Potassium dicyanoargentate

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian cell study: DNA damage and/or repair

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 6 May 2022 to 23 December 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

The study has been performed in accordance with OECD test guideline and followed GLP principles

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2022

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian erythrocyte micronucleus test

Test material

Test material information

Constituent 1

Reference substance name:

Potassium dicyanoargentate

EC Number:

208-047-0

EC Name:

Potassium dicyanoargentate

Cas Number:

506-61-6

Molecular formula:

C2AgN2.K

IUPAC Name:

Potassium silver(1+) cyanide (1:1:2)

Test material form:

solid: particulate/powder

Remarks:

white powder

Details on test material:

- Lot/batch No.: 311-21
- pH: 9.27 - 9.60
- Solubility in vehicle: 145 g/L in water

Specific details on test material used for the study:

- Expiry date: 17 December 2026 (5 year expiry date applied)
- Storage condition: at room temperature

Test animals

Species:

rat

Strain:

Wistar

Remarks:

Crl:WI(Han)

Details on species / strain selection:

The Wistar Han rat was the species and strain of choice because it is readily available rodent, which is commonly used for genotoxicity testing, with documented susceptibility to a wide range of toxic materials. Moreover, historical control background data has been generated with this strain.

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Animal sex: since there were no substantial differences in toxicity between sexes only in males were used in the main study.

- Source: Charles River Deutschland, Sulzfeld, Germany
- Age at study initiation: 6-9 weeks
- Weight at study initiation: within 20% of sex mean
- Assigned to test groups randomly: yes - the animals were allocated at random to treatment groups. Males and females were randomized separately. Animals in poor health or at extremes of body weight range were not assigned to groups. Females were nulliparous and non-pregnant.
- Identification: rats were identified with a unique number on the tail written with marker pen.
- Fasting period before study:
- Housing: Polycarbonate cages (Makrolon MIV type or 2000P Tecniplast) 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.
- 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): SM R/M-Z from SSNIFF Spezialdiäten GmbH, Soest, Germany
- Type of diet: Pellets
- Frequency: Ad libitum, except during designed 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): Municipal tap water
- Frequency: 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 5 days before the commencement of dosing

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 to 24°C
- Humidity (%): 40 to 70%
- 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 designed procedures)

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: mili-Q water
- Concentration of test material in vehicle: 450mg/L - 900 mg/L - 1800 mg/L
- Amount of vehicle (if gavage or dermal): 10 mL/kg bw meaning 1.75 mL per animal

Preparation of test material: No correction factor was made for the purity / composition of test material.
A solubility test was performed based on visual assessment. The test material was dissolved in Milli-Q Water (Millipore Corp., Bedford, MA., USA). The specific gravity of Mili-Q water is 1.0g/mL. Test material concentrations were vortexed until the test material is completely dissolved.

This resulted in colourless solutions for all formulations. Test material concentrations were dosed within 4 hours after preparation.

Sample collection and Analysis: Dose formulation samples were collected for analysis as indicated in the section "Any other information on Materials and Methods"

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: The test item formulations were prepared and subsequently dosed within 4 hours.

Duration of treatment / exposure:

3 consecutive days

Frequency of treatment:

once per day

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

18 mg/kg bw/day

Doses / concentrations 2

Dose / conc.:

9 mg/kg bw/day

Doses / concentrations 3

Dose / conc.:

4.5 mg/kg bw/day

No. of animals per sex per dose:

In the dose range finding: 3 animal/dose/group (3 males and 3 females)
In the main study: 5 male rats per dose

Control animals:

yes, concurrent vehicle

Positive control(s):

The positive control for micronucleus test was Cyclophosphamide (CP; CAS 6055-19-2)
- Route of administration: oral gavage
- Frequency: twice (once daily)
- Method: a limited quantity of food was supplied during the night before last dosing (approximately 7g/rat). The dose was given using a plastic feeding tube. The dosing volume was 10mL/kg bw.
- Doses / concentrations: 19 mg/kg bw dissolved in physiological saline.

Examinations

Tissues and cell types examined:

1. Isolation of Cells:
Approximately 3-4 hours after the third treatment with the test material bone marrow was isolated for the micronucleus test

Details of tissue and slide preparation:

1. 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 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 and air-dried overnight. At least two slides were prepared per animal.

2. Staining of the bone marrow smears:
The slides were automatically stained using the "wright-stain-procedure" in a HEMA-tek slide stainer. This staining is based on Giemsa. The dry slides were automatically mounted with a coverslip with an automated coverslipper.

3. Analysis of the bone marrow smears for micronuclei:
To prevent bias, all slides were randomly coded before examination. 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 500 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:

Acceptance criteria:
A micronucleus test is considered acceptable if it meets the following criteria:
- 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.
- The concurrent positive controls should induce responses that are compatible with those generated in the historical positive control database.
- The positive control material induces a statistically significant increase in the frequency of micronucleated polychromatic erythrocytes.

Statistics:

ToxRat Professional v 3.3.0 (ToxRat Solution GmbH, Germany) was used for statistical analysis of the data.
A test material is considered positive in the micronucleus test if all of the following criteria are met:
- at least one of the treatment groups exhibits a statistically significant (one-sides, p< 0.05) increase in the frequency of micronucleated polychromatic erythrocytes compared with the concurrent negative control.
- The increase is dose related when evaluated with a trend test
- 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:
- None of the treatment groups exhibits a statistically significant increase in the frequency of micronucleated polychromatic erythrocytes compared with the concurrent negative control.
- There is no concentration-related increase when evaluated with a trend test.
- All results are within the 95% control limits of the negative historical control data range.

Results and discussion

Test results

Key result

Sex:

male

Genotoxicity:

negative

Remarks:

All test item dosed in animals showed increased levels of the test item in the blood, confirming systemic exposure.

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Any other information on results incl. tables

Dose Formulation sample collection schedule

Interval	Concentration (M)	Homogeneity (TMB)	Sampling From
Single Occasion	All groups 2x approximately 500mg	Groups 2 and 4 Approximately 500 mg	Dosing containing

M= sample collected from approximately Middle; TMB = sample collected from approximately Top, Middle and Bottom
The homogeneity results obtained from top, middle and bottom for groups 2 and 4 was averaged and utilized as the concentration results.

Accuracy: a small response was observed in the vehicle. It was considered to possibly derive from the vehicle since the response was approximately double that of the analytical blanks. However, the maximum contribution to the samples was 0.16%, therefore, this observed small response is not considered to have impacted the outcome of the study.

The concentration analyzed in the dose formulation samples were in agreement with target concentrations (i.e. mean sample concentration results were within or equal to 90%-110%)

Homogeneity: The dose formulation samples wre homogeneous

Unscheduled deaths:

In the Dose-range finding, one animal dosed with 20 mg/kg bw was euthanized for humane reasons.
In the Main study, one animal from the highest dosed group (18 mg/kg bw) euthanized for humane reasons.
Animals were deeply anaesthetized using isoflurane and subsequently exsanguinated.

Dose Range finding results:

During the dose-range finding study, at a dose level of 20 mg/kg rough coat and lethargy was observed in 1 male and 1 female, and the female animal showed so severe clinical symptoms after the third dosing it was prematurely euthanized.
At a dose level of 15 mg/kg no clinical symptoms were observed in 1 male and 1 female.
At a dose level of 18 mg/kg 1 male and 1 female had diarrhea after dosing and a rough coat on day three.
Two additional males had a rough coat after the first dosing. With these low dose levels and the small dosing steps, and the quick onset of severe symptoms, 18 mg/kg bw is determined to be the MTD.

In a dose-range finding study 10 animals (group 1 and 2: 1 male and 1 female, group 3: 3 males and 3 females) were dosed via oral gavage with 20, 15 and 18 mg/kg bw (group 1, 2 and 3, respectively). 

Mortality and Toxic Signs in the Dose-range Finding Study
				Toxic signs*
Group	Sex	Animal Number	Dose mg/kg	Day 1		Day 2		Day 3		Day4
				Post-dose	Pre-dose	Post-dose	Pre-dose	Post-dose	Post-dose (additional observation)
1	Male	101	20	F	B	F, N	F, N	F, N	F, N	B
1	Female	102	20	F, N	B	F, N	F, N	D, E, F, N, O, Q, X (1)	NA	NA
2	Male	103	15	B	B	B	B	B	2)	B
2	Female	104	15	B	B	B	B	B	2)	B
3	Male	105	18	N	B	N	B	N	2)	2)
3	Female	106	18	N	B	N	B	N	2)	2)
3	Male	107	18	N	B	B	B	B	2)	2)
3	Male	108	18	N	B	B	B	B	2)	2)
3	Female	109	18	B	B	B	B	B	2)	2)
3	Female	110	18	B	B	B	B	B	2)	2)

* Legend "mortality and toxic signs"

NA= Not Applicable; B= showed no abnormalities; D= convulsions; E= tremors; F= lethargy; N= rough coat; O= pale skin; Q= quick breathing; X= pinched eyes

(1)= Sacrificed for humane reasons

2) Animal not observed

Main study:

Mean body weight per group recorded prior to dosing:

Group code	dose (mg/kg bw	Day 1 Body weight gram (Mean +/- S.D)	Day 2 Body weight gram (Mean +/-SD)	Day 3 Body weight gram (Mean +/- SD)
1	0	176,4 +/- 4,2	180 +/- 4,4	173,2 +/- 3,1
2	4,5	173,8 +/- 10,5	175,2 +/- 13,0	171,6 +/- 8,7
3	9	176,4 +/- 11,6	175,8 +/- 15,7	172,6 +/- 13,5
4	18	179,1 +/- 9,3	184,9 +/-9,5	180,7 +/- 9,5
TK 1	0	178,3 +/- 6,4	182,7 +/- 3,5	not dosed
TK 4	18	171,7 +/- 16,2	174,3 +/- 15,9	not dosed

Group 1: control - Group 2: 4.5 mg/kg bw - Group 3: 9 mg/kg bw - Group 4: 18 mg/kg bw

Mortality and Toxic Signs after treatment in the main study

Mortality and Toxicity Signs after Treatment in the Main Study
				Toxic signs
Group	Sex	Animal Number	Dose mg/kg	day 1 post-dose within	day 2 pre-dose	day 2 post-dose within	day 3 pre-dose	day 3 post-dose within
1	Male	1	0	B	B	B	B	B
1	Male	2	0	B	B	B	B	B
1	Male	3	0	B	B	B	B	B
1	Male	4	0	B	B	B	B	B
1	Male	5	0	B	B	B	B	B
2	Male	6	4,5	B	B	B	B	B
2	Male	7	4,5	B	B	B	B	B
2	Male	8	4,5	B	B	B	B	B
2	Male	9	4,5	B	B	B	B	B
2	Male	10	4,5	B	B	B	B	B
3	Male	11	9	B	B	B	B	B
3	Male	12	9	B	B	B	B	B
3	Male	13	9	B	B	B	B	B
3	Male	14	9	B	B	B	B	B
3	Male	15	9	B	B	B	B	B
4	Male	16	18	B	B	B	B	B
4	Male	17	18	B	B	B	B	B
4	Male	18	18	B	B	B	B	B
4	Male	19	18	B	B	B	B	B
4	Male	20	18	B	B	B	B	B
4	Male	21	18	B	B	B	B	B
4	Male	22	18	B	B	B	B	B
4	Male	23	18	D, E, F, G, O	NA	NA	NA	NA
TK 1	Male	27	0	B	B	B	B	B
TK 1	Male	28	0	B	B	B	B	B
TK 1	Male	29	0	B	B	B	B	B
TK 4	Male	30	18	B	B	F	B	B
TK 4	Male	31	18	B	B	F	B	B
TK 4	Male	32	18	B	B	F	B	B

Mean Number of Micronucleated Polychromatic Erythrocytes and Ratio of Polychromatic/Normochromatic Erythrocytes

Group	Treatment	Number of Animals	Dose (mg/kg bw/day)	Number of micronucleated polychromatic erythrocytes (mean +/-SD)	Ration polychromatic/normochromatic erythrocytes (mean +/-SD)
1	vehicle control	5	0	4,2 +/- 2,5	0,94 +/- 0,07
2	test item	5	4,5	2,8 +/- 0,8	0,94 +/- 0,06
3	test item	5	9	1,6 +/- 0,5	0,95 +/- 0,02
4	test item	5	18	2 +/- 1,2	0,97 +/- 0,03
5	CP	3	19	71 +/- 14,8	0,28 +/- 0,01

Individual Data Micronucleus Assay

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	498	503	0,99	2	4005
1	2	499	505	0,99	5	4010
1	3	451	553	0,82	4	4014
1	4	497	509	0,98	2	4001
1	5	494	528	0,94	8	4000
2	6	483	518	0,93	4	4001
2	7	498	503	0,99	3	4006
2	8	468	550	0,85	2	4008
2	9	492	513	0,96	2	4000
2	10	497	507	0,98	3	4020
3	11	486	518	0,94	1	4000
3	12	492	526	0,94	2	4006
3	13	493	507	0,97	2	4026
3	14	496	509	0,97	1	4004
3	15	491	516	0,95	2	4013
4	16	496	506	0,97	1	4002
4	17	499	501	1	4	4010
4	18	484	522	0,93	2	4027
4	19	498	506	0,98	1	4005
4	20	493	507	0,97	2	4004
(2)	113	223	778	0,29	78	4000
(2)	114	208	792	0,26	81	4000
(2)	115	218	789	0,28	54	4000

(2) Positive control slides taken from male animals previously dosed with CP were added to the study slides for evaluation as scoring controls.

	Historical Positive control data for Micronucleus
			Male
Mean Number of micronucleated cells per 4000 cells			34,6
SD			23,6
n			55
Lower control limit (95% control limit)			-12
Upper control limit (95% control limit)			81
SD: standard deviation
n= number of observations
HCD from experiments in May 2019 to May 2022

Micronucleated Polychromatic Erythrocytes:

No increase in the mean frequency of micronucleated polychromatic erythrocytes was observed in the bone marrow of test item treated animals compared to the vehicle treated animals.

The incidence of micronucleated polychromatic erythrocytes in the bone marrow of all negative control animals was within the 95% control limites 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.

Applicant's summary and conclusion

Conclusions:

In conclusion, the test item is not clastogenic or aneugenic in the bone marrow micronucleus test of male rats up to a dose of 18 mg/kg bw (MTD) under the experimental conditions.
All criteria for an acceptable assay were met.

Executive summary:

The test item was administered to Wistar Han rat (male) at the maximum recommended dose in accordance with OECD 474 guideline and followed GLP principles. The potential genotoxicity was assessed by measuring the increase in the number of micronucleated polychromatic erythrocytes per 4000 polychromatic erythrocytes in rat bone marrow..

Based on the results of the dose-range finding study test concentrations of 18 mg/kg bw/day for male animals was selected as maximum tolerated dose for the main test. The test item was dissolved in Milli-Q water.

In the main study male animals were dosed three times by oral gavage with vehicle or with 4.5, 9 and 18 mg test material per kg body weight for three consecutive days. A positive control group slides from male animals previously dosed with 19 mg cyclophosphamide were added to this study for evaluation as scoring controls. In total 5 treatment groups were used, each consisting of 5 animals with exception of positive control and TK animals and high dose group (8 animals).

Clinical signs of toxicity were limited to the high dose groupo and included lethargy. Only one animal showed signs of toxicity and included convulsions, tremors, lethargy, no reaction to stimulus and pale skin.

Approximately 3-4 hours after the last dose the animals were sacrified by abdominal aorta bleeding under isoflurane anesthesia. Bone marrow smears were prepared for micronucleus analysis.

No increase in the mean frequency of micronucleated polychromatic erythrocytes was observed in the bone marrow of animals treated with the test item compared to the vehicle treated animals. The incidence of micronucleated polychromic erythrocytes in the bone marrow of all negative control animals was 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 micronuclei. In addition, the number of micronuclei found in the positive control animals was within 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 item showed no decrease in the ratio of polychromatic to normochromatic erythrocytes compared to the concurrent vehicle control group, indicating a lack of toxic effects of this test item on erythropoiesis. The group that was treated with cyclophosphamide showed an expected decrease in the ratio of polychromatic to normochromatic erythrocytes compared to the vehicle control, demonstrating toxic effect on erythropoiesis.

In conclusion, the test item is not clastogenic or aneugenic in the bone marrow micronucleus test of male rats up to a dose of 18 mg/kg bw (MTD) under the experimental conditions.