Silver cyanide

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames test (Screening study and Main study) conducted in bacterial cells in accordance with OECD471 guideline and to GLP In vitro mammalian cell mutagenicity test conducted in mouse lymphoma cells in accordance with OECD476 guideline and to GLP In vitro micronucleus assay conducted in human lymphocytes in accordance with OECD487 guideline and to GLP

Link to relevant study records

Reference

Reference 1

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.

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

Target gene:

Cells with micronuclei

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

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.

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.

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.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

3rd February 2015 to 15th June 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted in accordance with test guideline OECD476 and to GLP

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Target gene:

Hypoxanthine-guanine phosphoribosyl transferase (hprt) locus (6-thioguanine [6TG] resistance) in mouse lymphoma cells

Test concentrations with justification for top dose:

Range-Finder:
0.4688, 0.9375, 1.875, 3.750, 7.500, 15.00, 30.00 and 60.00 ug/mL with and without S-9

Experiment 1:
5, 10, 15, 18, 21, 24, 27, 30, 35, 40 and 60 ug/mL without S-9
5, 10, 15, 20, 30, 40, 50, 60, 70, 80 and 100 ug/mL with S-9

Experiment 2:
4, 8, 12, 14, 16, 17, 18, 20, 30 and 40 ug/mL without S-9
5, 10, 20, 3040, 50, 60, 70, 80 and 100 ug/mL with S-9

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: 1% Methyl Cellulose
- Justification for choice of solvent/vehicle: The use of 1% Methyl Cellulose with the aid of Silverson mixing under subdued lighting gave the maximum required concentrations. The solubility of Silver cyanide was previously assessed in Covance Study Number 8288362 (Mc Garry, 2014). The test article was insoluble in the vehicles commonly used in this laboratory including water, acetone, dimethyl sulphoxide (DMSO), ethanol, tetrahydrofuran, dimethylformamide and acetonitrile and did not form a satisfactory suspension in 0.5% Methyl cellulose, but formed a homogenous suspension at approximately 10 mg/mL in 1% high viscosity methyl cellulose (1% MC) which was considered acceptable for use in the assay.

Untreated negative controls:

yes

Remarks:

Culture medium alone

Negative solvent / vehicle controls:

yes

Remarks:

1% Methyl Cellulose diluted 10-fold in the treatment medium

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

benzo(a)pyrene

Remarks:

Both positive control substances were used as supplied with a 100-fold dilution

Details on test system and experimental conditions:

Mutation Assays
Treatment of Cell Cultures:
At least 107 cells in a volume of 17.0 mL of RPMI 6 (cells in RPMI 10 diluted with RPMI A [no serum] to give a final concentration of 6% serum) were placed in a series of sterile disposable 50 mL centrifuge tubes. For all treatments 2.0 mL vehicle control, culture medium (for the UTC) or test article formulation, or 0.2 mL positive control solution (plus 1.8 mL 1% MC) was added. S-9 mix or 150 mM KCl was added as described. Each treatment, in the absence or presence of S-9, was in duplicate (single cultures only used for positive control treatments) and the final treatment volume was 20 mL in RPMI 5 (final concentration of 5% serum). After 3 hours’ incubation at 37±1°C with gentle agitation, cultures were centrifuged (200 g) for 5 minutes, washed with the appropriate tissue culture medium, centrifuged again (200 g) for 5 minutes and resuspended in 20 mL RPMI 10 medium. Cell densities were determined using a Coulter counter and the concentrations adjusted to 2 x 105 cells/mL. Cells were transferred to flasks for growth throughout the expression period or were diluted to be plated for survival.
Plating for Survival:
Following adjustment of the cultures to 2 x 105 cells/mL after treatment, samples from these were diluted to 8 cells/mL. Using a multichannel pipette, 0.2 mL of the final concentration of each culture was placed into each well of 2 x 96-well microtitre plates (192 wells, averaging 1.6 cells/well). The plates were incubated at 37±1ºC in a humidified incubator gassed with 5±1% v/v CO2 in air until scoreable (7 days). Wells containing viable clones were identified by eye using background illumination and counted.
Expression Period:
Cultures were maintained in flasks for a period of 7 days during which the hprt mutation would be expressed. Sub-culturing was performed as required with the aim of retaining an appropriate concentration of cells/flask. From observations on recovery and growth of the cultures during the expression period, the following cultures were selected to be plated for viability and 6TG resistance.
Plating for Viability:
At the end of the expression period, cell concentrations in the selected cultures were determined using a Coulter counter and adjusted to give 1 x 105 cells/mL in readiness for plating for 6TG resistance. Samples from these were diluted to 8 cells/mL. Using a multichannel pipette, 0.2 mL of the final concentration of each culture was placed into each well of 2 x 96-well microtitre plates (192 wells averaging 1.6 cells/well). The plates were incubated at 37±1ºC in a humidified incubator gassed with 5±1% v/v CO2 in air until scoreable (8 days). Wells containing viable clones were identified by eye using background illumination and counted.
Plating for 6TG Resistance:
At the end of the expression period, the cell densities in the selected cultures were adjusted to 1 x 105 cells/mL. 6TG (1.5 mg/mL) was diluted 100-fold into these suspensions to give a final concentration of 15 μg/mL. Using a multichannel pipette, 0.2 mL of each suspension was placed into each well of 4 x 96-well microtitre plates (384 wells at 2 x 104 cells/well). Plates were incubated at 37±1ºC in a humidified incubator gassed with 5±1% v/v CO2 in air until scoreable (12 to 14 days) and wells containing clones were identified as above and counted.

Evaluation criteria:

For valid data, the test article was considered to induce forward mutation at the hprt locus in mouse lymphoma L5178Y cells if:
1. The Mutant Frequency at one or more concentrations was significantly greater than that of the vehicle control (p≤0.05)
2. There was a significant concentration-relationship as indicated by the linear trend analysis (p≤0.05)
3. The effects described above were reproducible.
Results that only partially satisfied the assessment criteria described above were considered on a case-by-case basis. The assay was considered valid if both of the following criteria were met:
1. The MF in the vehicle control cultures fell within the normal range (not more than three times the historical mean value)
2. At least one concentration of each of the positive control chemicals induced a clear, unequivocal increase in mutant frequency.

Statistics:

Statistical significance of mutant frequencies was carried out according to the UKEMS guidelines. The control log mutant frequency (LMF) was compared with the LMF from each treatment concentration and the data were checked for a linear trend in mutant frequency with test article treatment. These tests require the calculation of the heterogeneity factor to obtain a modified estimate of variance.

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Concentrations determined in the Range Finder test

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Additional information on results:

A 3 hour treatment incubation period was used for all experiments.
In the first cytotoxicity Range-Finder Experiment, six concentrations were tested in the absence and presence of S-9, ranging from 41.84 to 1339 μg/mL (10 mM). Extreme or complete toxicity was observed at all concentrations analysed. In the second Range-Finder, eight concentrations were tested in the absence and presence of S-9, ranging from 0.4688 to 60.00 μg/mL (limited by the toxicity seen in the first Range-Finder). The highest concentration to provide >10% relative survival (RS) in the absence of S-9 was 15 μg/mL, which gave 53% RS. The highest concentration
tested in the presence of S-9 (60 μg/mL) gave 49% RS and post-treatment precipitate/undissolved test article was seen at this concentration.
In Experiment 1, eleven concentrations, ranging from 5 to 60 μg/mL in the absence of S-9 and from 5 to 100 μg/mL in the presence of S-9, were tested. Seven days after treatment the highest concentration selected to determine viability and 6TG resistance in the absence of S-9 was 18 μg/mL, which gave 3% RS. Steep concentration-related toxicity was observed between 15 and 18 μg/mL in the absence of S-9, giving 28% and 3% RS, respectively, therefore both concentrations were analysed. The highest concentration selected in the presence of S-9 was 60 μg/mL (limited by the appearance of post-treatment precipitate/undissolved test article), which gave 43% RS.
In Experiment 2, ten concentrations, ranging from 4 to 40 μg/mL in the absence of S-9 and from 5 to 100 μg/mL in the presence of S-9, were tested. Seven days after treatment the highest concentrations analysed to determine viability and 6TG resistance were 20 μg/mL in the absence of S-9 and 50 μg/mL in the presence of S-9, which gave 17% and 21% RS, respectively. The RS value of 21% observed at 50 μg/mL in the presence of S-9 was sufficiently close to 10-20% RS to be considered an acceptable maximum concentration for analysis.
When tested up to toxic or precipitating concentrations in the absence and presence of S-9 in Experiments 1 and 2, no statistically significant increases in mutant frequency were observed with Silver cyanide at any concentration analysed and there were no statistically significant linear trends.
It is concluded that Silver cyanide did not induce mutation at the hprt locus of L5178Y mouse lymphoma cells when tested up to toxic and/or precipitating concentrations in the absence and presence of a rat liver metabolic activation system (S-9).

Remarks on result:

other: strain/cell type: L5178Y cells

Remarks:

Migrated from field 'Test system'.

Conclusions:

Interpretation of results (migrated information):
negative

It is concluded that Silver cyanide did not induce mutation at the hprt locus of L5178Y mouse lymphoma cells when tested up to toxic and/or precipitating concentrations in the absence and presence of a rat liver metabolic activation system (S-9).

Executive summary:

Silver cyanide was assayed for the ability to induce mutation at the hypoxanthine-guanine phosphoribosyl transferase (hprt) locus (6-thioguanine [6TG] resistance) in mouse lymphoma cells using a fluctuation protocol. The study consisted of two cytotoxicity Range-Finder Experiments followed by two independent Mutation Experiments, each conducted in the absence and presence of metabolic activation by an Aroclor 1254-induced rat liver post-mitochondrial fraction (S-9). All Silver cyanide treatments in this study were performed using formulations prepared in 1% high viscosity methyl cellulose (1% MC). As the Silver cyanide stock formulations were in suspension, all concentrations cited in this report may therefore be regarded as nominal. A 3 hour treatment incubation period was used for all experiments.

From the two Range-Finder experiments, the highest concentration to provide >10% relative survival (RS) in the absence of S-9 was 15 μg/mL, which gave 53% RS. The highest concentration tested in the presence of S-9 (60 μg/mL) gave 49% RS and post-treatment precipitate/undissolved test article was seen at this concentration. In Experiment 1, eleven concentrations, ranging from 5 to 60 μg/mL in the absence of S-9 and from 5 to 100 μg/mL in the presence of S-9, were tested and in Experiment 2, ten concentrations, ranging from 4 to 40 μg/mL in the absence of S-9 and from 5 to 100 μg/mL in the presence of S-9, were tested.

When tested up to toxic or precipitating concentrations in the absence and presence of S-9 in Experiments 1 and 2, no statistically significant increases in mutant frequency were observed with Silver cyanide at any concentration analysed and there were no statistically significant linear trends. It is concluded that Silver cyanide did not induce mutation at the hprt locus of L5178Y mouse lymphoma cells when tested up to toxic and/or precipitating concentrations in the absence and presence of a rat liver metabolic activation system (S-9).

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

11 September 2013 to14 September 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP compliant, guideline study, available as an unpublished report.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

not specified

Principles of method if other than guideline:

Silver cyanide was tested for mutation (and toxicity) in three histidine-requiring strains of Salmonella typhimurium (TA98, TA100 and TA102) using a plate incorporation treatment methodology, in the absence and presence of S-9.

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Target gene:

histidine

Species / strain / cell type:

S. typhimurium TA 98

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

not applicable

Species / strain / cell type:

S. typhimurium TA 100

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

not applicable

Species / strain / cell type:

S. typhimurium TA 102

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

rat liver metabolising system (S-9)

Test concentrations with justification for top dose:

1, 3.162, 10, 31.62, 100, 316.2, 1000 μg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Methyl cellulose (1 %)
- Justification for choice of solvent/vehicle: Preliminary solubility data indicated that Silver cyanide was insoluble in several commonly used solvents including water,acetone, DMSO, ethanol, tetrahydrofuran and dimethylformamide (DMF). The test article formed a homogenous suspension at approximately 10 mg/mL high viscosity Methyl cellulose (1% MC).

Untreated negative controls:

yes

Remarks:

TA98

Negative solvent / vehicle controls:

yes

Remarks:

methyl cellulose

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

without S-9

Untreated negative controls:

yes

Remarks:

TA98

Negative solvent / vehicle controls:

yes

Remarks:

methyl cellulose

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

with S-9

Untreated negative controls:

yes

Remarks:

TA100

Negative solvent / vehicle controls:

yes

Remarks:

methyl cellulose

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

without S-9

Untreated negative controls:

yes

Remarks:

TA100

Negative solvent / vehicle controls:

yes

Remarks:

methyl cellulose

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

with S-9

Untreated negative controls:

yes

Remarks:

TA102

Negative solvent / vehicle controls:

yes

Remarks:

methyl cellulose

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

without S-9

Untreated negative controls:

yes

Remarks:

TA102

Negative solvent / vehicle controls:

yes

Remarks:

methyl cellulose

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

with S-9

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Preincubation period: no
- Exposure duration: not reported
- Expression time (cells in growth medium): not applicable
- Selection time (if incubation with a selection agent): not applicable

NUMBER OF REPLICATIONS: triplicate plates. Negative controls were included in quintuplicate and positive controls in triplicate.

DETERMINATION OF CYTOTOXICITY
- Method: plates were assessed for numbers of revertant colonies and examined for effects on the growth of the bacterial background lawn.

Statistics:

Standard deviation, Dunnett's test

Species / strain:

S. typhimurium TA 98

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

Species / strain:

S. typhimurium TA 100

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

Species / strain:

S. typhimurium TA 102

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:

TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: The test article was completely soluble in the aqueous assay system at all concentrations tested.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Toxicity

Following treatments of all the test strains evidence of toxicity ranging from a slight thinning of the background bacterial lawn, with or without a marked reduction in revertant numbers, to a complete killing of the test bacteria, was observed at 100 μg/plate and above in all strains in the absence of S-9 and at 316.2 and/or 1000 μg/plate in all strains in the presence of S-9.

Mutation

Following Silver cyanide treatments of all the test strains in the absence and presence of S-9, no increases in revertant numbers were observed that were statistically significant when the data were analysed atthe 1% level using Dunnett’s test.

Conclusions:

Interpretation of results (migrated information):
negative

The test item, silver cyanide, was considered to be non-mutagenic under the conditions of the test.

Executive summary:

Objective

The objective of the study was to evaluate the potential mutagenic activity of silver cyanide by examining its ability to revert three histidine-requiring strains of Salmonella typhimurium in the absence and presence of a rat liver metabolising system (S-9).

Method

Silver cyanide was tested for mutation (and toxicity) using a plate incorporation treatment methodology, in the absence and presence of S-9. Appropriate negative (vehicle) and positive controls were included.

Conclusion

It was concluded that silver cyanide did not induce mutation in three histidine-requiring strains (TA98, TA100 and TA102) of Salmonella typhimurium when tested under the conditions of this study. These conditions included treatments up to

1000 μg/plate (the maximum achievable concentration) in the absence and in the presence of a rat liver metabolic activation system (S-9).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vitro:

Silver cyanide was tested for its ability to induce reverse mutations in a screening and definitive Ames assay (McGarry 2013, McGarry 2014) in five strains of Salmonella typhimurium (TA98, TA100, TA102, TA1535 and TA1537). Two experiments were carried out (a screening study followed by a main study) with 7 to 8 test concentrations. There was no mutation induced at concentrations up to toxic concentrations in the presence and absence of rat liver S9 metabolic activation. Cytotoxicity was observed in various strains at various concentrations with and without metabolic activation.

The in vitro mammalian cell mutation assay (Lloyd 2015) was conducted in mouse lymphoma cells in the presence and absence of rat liver S9 metabolic activation, assaying for the ability of silver cyanide to induce mutation at the hypoxanthine-guanine phosphoribosyl transferase (hprt) locus. When tested up to toxic or precipitating concentrations in two consecutive experiments, no statistically significant increases in mutant frequency were observed with Silver cyanide at any concentration analysed and there were no statistically significant linear trends. It is concluded that Silver cyanide did not induce mutation at the hprt locus of L5178Y mouse lymphoma cells.

The in vitro experiment for chromosomal damage using human lymphocytes the cell cultures were exposed to 8 concentrations of silver cyanide with and without activation for 4 and 24 hours (Morris 2015). Silver cyanide was found to be non-clastogenic and non-aneugenic to human lymphocytes.

Whilst all three tests were conducted in accordance with GLP and to appropriate test guidelines, it is the chromosome damage study using human lymphocytes that is selected as the key study for this endpoint.

Overall, there is no consistent evidence of genotoxicity below the cytotoxic concentration for silver cyanide.

Justification for selection of genetic toxicity endpoint
The in vitro micronucleus assay for detection of chromosome damage in a human cell line was selected as representative of the Annex VII and Annex VIII gentic toxicity battery of tests since it is conducted on human cells and is a regulatory design study (OECD487) conducted in accordance with GLP.

Justification for classification or non-classification

No genotoxicity would be expected from exposure to silver cyanide, as negative results were observed in all genotoxicity studies. In consequence, no classification is required.