254 rat liver microsomes
Test concentrations with justification for top dose:
The dose-levels for the main study were 400, 800, 1000, 2000 and 3000 μg/ml without S9 mix and 1000, 2000, 3000, 4000, 6000, 8000 and 10 000 μg/ml with S9 mix.
Vehicle / solvent:
Controlsopen allclose all
Negative solvent / vehicle controls:
Positive controls:
Positive control substance:
Migrated to IUCLID6: 600 and 700 microgram/ml
Negative solvent / vehicle controls:
Positive controls:
Positive control substance:
Migrated to IUCLID6: 20 and 30 microgram/ml
Details on test system and experimental conditions:
Cells and tissue culture media:
V79 cells are maintained in Dulbecco's modified Eagle-medium supplemented with 10% fetal calf serum, penicillin and streptomycin, called DMEM-FCS. Two independent experiments each with and without S9 mix were carried out.
Cultures are incubated at 37oC in a humidified atmosphere (90%) containing 10% CO2. Exposure to the test compound in the presence of S9 mix is performed in Dulbecco’s phosphate buffered saline (PBS) which additionally contains 20 mM HEPES, pH 7.4 (PBS-HEPES).
Preparation of the Aroclor 1254 S9 mix was according to standard protocol (Maron and Ames, 1983).
Cytotoxicity Experiment.: On the day following subculturing of V79 cells the cells are exposed to a wide range of concentrations of concentrations of the test compound. In the absence of S9 mix, the cells are exposed in DMEM-FCS to the test compound for 24 hours. In the experiments with S9 mix, the medium is replaced by 18 ml S9 mix and the exposure limited to 2 hours. After removal of the test compound and washing of the plates with PBS, the cells are trypsinized and the relative plating efficiency. Three replicate plates are used with a known number of cells. After about 8 days, the cells are fixed and stained with methylene blue in ethanol. The colonies are then counted.
A concentration of the test compound which produces a low level of survival (10-60%) is used as the highest concentration.
Mutagenicity experiments:
On the day following plating, the cells are exposed to the test compound. After removal of the test compound after the specified time, and washing of the cells with PBS, the cells are trypsinized and a relative plating efficiency (PE1) is determined for each dose. The remaining cells are replated and the culture incubation continued until day 8 with normal DMEM-FCS with one subcultivation on day 5. After 8-12 days, the cells are harvested by trypsinization and replated at 1.0E6 per 150 nm in DMEM-FCS in medium without 6-thioguanine for the estimation of plating efficiencies (PE2). The plates are fixed and stained after 8-12 days.
The concentrations of positive controls EMS were 600 and 700 microgram/ml and DMBA were 20 and 20 microgram/ml.
Evaluation criteria:
For the compound to be considered negative, solvent and positive controls show results within the norm and if the test compound does not increase the mutation frequency 2-fold above the mean of the solvent controls under any condition, or if the mutation frequency is always lower than 20.0E-6, and if at least 1.0E6 cells per condition have been evaluated.
For the compound to be considered positive, the dose-dependent increase of the mutation frequency in both independent experiments to at least 2-fold times the solvent control and at least 20.0E-6 both in the presence and/or absence of S9 mix.
not applicable

Results and discussion

Test results
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Cytotoxicity / choice of top concentrations:
Vehicle controls validity:
Positive controls validity:
Remarks on result:
other: all strains/cell types tested
Migrated from field 'Test system'.

Any other information on results incl. tables

The mutation frequencies of the solvent controls ranged from 7.3 to 15.0E-6 clonable cells, and were within the range of historical controls (1 to 44 mutants per 1.0E6 cells). The mutation frequencies of the KCN treated cells (without S9) ranged from 0.4 to 14.9E-6 clonable cells, and were within the range of historical controls (1 to 44 mutants per 1.0E6 cells). In the presence of S9 mix, the mutation frequencies of the KCN treated cells ranged from 3.0 to 24.4E-6 clonable cells, and were also within the range of historical controls (1 to 44 mutants per 1.0E6 cells). The mutation frequency of 24.4E-6 is probably related to extremely high cytotoxicity (plating efficiency 0.00%).

Positive control EMS in the direct test, and DMBA in the activated test, caused a pronounced increase in the mutation frequencies, ranging from 745 to 1495E-6 clonable cells, and 333 to 539E-6 cells, respectively.

Applicant's summary and conclusion

Interpretation of results (migrated information):

Potassium cyanide, tested up to a dose-related high cytotoxicity in the absence and presence of metabolic activation, was negative (non-mutagenic) at the HGPRT locus in the in-vitro mammalian gene mutation assay using V79 cells. Hydrogen cyanide (Index No.006-006-00-X) and salts of hydrogen cyanides (Index No.006-007-00-5) are both listed in Annex VI, Table 3.1 of Regulation (EC) No. 1272/2008, entry 006-007-00-5, and are restricted in comparable ways taking into account physical characteristics. Thus, the assignment of potassium cyanide and sodium cyanide to a chemical category does not result in a less protective regulatory status.