Silver cyanide

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / micronucleus study

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Reliable without restriction - guideline study, GLP-compliant.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

Test material

Test material information

Constituent 1

Reference substance name:

Silver cyanide

EC Number:

208-048-6

EC Name:

Silver cyanide

Cas Number:

506-64-9

Molecular formula:

CAgN

IUPAC Name:

silver(1+) cyanide

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

- Physical state: White solid
- Storage condition of test material: Stored at room temperature, in the dark.

Method

Target gene:

Cells with micronuclei

Species / strain

Species / strain / cell type:

human lymphoblastoid cells (TK6)

Details on mammalian cell type (if applicable):

- Type and identity of media: Eagle#s minimal essential medium with HEPES buffer, supplemented with L-glutamine, penicillin/streptomycin, amphotericin B and 10 % foetal bovine serum
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: no
- Periodically checked for karyotype stability: no
- Periodically "cleansed" against high spontaneous background: no

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9

Test concentrations with justification for top dose:

Experiment 1a: Nominal concentrations of 5, 10, 20, 30, 40, 60, 80 and 100 ug/mL
Experiment 1b: Nominal concentrations of 5, 10, 20, 40, 80, 100, 120 and 160 ug/mL
Experiment 2: Nominal concentrations of 5, 10, 20, 40, 80, 100, 120 and 160 ug/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Minimal essential medium (MEM)
- Justification for choice of solvent/vehicle: Maximum test item solubility was 13.4 mg/mL in MEM

Controls

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

Remarks:

Minimal essential medium

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

other: demecolcine

Remarks:

Mitomycin C was dissolved in MEM and was tested in the absence of S9 at 0.2 ug/mL. Cyclophosphamid was dissolved in DMSO and was tested in the presence of S9 at 5 ug/mL. Demecolcine was dissolved in water and tested in the absence of S9 at 0.075 ug/mL.

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 48 hours
- Exposure duration: 4 hours (Exp. 1); 24 hours (Exp. 2)
- Expression time (cells in growth medium): 28 hours

SPINDLE INHIBITOR (cytogenetic assays): Cytochalasin B
STAIN (for cytogenetic assays): 5 % Giemsa

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: A minimum of approximately 500 cells per culture for cytoxicity and 1000 binucleated cells per culture (two cultures per concentration) for micronucleus frequency.

DETERMINATION OF CYTOTOXICITY
- Method: Cytokinesis Block Proliferation Index (CBPI; the number of cell cycles per cell during the period of exposure to Cytochalasin B)

OTHER EXAMINATIONS:
- Determination of polyploidy: Micronucleus frequency.

Evaluation criteria:

Negative control - the frequency of binucleate cells with micronuclei in the vehicle control cultures should be within the range of the laboratory historical control data.

Positive control - the positive control chemicals must induce positive responses (p=<0.01)

Statistics:

Treatment data were compared against the vehicle control using the Chi-squared Test on observed numbers of cells with micronuclei. Significance was determined at p < 0.05 and the presence of a reproducible dose-response relationship.

Results and discussion

Test results

Species / strain:

human lymphoblastoid cells (TK6)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

No surviving cells above 40 ug/mL in the absence of S9 and 80 ug/mL in the presence of S9 in Experiment 1. No surviving cells above 80 ug/mL in Experiment 2.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No significant change in pH when the test item was dosed into media (range: 7.31 to 7.37).
- Effects of osmolality: Osmolality did not increase by more than 50 mOsm.
- Evaporation from medium: Treatment solutions were formulated within two hours of being applied to the test system and it is assumed that the test item formulation was stable for this duration.
- Water solubility: Silver cyanide is insoluble in water.
- Precipitation: No precipitate was observed at the end of the exposure period in all experiments.

RANGE-FINDING/SCREENING STUDIES:
The dose range for the Preliminary Toxicity Test was 0.65, 1.31, 2.62, 5.23, 10.47, 20.94, 41.88, 83.75, 167.5, 335 and 1340 ug/mL. In the 4-hour exposure group, binucleate cells were present at up to 41.88 ug/mL and 83.75 ug/mL in the absence and presence of metabolic activation, respectively. The selection of the maximum dose level for the main study was based on toxicity.

COMPARISON WITH HISTORICAL CONTROL DATA:
The results for the vehicle control and positive control were within the historical range.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
No surviving cells were present at concentrations above 40 ug/mL in all tests.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

Silver cyanide is considered to be non-clastogenic and non-aneugenic to human lymphocytes in vitro.

Executive summary:

The potential for silver cyanide to cause chromosome aberrations was tested in a micronucleus test in human lymphocytes in vitro. Cells were exposed to nominal concentrations of 5, 10, 20, 30, 40, 60, 80 and 100 ug/mL silver cyanide in the absence of S9 for 4 hours and nominal concentrations of 5, 10, 20, 40, 80, 100, 120 and 160 ug/mL silver cyanide for 4 -hours in the presence of S9 for 4 -hours and for 24 -hours in the absence of S9. Exposure periods were followed by a 28 -hour incubation period in treatment-free media, in the presence of Cytochalasin B. Cytokinesis Block Proliferation Index and micronucleus frequency were calculated. Silver cyanide was determined to be non-clastogenic and non-aneugenic to human lymphocytes in vitro. This study is reliable without restriction as it was GLP-compliant and was conducted according to guideline.