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Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In vitro micronucleus assay (OECD487) using human lymphocytes. AMES reverse mutation assay (OECD471) in bacterial cells.

Link to relevant study records

Referenceopen allclose all

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other: GLP compliant, guideline study, available as an unpublished report.
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
Target gene:
histidine
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).
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
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
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
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
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
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
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
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
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
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.
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
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
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: 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'.

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.

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.

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 0.030, 0.063, 0.125, 0.25, 0.5, 1, 2 and 3 ug/mL
Experiment 1b: Nominal concentrations of 0.063, 0.125, 0.25, 0.5, 0.75, 1, 1.5 and 3 ug/mL
Experiment 2: Nominal concentrations of 0.063, 0.125, 0.25, 0.5, 0.75, 1, 1.5 and 3 ug/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Minimal essential medium (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:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
No surviving cells above 1 ug/mL in the absence of S9 and 1.5 ug/mL in the presence of S9 in Experiment 1. No surviving cells above 1 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: 7.30 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: The test item is soluble 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 0.70, 1.41, 2.81, 5.63, 11.25, 22.5, 45, 90, 360, 1440 and 2880 ug/mL. In the 4-hour exposure group, binucleate cells were present at up to 2.81 ug/mL in both the absence and presence of metabolic activation. In the 24-hour exposure group, binucleate cells were found up to 1.41 ug/mL.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.
Conclusions:
Interpretation of results (migrated information):
positive

Potassium dicyanoaurate is considered to be mutagenic to human lymphocytes in vitro.
Executive summary:

The potential for potassium dicyanoaurate to cause chromosome aberrations was tested in a micronucleus test in human lymphocytes in vitro. Cells were exposed to nominal concentrations of 0.030, 0.63, 0.125, 0.25, 0.5, 1, 2 and 3 ug/mL potassium dicyanoaurate in the absence of S9 for 4 hours and nominal concentrations of 0.063, 0.125, 0.25, 0.5, 0.75, 1, 1.5 and 3 ug/mL potassium dicyanoaurate for 4 -hours in the presence of S9 and for 4 -hours and 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. Potassium dicyanoaurate is considered to be mutagenic to human lymphocytes in vitro.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

Potassium Dicyanoaurate is not clastogenic or aneugenic in the bone marrow micronucleus test of male rats (according to OECD474, GLP compliant) up to a dose of 20 mg/kg body weight (the maximum tolerated dose in accordance with current regulatory guidelines).

Link to relevant study records
Reference
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Study period:
18 Nov 2021 - 9 May 2022
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
29 July 2016
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
14 Feb 2017
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
mammalian erythrocyte micronucleus test
Specific details on test material used for the study:
purity: 68.14% Au
white powder
expiry date: 19 Aug 2026
pH 10.2 at 5% concentration
Species:
rat
Strain:
Wistar
Remarks:
Crl: WI(Han)
Details on species / strain selection:
The Wistar-Han rat was chosen as the animal model for this study as it is an accepted rodent species for nonclinical toxicity test by regulatory agencies.
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany.
- Age at study initiation: 5-6 weeks
- Weight at study initiation: The body weights of the rats at the start of the treatment were within 20% of the sex mean. The mean body weights were for males 180.1 ± 7.9 g and the range for males 165 - 193 g.
- Assigned to test groups randomly: yes, the animals were allocated at random to treatment groups. Animals in poor health or at
extremes of body weight range were not assigned to groups.
- Fasting period before study: no
- Housing:
°Caging: Polycarbonate cages (Makrolon type IV, height 18 cm) containing sterilized sawdust as bedding material (Lignocel S 8-15, JRS -
J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany) equipped with water bottles.
During treatment in the dose-range finding study, polycarbonate cages (Makrolon type MIII, height 18 cm) containing sterilized sawdust as
bedding material (Lignocel S 8-15, JRS - J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany) equipped with water bottles.
Up to 5 animals of the same sex and same dosing group were housed together.
The animals were housed in room number R.20 (DRF) or R.11 (main).
°Cage Identification: Colour-coded cage card indicating Test Facility Study No., group, animal number(s).
°Animal Enrichment: Animals were socially housed for psychological/environmental enrichment and were provided with materials such as devices for hiding in, paper and/or objects for chewing, except when interrupted by study procedures/activities.
- Diet (e.g. ad libitum):
°Diet: SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany
°Type: Pellets
°Frequency: Ad libitum, except during designated procedures.
°Analysis: Results of analysis for nutritional components and environmental contaminants were provided by the supplier and are on file at the Test Facility. It is considered that there were no known contaminants in the feed that would interfere with the objectives of the study.
- Water (e.g. ad libitum):
°Type: Municipal tap water.
°Frequency/Ration: Freely available to each animal via water bottles.
°Analysis: Periodic analysis of the water was performed, and results of these analyses are on file at the Test Facility. It is considered that there were no known contaminants in the water that could interfere with the outcome of the study.
- Acclimation period: The animals were allowed to acclimate to the Test Facility toxicology accommodation for at least 6 days before the commencement of dosing.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 to 24°C (actual daily mean temperature during the study period was 20 to 22°C)
- Humidity (%): 40 to 70% (actual daily mean relative humidity of 44 to 51%)
- Air changes (per hr): Ten or more air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours light and 12 hours dark (except during designated procedures)

IN-LIFE DATES: 22-24 Nov 2021 (in-life DRF) and 21-23 Dec 2021 (in-life main study)
Route of administration:
oral: gavage
Vehicle:
The vehicle of the test item was Milli-Q water (Millipore Corp., Bedford, MA., USA). The vehicle was determined in a non-glp solubility test at the CRL lab (data retained in lab archives).
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
-No correction was made for the purity/composition of the test material.
-The test material was dissolved in Milli-Q water. The density of Milli-Q water is 1.0 g/mL. Test material concentrations were vortexed until the test material was completely dissolved, and a magnetic stirring bar was added during the DRF.
-This resulted in solutions for all formulations. Test material concentrations were dosed within 3.5 hours after preparation.
-Concentration and Homogeneity Analysis:
°Storage Conditions: Room temperature set to maintain 21°C
°Acceptance Criteria: For concentration:, mean sample concentration results within or equal to
+/- 10% of theoretical concentration. For homogeneity, relative standard deviation (RSD) of concentrations of +/- 10% for each group.
-stability analysis: the validated analytical method was based on inductively coupled plasma - mass spectrometry (ICP-MS) for quantitative analysis on the gold constituent of the test item. Test item formulations were prepared and subsequently dosed within 4 hours, during this time period samples were taken from the formulations and analyzed. The highest selected dose level was the MTD and systemic exposure were determined in plasma of the rats. The 24-hour stability of the test item in water has been confirmed in a study performed at another CRO (Alessa Chemie, Doc No UB-PCL 105/2013). Therefore, it was concluded that not having additional stability data has no impact on the integrity of the study.
Duration of treatment / exposure:
twice dosing with a 24h interval
Frequency of treatment:
once daily for 2 days
Post exposure period:
no
Dose / conc.:
5 mg/kg bw/day (nominal)
Remarks:
The concentration analyzed in the dose formulation samples was in agreement with target
concentrations (i.e., mean accuracies between 90% and 110%).
Dose / conc.:
10 mg/kg bw/day (nominal)
Remarks:
The concentration analyzed in the dose formulation samples was in agreement with target
concentrations (i.e., mean accuracies between 90% and 110%).
Dose / conc.:
20 mg/kg bw/day (nominal)
Remarks:
The concentration analyzed in the dose formulation samples was in agreement with target
concentrations (i.e., mean accuracies between 90% and 110%).
No. of animals per sex per dose:
5 animals per dose (except for high dose: 8 animals used to correct for possible deaths - cfr. outcome dose-range finding study)
3 additional animals for control and high dose for demonstration of systemic exposure to the test item (bioanalyticalsample collection at 0, 1, 2, 4, 6 and 24 h post-dose on day 2)
3 animals for positive control treatment
(32 animals in total for the main study)
Control animals:
yes, concurrent vehicle
Positive control(s):
The positive control used was cyclophosphamide (CP; CAS No. 6055-19-2; Sigma Aldrich Chemie GmbH, Steinheim, Germany) dissolved in physiological saline (Eurovet Animal Health, Bladel, the Netherlands).
Oral gavage, once, 10 ml/kg(bw)
Tissues and cell types examined:
Isolation of Bone Marrow:
Bone marrow was sampled 48 hours after the first dosing (i.e., 24h after the 2nd dosing). Both femurs were removed and freed of blood and muscles. Both ends of the bone were shortened until a small opening to the marrow canal became visible. The bone was flushed with approximately 4 mL of fetal calf serum (Invitrogen Corporation, Breda, The Netherlands). The cell suspension was collected and centrifuged at 216 g for 5 min.
Details of tissue and slide preparation:
DETAILS OF SLIDE PREPARATION:
°Preparation of Bone Marrow Smears
The supernatant was removed with a Pasteur pipette. Approximately 500 μl serum was left on
the pellet. The cells in the sediment were carefully mixed with the remaining serum. A drop
of the cell suspension was placed on the end of a clean slide, which was previously immersed
in a 1:1 mixture of 96% (v/v) ethanol (Merck, Darmstadt, Germany)/ether (Merck) and
cleaned with a tissue. The slides were marked with the study identification number and the
animal number. The drop was spread by moving a clean slide with round-whetted sides at an
angle of approximately 45° over the slide with the drop of bone marrow suspension. The
preparations were air-dried, fixed for 5 min in 100% methanol (Merck) and air-dried
overnight. At least two slides were prepared per animal.
°Staining of the Bone Marrow Smears
The slides were automatically stained using the "Wright-stain-procedure" in a HEMA-tek
slide stainer (Hematek 3000, Siemens Healthcare, Den Haag, The Netherlands). This staining
is based on Giemsa. The dry slides were automatically embedded in a 1:10 mixture of xylene
(Klinipath, Duiven, The Netherlands)/pertex (Klinipath) and mounted with a coverslip in an
automated cover slipper (Leica Microsystems B.V., Rijswijk, The Netherlands).

METHOD OF ANALYSIS:
Analysis of the Bone Marrow Smears for Micronuclei
To prevent bias, all slides were randomly coded before examination. An adhesive label with
study identification number and code was stuck over the marked slide. At first the slides were
screened at a magnification of 100 x for regions of suitable technical quality, i.e. where the
cells were well spread, undamaged and well stained. Slides were scored at a magnification of
1000 x. The number of micronucleated polychromatic erythrocytes was counted in at least
4000 polychromatic erythrocytes (with a maximum deviation of 5%). The ratio of
polychromatic to normochromatic erythrocytes was determined by counting and
differentiating at least the first 1000 erythrocytes at the same time. Micronuclei were only
counted in polychromatic erythrocytes. Averages and standard deviations were calculated.
Parts on the slides that contained mast cells that might interfere with the scoring of
micronucleated polychromatic erythrocytes were not used for scoring.
Evaluation criteria:
ACCEPTABILITY CRITERIA
A micronucleus test is considered acceptable if it meets the following criteria:
a) The concurrent negative control data are considered acceptable when they are within the 95% control limits of the distribution of the historical negative control database.
b) The concurrent positive controls should induce responses that are compatible with those generated in the historical positive control database.
c) The positive control material induces a statistically significant increase in the frequency of micronucleated polychromatic erythrocytes.

A test material is considered positive in the micronucleus test if all of the following criteria are met:
a) At least one of the treatment groups exhibits a statistically significant (one-sided, p < 0.05) increase in the frequency of micronucleated polychromatic erythrocytes compared with the concurrent negative control
b) The increase is dose related when evaluated with a trend test.
c) Any of the results are outside the 95% control limits of the historical control data range.

A test material is considered negative in the micronucleus test if:
a) None of the treatment groups exhibits a statistically significant (one-sided, p < 0.05) increase in the frequency of micronucleated polychromatic erythrocytes compared with
the concurrent negative control.
b) There is no concentration-related increase when evaluated with a trend test.
c) All results are within the 95% control limits of the negative historical control data range.
Statistics:
ToxRat Professional v 3.2.1 (ToxRat Solutions® GmbH, Germany) was used for statistical analysis of the data.
Key result
Sex:
male
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
-Mortality and Toxic Signs: The animals of the groups treated test material/kg body weight and the animals of the negative and positive control groups showed no treatment related clinical signs of toxicity or mortality. Except for a few incidental symptoms in a single animal at the highest dose group:
°animal 22: lethargy, squinted eyes and drooling immediately after dosing (technician assessed the animals as ‘not feeling well’) at day2 post-dose (within 1 h), lethargy at day3)
°animal 32: drooling immediately after dosing (technician assessed the animals as ‘not feeling well’) at day2 post-dose (within 1 h))
Animal 10 has a bulging right eye as from day 2 pre-dose onwards.
Not other toxic signs (or mortality) observed during the main study.

-Micronucleated polychromatic erythrocytes:
No increase in the mean frequency of micronucleated polychromatic erythrocytes was observed in the bone marrow of the test material treated animals compared to the vehicle treated animals. In three animals (2 low dose and 1 high dose), the observed number of micronucleated polychromatic erythrocytes was just above the 95% control limits of the distribution of the historical negative control database (8 vs 7). However, these were within the min-max range observed in our historical control database and therefore accepted.
The incidence of micronucleated polychromatic erythrocytes in the bone marrow of all negative control animals was within the within the 95% control limits of the distribution of the historical negative control database.
Cyclophosphamide, the positive control material, induced a statistically significant increase in the number of micronucleated polychromatic erythrocytes. In addition, the number of micronucleated polychromatic erythrocytes found in the positive control animals was within the 95% control limits of the distribution of the historical positive control database. Hence, all criteria for an acceptable assay were met.

-Ratio Polychromatic to Normochromatic Erythrocytes:
The animals of the groups, which were treated with the test material showed no decrease in the ratio of polychromatic to normochromatic erythrocytes, which indicated a lack of toxic effects of this test material on the erythropoiesis. The animals of the groups treated with cyclophosphamide showed an expected decrease in the ratio of polychromatic to normochromatic erythrocytes, demonstrating toxic effects on erythropoiesis.

-Bioanalysis:
°The rat plasma (EDTA) samples have been analyzed by ICP-MS/MS for the quantification of gold using a fit for purpose method. Gold was used as analyte representative of Potassium Dicyanoaurate. The analytical batches from which results have been reported met the acceptance criteria.
Analyte was detected in rat plasma samples from all test material-dosed animals after dose administration. No measurable amount of analyte was detected in control rat plasma samples.
°Summary plasma gold concentration (ng/mL) on study day2 in rats:
Range of Au concentration from 6 sampling points between 0-24 h post-dose Group 1 (control animals)
animal 27: all animal 28: all animal 29: all Range of Au concentration from 6 sampling points between 0-24 h post-dose Group 4 (20 mg/kg (bw))
animal 30: 5220-8130
animal 31: 7990-11900
animal 32: 6060-8770
(LLOQ=50 ng/mL)
































































Table: Mean number of micronucleated plychromatic erythrocytes and ratio of polychromatic/normochromatic erythrocytes
grouptreatmentnumber of animalsdose (mg/kg(bw))number of micronucleated polychromatic erythrocytes (mean +/- SD) *ratio polychromatic/normochromatic erythrocytes (mean +/- SD) **
1Vehicle control504.0 ± 2.70.94 ± 0.06
2Test material555.4 ± 3.00.81 ± 0.13
3Test material5104.6 ± 2.10.84 ± 0.12
4Test material5205.2 ± 1.90.79 ± 0.12
5CP31938.0 ± 6.2***0.38 ± 0.08 ***
*: at least 4000 polychromatic erythrocytes were evaluated with a maximum deviation of 5%
**: the ratio was determined from at least the first 500 erythrocytes counted 
***: significantly different from corresponding control group (Student's t-test, P<0.001)

 






































































































































































































































Table: Individual number of micronucleated polychromatic erythrocytes and ratio of polychromatic/normochromatic erythrocytes
groupanimal numbernumber of polychromatic erythrocytes*number of normochromatic erythrocytes*ratio polychromatic/normochromatic erythrocytesnumber of micronucleated polychromatic erythrocytesnumber of polychromatic erythrocytes scored for micronuclei
114975060.9814006
124615520.8464009
134965080.9814001
144895120.9664010
154855190.9364003
264845260.9264013
274625570.8384011
284995200.9614014
294166160.6844007
2104086020.6884000
3114955130.9634010
3124495790.7864010
3134875270.9254009
3144745430.8724005
3154126140.6774026
4164475700.7864009
4174275750.7454006
4184715530.8534021
4194945250.9484014
4203916240.6344000
5242437860.31454002
5253186810.47364003
5262747270.38334037
*: the ratio was determined from the first 500 erythrocytes counted   

 







































Table: Historical negative control data for micronucleus studies (male)
mean number of micronucleated cells per 4000 cells3.9
Standard deviation (SD)1.5
Number of observations (n)50
Lower control limit (95% control limits)1
Upper control limit (95% control limits)7
minimum1
maximum8
Distribution historical negative control date from experiments performed between November 2018 and November 2021

 































Table: Historical positive control data for micronucleus studies (male)
mean number of micronucleated cells per 4000 cells36.1
Standard deviation (SD)26.5
Number of observations (n)47
Lower control limit (95% control limits)-16
Upper control limit (95% control limits)88
Distribution historical negative control date from experiments performed between November 2018 and November 2021






































































































Statistics Micronucleus Test    
Test Material:     
comparison with the corresponding vehicle control group by using the Dunnett's t test, no significant differences
Positive control*:    
GroupTreatmentDose (mg/kg bw)sexP-value (one-sided)Decision at 95% confidence level
5cyclophosphamide19males<0.001significant
*: comparicson with the corresponding vehicle control group by using the Student's t test 
      
Statistics polychromatic/normochromatic erythrocytes   
Test Material:     
comparison with the corresponding vehicle control group by using the Dunnett's t test, no significant differences
Positive control:     
GroupTreatmentDose (mg/kg bw)sexP-value (one-sided)Decision at 95% confidence level
5cyclophosphamide19males<0.001significant
*: comparicson with the corresponding vehicle control group by using the Student's t test 

 

Conclusions:
Potassium Dicyanoaurate is not clastogenic or aneugenic in the bone marrow micronucleus test of male rats (according to OECD474, GLP compliant) up to a dose of 20 mg/kg body weight (the maximum tolerated dose in accordance with current regulatory guidelines) under the experimental conditions described in this report.
Executive summary:

The clastogenicity and aneugenicity of Potassium Dicyanoaurate was assessed by administration of the substance to rats at a maximum tolerated acute dose, by measuring the increase in the number of micronucleated polychromatic erythrocytes per 4000 polychromatic erythrocytes in rat bone marrow (in compliance with the most recent OECD 474 and EC guidelines, GLP-compliant).
The test material was dissolved in Milli-Q water and administered via gavage. The concentrations analyzed in the dose formulation samples were in agreement with target
concentrations and homogeneous.
A vehicle control and positive control group was included.
No treatment related clinical signs or mortality were noted in any animal treated with the test material or control animals receiving vehicle or positive control, except for two animals in the highest dose group showing mild symptoms.
Bone marrow was sampled 48 hours after the first dosing.
No increase in the mean frequency of micronucleated polychromatic erythrocytes was observed in the bone marrow of animals treated with the test material compared to the vehicle treated animals. The incidence of micronucleated polychromatic erythrocytes in the bone marrow of negative control animals was within the 95% control limits of the distribution of the historical negative control database. The positive control material induced a statistically significant increase in the number of micronucleated polychromatic erythrocytes. In addition, the number of micronucleated polychromatic erythrocytes found in the positive control animals was within the 95% control limits of the distribution of the historical positive control database. Hence, all criteria for an acceptable assay were met.
The groups that were treated with the test material showed no decrease in the ratio of polychromatic (immature) to normochromatic (mature) erythrocytes compared to the concurrent vehicle control group, indicating a lack of toxic effects of this test material on erythropoiesis. The group that was treated with the positive control showed an expected decrease in the ratio of polychromatic to normochromatic erythrocytes compared to the vehicle control, demonstrating toxic effects on erythropoiesis.


Blood was sampled at six timepoints between 0 - 24 h after the second dose of satellite animals dosed with the vehicle and the highest concentration of the test material. Vehicle dosed animals showed levels below the lower limit of quantification in the plasma. All test material dosed animals showed increased levels of the test material in the plasma, confirming systemic exposure.
In conclusion, Potassium Dicyanoaurate is not clastogenic or aneugenic in the bone marrow micronucleus test of male rats up to a dose of 20 mg/kg (the maximum tolerated dose in accordance with current regulatory guidelines) under the experimental conditions described in this report.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vitro:
Potassium dicyanoaurate 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), following OECD guideline 471. Two experiments were carried out with 7 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.


In an in vitro experiment for chromosomal damage, human lymphocytes were exposed to 8 concentrations of potassium dicyanoaurate with and without activation for 4 and 24 hours (Bowles 2015). Potassium dicyanoaurate was determined to be mutagenic to human lymphocytes under the conditions of this test.


Additional information from genetic toxicity in vivo:


A follow-up in vivo assay according to OECD474 (GLP-compliant was performed). The clastogenicity and aneugenicity of Potassium Dicyanoaurate was assessed by administration of the substance to rats at a maximum tolerated acute dose, by measuring the increase in the number of micronucleated polychromatic erythrocytes per 4000 polychromatic erythrocytes in rat bone marrow. 
No increase in the mean frequency of micronucleated polychromatic erythrocytes was observed in the bone marrow of animals treated with the test material compared to the vehicle treated animals. The groups that were treated with the test material showed no decrease in the ratio of polychromatic (immature) to normochromatic (mature) erythrocytes compared to the concurrent vehicle control group. All criteria for an acceptable assay were met.


Blood was sampled at six timepoints between 0 - 24 h after the second dose of satellite animals dosed with the vehicle and the highest concentration of the test material. Vehicle dosed animals showed levels below the lower limit of quantification in the plasma. All test material dosed animals showed increased levels of the test material in the plasma, confirming systemic exposure.
In conclusion, Potassium Dicyanoaurate is considered not clastogenic or aneugenic in the bone marrow micronucleus test of male rats up to a dose of 20 mg/kg (the maximum tolerated dose in accordance with current regulatory guidelines) under the experimental conditions described in this report.

Justification for classification or non-classification

A recent in vivo assay (according to OECD guidelines, GLP-compliant) demonstrates that potassium dicyanoaurate is not clastogenic or aneugenic in the bone marrow micronucleus test up to 20 mg/kg (bw) (identified as the maximum tolerated dose). No classification for genetic toxicity is needed.