Potassium dicyanoaurate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

10 September 2013 to 29 September 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP compliant, guideline study, available as an unpublished report.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

The test article stock solution prepared in Experiment 2 was not filter sterilised. As there were no contaminations on the test plates following the three day incubation period, this is considered to have had no affect on the integrity of the study.

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Potassium dicyanoaurate

EC Number:

237-748-4

EC Name:

Potassium dicyanoaurate

Cas Number:

13967-50-5

Molecular formula:

C2AuN2.K

IUPAC Name:

potassium dicyanoaurate

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

- Name of test material: Potassium dicyanoaurate
- Other test material name: Umicore Potassium Gold Cyanide 68.1 %
- Substance type: white powder
- Physical state: solid
- Stability under test conditions: Stable for the duration of the study.
- Storage condition of test material: Stored at 15 - 25 °C protected from light
- Other: pH 10.2 (50 g/L)

Method

Target gene:

histidine

Species / strain

Species / strain / cell type:

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

mammalian liver post-mitochondrial fraction (S-9)

Test concentrations with justification for top dose:

Experiment 1 (with and without S-9): 0.01, 0.03162, 0.1, 0.3162, 1.0, 3.162 and 10 µg potassium dicyanoaurate/plate for strains TA98 and TA100 and 0.003906, 0.1563, 0.625, 2.5, 10, 40 and 160 μg/plate for strains TA1535, TA1537 and TA102, plus negative (vehicle) and positive controls
Experiment 2: 0.01563, 0.03125, 0.0625, 0.1250, 0.2500, 0.5000, 1.000 µg potassium dicyanoaurate/plate for strain TA98 (without S-9); 0.1563, 0.3125, 0.6250, 1.250, 2.5, 5.0, 10.0 µg potassium dicyanoaurate/plate for strains TA100, TA1535 and TA1537 (without S-9); 2.5, 5.0, 10.0, 20.0, 40.0, 80.0 and 160.0 µg potassium dicyanoaurate/plate for strain TA102 (without S-9); 0.1563, 0.3125, 0.6250, 1.250, 2.5, 5.0 and 10.0 µg potassium dicyanoaurate/plate for strains TA98, TA100and TA1537 (with S-9); 2.5, 5.0, 10.0, 20.0, 40.0, 80.0 and 160.0 µg potassium dicyanoaurate/plate for strains TA102 and TA1535 (with S-9).

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: purified water
- Justification for choice of solvent/vehicle: Potassium dicyanoaurate was soluble in water for irrigation (purified water) at concentrations equivalent to at least 50 mg/mL. According to current regulatory guidelines the maximum recommended test concentration for this assay is 5000 μg/plate (OECD, 1997). Based on toxicity data obtained during a previous screening study (Mc Garry, 2013), the maximum concentration tested for each strain in Experiment 1 were reduced to 10 µg/plate (strains TA98 and TA100) or 160 µg/plate (strains TA1535, TA1537 and TA102).

Controls

Controls 1

Untreated negative controls:

yes

Remarks:

TA98 without S-9

Negative solvent / vehicle controls:

yes

Remarks:

purified water

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

without S-9

Controls 2

Untreated negative controls:

yes

Remarks:

TA100

Negative solvent / vehicle controls:

yes

Remarks:

purified water

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

without S-9

Controls 3

Untreated negative controls:

yes

Remarks:

TA1535

Negative solvent / vehicle controls:

yes

Remarks:

purified water

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

without S-9

Controls 4

Untreated negative controls:

yes

Remarks:

TA1537

Negative solvent / vehicle controls:

yes

Remarks:

purified water

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

without S-9

Controls 5

Untreated negative controls:

yes

Remarks:

TA102

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

without S-9

Controls 6

Untreated negative controls:

yes

Remarks:

TA98

Negative solvent / vehicle controls:

yes

Remarks:

purified water

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

with S-9

Controls 7

Untreated negative controls:

yes

Remarks:

TA100, TA1535, TA1537

Negative solvent / vehicle controls:

yes

Remarks:

purified water

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

with S-9

Controls 8

Untreated negative controls:

yes

Remarks:

TA1535

Negative solvent / vehicle controls:

yes

Remarks:

purified water

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

with S-9

Controls 9

Untreated negative controls:

yes

Remarks:

TA1537

Negative solvent / vehicle controls:

yes

Remarks:

purified water

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

with S-9

Controls 10

Untreated negative controls:

yes

Remarks:

TA102

Negative solvent / vehicle controls:

yes

Remarks:

purified water

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: (AAN)

Remarks:

with S-9

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Preincubation period: Experiment 1: none; Experiment 2: 20 minutes at 37 ± 1 °C, with shaking.
- Exposure duration: 3 days at 37 ± 1 °C
- Expression time (cells in growth medium): Not applicable
- Selection time (if incubation with a selection agent): Not applicable

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

DETERMINATION OF CYTOTOXICITY
- Method: Plates were assessed for numbers of revertant colonies using aa electronic colony counter and examined for effects on the growth of the bacterial background lawn.

Evaluation criteria:

Acceptance Criteria
The assay was to be considered valid if the following criteria were met:
1. The vehicle control counts fell within the laboratory’s historical control ranges.
2. The positive control chemicals induced increases in revertant numbers of ≥2-fold (in strains TA98, TA100, or TA102) or ≥3-fold (in strains TA1535 or TA1537) the concurrent vehicle control confirming discrimination between different strains, and an active S-9 preparation.

Evaluation Criteria
For valid data, the test article was considered to be mutagenic if:
1. When assessed using Dunnett's test, an increase in revertant numbers gave a significant response (p≤0.01) which was concentration related.
2. The positive trend/effects described above were reproducible.

The test article was considered positive in this assay if all of the above criteria were met. The test article was considered negative in this assay if none of the above criteria were met.

Statistics:

Individual plate counts were recorded separately and the mean and standard deviation of the plate counts for each treatment were determined.

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 1535

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

Test results 2

Species / strain:

S. typhimurium TA 1537

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

Test results 3

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

Test results 4

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

Test results 5

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: Potassium dicyanoaurate was soluble in water for irrigation (purified water) at concentrations equivalent to at least 50 mg/mL.

COMPARISON WITH HISTORICAL CONTROL DATA: The mean vehicle control counts were comparable with the laboratory’s historical ranges.

ADDITIONAL INFORMATION ON CYTOTOXICITY: In Experiment 1 the evidence of toxicity ranging from a slightthinning of the background bacterial lawn, and/or a concurrent marked reduction in revertant numbers, to a complete killing of the test bacteria was observed at 0.3162 μg/plate and above in strain TA98 in the absence of S-9; 2.5 μg/plate and above in strains TA1535 and TA1537 in the absence of S-9; 3.162 μg/plate and above in strain TA98 inthe presence of S-9 only and in strain TA100 in the absence and presence of S-9; 10 μg/plate and above in strain TA1537 in the presence of S-9 and 40 and/or 160 μg/plate and above in strain TA1535 in the presence of S-9 and strain TA102 in the absence and presence of S-9.
In Experiment 2, the evidence of toxicity ranging from a slight thinning of the background bacterial lawn and/or a concurrent marked reduction in revertant numbers, to a complete killing of the test bacteria was observed at 1 μg/plate in strain TA98 inthe absence of S-9; 5 μg/plate and above in strains TA100 and TA1537 in the absence of S-9; 10 μg/plate in strains TA98, TA100 and TA1537 in the presence of S-9 and strain TA1535 in the absence of S-9; 40 µg/plate and above in strain TA1535 in the presence of S-9; 80 μg/plate and above in strain TA102 in the absence of S-9, and at 160 μg/plate in strain TA102 in the presence of S-9.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Data acceptability and validity

It was demonstrated from the data that mean vehicle control counts were comparable with the laboratory’s historical ranges. The positive control chemicals all induced increases in revertant numbers of ≥2-fold (in strains TA98, TA100 or TA102) or ≥3-fold (in strains TA1535 or TA1537) the concurrent vehicle controls confirming discrimination between different strains, and an active S-9 preparation. The study therefore demonstrated correct strain and assay functioning and was accepted as valid.

Applicant's summary and conclusion

Conclusions:

The test item, potassium dicyanoaurate, was considered to be non-mutagenic under the conditions of this test.

Executive summary:

Introduction

Potassium dicyanoaurate was assayed for mutation in five histidine-requiring strains (TA98, TA100, TA1535, TA1537 and TA102) of Salmonella typhimurium, both in the absence and in the presence of metabolic activation by an Aroclor 1254-induced rat liver post-mitochondrial fraction (S-9), in two separate experiments. the test was conducted according to OECD 471 guideline.

Method

Salmonella typhimurium strains TA1535, TA1537, TA98, TA100 and TA102 were treated with suspensions of the test item, Potassium dicyanoaurate using the Ames plate incorporation method, in triplicate, both with and without the addition of a rat liver homogenate metabolising system (10% liver S9 in standard co-factors).  As the results of Experiment 1 were negative, treatments in the presence of S-9 in Experiment 2 included a pre-incubation step.

Results

Negative (vehicle) and positive control treatments were included for all strains in both experiments. The mean numbers of revertant colonies all fell within acceptable ranges for negative control treatments, and were elevated by positive control treatments.

Following Potassium dicyanoaurate treatments of all the test strains in the absence and presence of S-9, no increases in revertant numbers were observed. No statistically significant increases in revertants (when the data were analysed at the 1% level using Dunnett’s test) were observed in either Experiment 1 or Experiment 2, where treatments were performed up to toxic concentrations.

Conclusion

It was concluded that Potassium dicyanoaurate did not induce mutation in five histidine-requiring strains (TA98, TA100, TA1535, TA1537 and TA102) of Salmonella typhimurium when tested under the conditions of this study.