Potassium dicyanoaurate

Genetic toxicity: in vivo

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2022

Report date:

2022

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

29 July 2016

Deviations:

no

Test guideline 2

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

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

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

Test animals

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)

Administration / exposure

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

Doses / concentrations

Doses / concentrations 1

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%).

Doses / concentrations 2

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%).

Doses / concentrations 3

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)

Examinations

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.

Results and discussion

Test results

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)

Any other information on results incl. tables

Table: Mean number of micronucleated plychromatic erythrocytes and ratio of polychromatic/normochromatic erythrocytes
group	treatment	number of animals	dose (mg/kg(bw))	number of micronucleated polychromatic erythrocytes (mean +/- SD) *	ratio polychromatic/normochromatic erythrocytes (mean +/- SD) **
1	Vehicle control	5	0	4.0 ± 2.7	0.94 ± 0.06
2	Test material	5	5	5.4 ± 3.0	0.81 ± 0.13
3	Test material	5	10	4.6 ± 2.1	0.84 ± 0.12
4	Test material	5	20	5.2 ± 1.9	0.79 ± 0.12
5	CP	3	19	38.0 ± 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
group	animal number	number of polychromatic erythrocytes*	number of normochromatic erythrocytes*	ratio polychromatic/normochromatic erythrocytes	number of micronucleated polychromatic erythrocytes	number of polychromatic erythrocytes scored for micronuclei
1	1	497	506	0.98	1	4006
1	2	461	552	0.84	6	4009
1	3	496	508	0.98	1	4001
1	4	489	512	0.96	6	4010
1	5	485	519	0.93	6	4003
2	6	484	526	0.92	6	4013
2	7	462	557	0.83	8	4011
2	8	499	520	0.96	1	4014
2	9	416	616	0.68	4	4007
2	10	408	602	0.68	8	4000
3	11	495	513	0.96	3	4010
3	12	449	579	0.78	6	4010
3	13	487	527	0.92	5	4009
3	14	474	543	0.87	2	4005
3	15	412	614	0.67	7	4026
4	16	447	570	0.78	6	4009
4	17	427	575	0.74	5	4006
4	18	471	553	0.85	3	4021
4	19	494	525	0.94	8	4014
4	20	391	624	0.63	4	4000
5	24	243	786	0.31	45	4002
5	25	318	681	0.47	36	4003
5	26	274	727	0.38	33	4037
*: 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 cells	3.9
Standard deviation (SD)	1.5
Number of observations (n)	50
Lower control limit (95% control limits)	1
Upper control limit (95% control limits)	7
minimum	1
maximum	8
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 cells	36.1
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*:
Group	Treatment	Dose (mg/kg bw)	sex	P-value (one-sided)	Decision at 95% confidence level
5	cyclophosphamide	19	males	<0.001	significant
*: 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:
Group	Treatment	Dose (mg/kg bw)	sex	P-value (one-sided)	Decision at 95% confidence level
5	cyclophosphamide	19	males	<0.001	significant
*: comparicson with the corresponding vehicle control group by using the Student's t test

 

Applicant's summary and conclusion

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.