Potassium dicyanoargentate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

OECD471 in vitro reverse mutation test in bacterial cells (AMES test), designed to detect frameshift mutations or base pair substitutions in four strains of Salmonella typhimurium (TA98, TA100, TA1535, TA1537) and one Escherichia coli strain (WP2uvrA).

OECD487 in vitro micronucleus test in human lymphocytes designed to detect potential increases in mono- and binucleated cells with micronuclei in the absence and presence of metabolic activation as an indication of chromosome damage.

OECD490 in vitro mammalian cell gene mutation test designed to detect the potential for inducting forward mutations at the thymidine-kinase locus (TK-locus) in L5178Y mouse lymphoma cells.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

15 January 2016 - 24 March 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Mutagenicity refers to the induction of permanent transmissible changes in the amount or structure of the genetic material of cells or organisms. These changes may involve a single gene or a gene segment, a block of genes or chromosomes (i.e. cytogenicity). Genetic toxicity is a broader term and refers to processes which alter the structure, information content or segregation of DNA and are not necessarily associated with mutagenicity. The in vitro micronucleus test (OECD 487) is required to detect structural chromosomal aberrations, and is required to fulfil VIII information requirements.

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Version / remarks:

Adopted 26 September 2014

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Identification: Potassium dicyanoargentate
- Source and lot/batch No. of test material: Batch 112415
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature
TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: No correction was made for the purity/composition of the test item. A solubility test was performed, consequently, Potassium dicyanoargentate was dissolved in dimethyl sulfoxide (DMSO) of spectroscopic quality. Potassium dicyanoargentate concentrations were used within 1 hour after preparation. The final concentration of the solvent in the culture medium was 1.0% (v/v).

Target gene:

The in vitro micronucleus test (OECD 487) is a genotoxicity test for the detection of micronuclei in the cytoplasm of interphase cells, which originate from acentric chromosome fragments (i.e. lacking a centromere) or whole chromosomes that failed to migrate during anaphase. Thus, the in vitro micronucleus test does not detect specific mutations at a target loci, but rather a comprehensive basis for investigating chromosome damaging potential in vitro (i.e. both aneugens and clastogens).

Species / strain / cell type:

lymphocytes:

Details on mammalian cell type (if applicable):

Cultured peripheral human lymphocytes, obtained from blood collected from healthy adult, non-smoking volunteers (aged 18 - 35 years), were used as a test system. The Average Generation Time (AGT) ranged from 12.7 - 13.5 hours. Lymphocyte cultures were started upon collection - 0.4 mL whole blood treated with heparin was added to 5 mL or 4.8 mL supplemented culture medium.

Additional strain / cell type characteristics:

not applicable

Cytokinesis block (if used):

Cytochalasin B was added to cultures to arrest teh formation of actin filaments, in order to discriminate between binucleated and mononucleated cells.

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

In order to select the appropriate dose levels for the in vitro micronucleus, the cytotoxicity of Potassium dicyanoargentate was tested in the presence and absence of metabolic activation. The highest tested concentration was 1990 µg/mL (=0.01 M). Following 24-27 hours exposure, the cytotoxicity of Potassium dicyanoargentate in the lymphocyte cultures was determined using the cytokinesis-block proliferation index (CBPI index). A second dose ranging test informed the highest dose level presenting severe cytotoxicity.
The following dose levels were selected for the first cytogenetic assay: 1, 5, 7.5, 10, 12.5, 15 and 17.5 µg/mL culture medium (with and without S9-mix).
The following dose levels were selected for the second cytogenetic assay: 1, 3, 5, 7.5, 10, 12.5 and 15 µg/mL culture medium (without S9-mix).

Vehicle / solvent:

DMSO

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

other:

Details on test system and experimental conditions:

METHOD OF APPLICATION: in suspension

DURATION
- Exposure duration: 4 hours
- Fixation time (start of exposure up to fixation or harvest of cells): 24-27 hours

SPINDLE INHIBITOR (cytogenetic assays): Cytochalasin B

STAIN (for cytogenetic assays): 5% (v/v) Giemsa (Merck) solution in Sörensenbuffer pH 6.8

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: Cells were collected by centrifugation (5 min, 250 g) and cells in the pellet were fixated carefully with 3 changes of ethanol: acetic acid fixative (3:1 v/v). Fixed cells were dropped onto cleaned slides. At least two slides were prepared per culture. Slides were allowed to dry and thereafter stained for 10 - 30 min with 5% (v/v) Giemsa (Merck) solution in Sörensenbuffer pH 6.8. Thereafter slides were rinsed in water and allowed to dry. The dry slides were automatically embedded in a 1:10 mixture of xylene and mounted with a coverslip.

NUMBER OF CELLS EVALUATED: 1000

CRITERIA FOR MICRONUCLEUS IDENTIFICATION:
The following criteria for scoring of binucleated cells were used (1 - 2, 6):
- Main nuclei that were separate and of approximately equal size.
- Main nuclei that touch and even overlap as long as nuclear boundaries are able to be distinguished.
- Main nuclei that were linked by nucleoplasmic bridges.

The following cells were not scored:
- Trinucleated, quadranucleated, or multinucleated cells.
- Cells where main nuclei were undergoing apoptosis (because micronuclei may be gone already or may be caused by apoptotic process).

The following criteria for scoring micronuclei were adapted from Fenech, 1996 (1):
- The diameter of micronuclei should be less than one-third of the main nucleus.
- Micronuclei should be separate from or marginally overlap with the main nucleus as long as there is clear identification of the nuclear boundary.
- Micronuclei should have similar staining as the main nucleus.

DETERMINATION OF CYTOTOXICITY
- A minimum of 500 cells per culture was counted, scoring cells with one, two or more nuclei (multinucleated cells). The cytostasis / cytotoxicity was determined by calculating the Cytokinesis-Block Proliferation Index (CBPI).

- OTHER:

Rationale for test conditions:

All incubations were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 - 100% (actual range 51 - 91%), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 35.0 - 37.2°C). Temperature and humidity were continuously monitored throughout the experiment. The CO2 percentage was monitored once on each working day. Temporary deviations from the temperature, humidity and CO2 percentage may occur due to opening and closing of the incubator door. Based on laboratory historical data these deviations are considered not to affect the study integrity.

Evaluation criteria:

ACCEPTABILITY
The in vitro 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 item colchicine induces a statistically significant increase in the number of mononucleated cells with micronuclei and the positive control items MMC-C and CP induces a statistically significant increase in the number of binucleated cells with micronuclei. The positive control data will be analysed by the Chi-square test (one-sider, p<0.05).
EVALUATION CRITERIA
A test item is considered positive (clastogenic or aneugenic) in the in vitro micronucleus test if all the following criteria are met:
- At least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control
- Any of the results are outside the 95% control limits of the historical control data range
A test item is considered negative (not clastogenic or aneugenic) in the in vitro micronucleus test if:
- None of the test concentrations exhibit a statistically significant increase compared with the concurrent negative control.
- All results are inside the 95% control limits of the negative historical control data range

Statistics:

GraphPad PRISM version 4.03 was used for statistical analysis of the data

Key result

Species / strain:

lymphocytes: Human peripheral lymphocytes from healthy donors

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

Conclusions:

OECD 487 detects micronuclei in the cytoplasm of interphase cells, which originate from acentric chromosome fragments (i.e. lacking a centromere) or whole chromosomes that failed to migrate during anaphase. Identifying structural chromosomal aberrations (aneugens and clastogens), which are not necessarily induced by mutagenicity, the genotoxicity test is required to fulfil VIII information requirements. Under the test conditions, Potassium dicyanoargentate did not present any genotoxic properties in the micronucleus test. Conducted according to the aforementioned guidelines and GLP, the study passed all validity criteria and was considered to be reliable without restriction (Klimisch 1).

Executive summary:

Mutagenicity refers to the induction of permanent transmissible changes in the amount or structure of the genetic material of cells or organisms. These changes may involve a single gene or a gene segment, a block of genes or chromosomes (i.e. cytogenicity). Genetic toxicity is a broader term and refers to processes which alter the structure, information content or segregation of DNA and are not necessarily associated with mutagenicity. The in vitro micronucleus test (OECD 487) is required to detect structural chromosomal aberrations, and is required to fulfil VIII information requirements. The possible clastogenicity and aneugenicity of Potassium dicyanoargentate was tested in two independent experiments, concentrations up to 8 µg/mL for 3 hours in the first cytogenetic assay, and up to 5 µg/mL for 24 hours in the second. Cells were harvested for analysis following a 24-27 hour incubation.

The number of mono- and binucleated cells with micronuclei found in the solvent control cultures was within the 95% control limits of the distribution of the historical negative control database. The positive control chemicals, mitomycin C and cyclophosphamide both produced a statistically significant increase in the number of binucleated cells with micronuclei. The positive control chemical colchicine produced a statistically significant increase in the number of mononucleated cells with micronuclei. In addition, the number of mono- and binucleated cells with micronuclei found in the positive control cultures was within the 95% control limits of the distribution of the historical positive control database. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly. Potassium dicyanoargentate did not induce a statistically significant or biologically relevant increase in the number of mono- and binucleated cells with micronuclei in the absence and presence of S9-mix, in either of the two experiments.

The In Vitro Micronucleus Assay (OECD 487) is an intentionally accepted in vitro test method to detect genotoxicity, as described in the Annex to the EU Test Methods (TM) Regulation (Council Regulation (EC) No 440/2008). Conducted according to the aforementioned guidelines and GLP, the test passed all validity criteria and was considered to be reliable without restriction (Klimisch 1). It is concluded that the test is valid and that Potassium dicyanoargentate is not clastogenic or aneugenic in human lymphocytes under the experimental conditions reported.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

06 July 2016 - 01 August 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Mutagenicity refers to the induction of permanent transmissible changes in the amount or structure of the genetic material of cells or organisms. Column 1 of REACH Annex VII and VIII inform on the standard mutagenicity information requirements, for substances produced or imported in quantities >1 tpa and >10 tpa, respectively. The in vitro mammalian cell gene mutation test - mouse lymphoma assay (OECD 490, EU B.17) detects is required to fulfil Annex VIII information requirements.

Qualifier:

according to guideline

Guideline:

OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)

Version / remarks:

Adopted 28 July 2015

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: In vitro mammalian cell gene mutation assay

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Identification: Potassium dicyanoargentate
- Source and lot/batch No. of test material: Batch 112415

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: No correction was made for the purity/composition of the test item. A solubility test was performed, consequently, Potassium dicyanoargentate was dissolved in dimethyl sulfoxide (DMSO). The final concentration of the solvent in the culture medium was 1.0% (v/v).

Target gene:

The L5178Y mouse lymphoma assay (OECD 490) detects forward mutations at the thymidine kinase (TK) locus in L5178Y cells, either r in the absence of presence of a metabolic system (S9-mix). The TK mutational system detects base pair mutations, frame shift mutations and small deletions. Cells deficient in the thymidine kinase (TK), due to the forward mutation (TK+/- to TK-/-) are resistant to the cytotoxic effects of the pyrimidine analogue trifluorothymidine (TFT).

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

L5178Y/TK+/- 3.7.2C mouse lymphoma cells were obtained from the American Type Culture Collection (ATCC)

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9-mix

Test concentrations with justification for top dose:

Significant toxicity was observed during an in vitro micronucleus assay in cultured peripheral human lymphocytes at 100 μg/ml exposure medium in the absence and presence of S9-mix. In order to select appropriate dose levels for mutagenicity testing, cytotoxicity data were obtained by treating varying concentrations of cells with test item in the presence or absence of S9-mix. In the first dose range finding test, L5178Y mouse lymphoma cells were treated with Potassium dicyanoargentate concentration range of 6.25 to 100 µg/mL in the absence and presence of S9-mix with a 3 hour treatment period, however, severe cytotoxicity was observed at almost all dose levels. A second dose ranging study at concentrations of 0.01 to 5 µg/mL in the absence of S9-mix and at a range of 0.5 to 10 µg/mL in the presence of S9-mix. The suspension growth expressed as the reduction in cell growth of the solvent control, was used to determine an appropriate dose range for the mutagenicity test.

In the absence of S9-mix, no toxicity in the suspension growth was observed up to and including the test item concentration of 0.5 µg/mL compared to the control. No (or hardly any) cell survival was observed at test item concentrations of 1 and 5 µg/mL. In the presence of S9-mix, the relative suspension growth was 29% at the test item concentration of 7.5 µg/mL compared to the relative suspension growth of the solvent control. Hardly any cell survival was observed at the test item concentration of 10 µg/mL. Based on the results of the dose-ranging test, the following dose range was selected for the mutagenicity test:
Without S9-mix: 0.005, 0.01, 0.05, 0.1, 0.25, 0.5, 0.6, 0.7, 0.8, 0.9, and 1.0 µg/mL exposure medium
With S9-mix: 0.05, 0.1, 0.5, 1, 2.5, 5, 6, 7, 8, 9 and 10 µg/mL exposure medium.

Vehicle / solvent:

Dimethyl sulfoxide (DMSO)

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

methylmethanesulfonate

Details on test system and experimental conditions:

PRE-TREATMENT
Prior to dose range finding and mutagenicity testing, the mouse lymphoma cells were grown for 1 day in R10 medium supplemented with hypoxanthine, aminopterine and thymidine to reduce the amount of spontaneous mutants, followed by a recovery period of 2 days on R10 containing hypoxanthine and thymidine only.

TREATMENT OF CELLS

Potassium dicyanoargentate was tested both in the absence and presence of S9-mix. Per culture 8 x 10^6 cells (10^6/ml) were used. Cell cultures were exposed for 3 hours to the test item in exposure medium in sterile 30 mL centrifuge tubes, and incubated in a shaking incubator at 37.0 ± 1.0 °C and 145 spm. Solvent and positive controls were included and the solvent control was tested in duplicate.
Cell cultures were exposed to the test item in exposure medium in the absence as well as in the presence of S9-mix. After exposure, the cells were separated from the treatment solutions by 2 centrifugation steps (216 g, 5 min). The first centrifugation step was followed by removal of the supernatant and resuspension of the cells in Hanks’ balanced salt solution and finally resuspension in 50 ml growth medium (R10).

EXPRESSION PERIOD

For expression of the mutant phenotype, the remaining cells were cultured for 2 days after the treatment period. During this culture period at least 4 x 10^6 cells (where possible) were subcultured every day in order to maintain log phase growth. Two days after the end of the treatment with the test item the cells were plated for determination of the cloning efficiency (CEday2) and the mutation frequency (MF).

DETERMINATION OF THE MUTATION FREQUENCY

For determination of the CEday2 the cell suspensions were diluted and seeded in wells of a 96-well dish. For determination of the mutation frequency (MF) a total number of 9.6 x 10^5 cells/concentration were plated in five 96-well microtiter plates, each well containing 2000 cells in selective medium (TFT-selection), with the exception of the positive control groups (MMS and CP) where a total number of 9.6 x 10^5 cells/concentration were plated in ten 96-well microtiter plates, each well containing 1000 cells in selective medium (TFT-selection). The microtiter plates for CEday2 and MF were incubated for 12 days. After the incubation period, the plates for the TFT-selection were stained for 2 hours, by adding 0.5 mg/ml 3-[4,5-dimethylthiazol-2-yl]-2,5-
diphenyltetrazolium bromide (MTT) (Sigma) to each well. The plates for the CE day2 and MF were scored with the naked eye or with the microscope.

DURATION
- Exposure duration:3 hours
- Expression time (cells in growth medium): 2 days
- Selection time (if incubation with a selection agent): 12 days
- Fixation time (start of exposure up to fixation or harvest of cells): 2 hours

SELECTION AGENT (mutation assays): TFT-Selection

NUMBER OF CELLS EVALUATED: 5 wells of 2000 cells per test item concentration

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency

Rationale for test conditions:

Test conditions were consistent with OECD TG 490. All incubations were carried out in a humid atmosphere (80 - 100%, actual range 54 – 100%) containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 34.0 – 37.9°C). Temperature and humidity were continuously monitored throughout the experiment. The CO2 percentage was monitored once on each working day. Temporary deviations from the temperature, humidity and CO2 percentage may occur due to opening and closing of the incubator door. Any variation to these conditions were evaluated and maintained in the raw data.

Evaluation criteria:

DETERMINATION OF MUTANT COLONIES
Small colonies are indicative of chromosomal mutations, as mutant cells that have suffered extensive genetic damage have prolonged doubling times and thus form small colonies, whereas less severely affected mutant cells grow at rates similar to the parental cells and form large colonies. The large colonies appear to result from mutants with single gene mutations (substitutions, deletions of base-pairs) affecting the TK gene. The small colonies are morphologically dense colonies with a sharp contour and with a diameter less than a quarter of a well. The large colonies are morphologically less dense colonies with a hazy contour and with a diameter larger than a quarter of a well.

CALCULATION OF THE SURVIVAL OR VIABILITY
Relative Suspension Growth (RSG) = (Suspension growth of test / Suspension growth controls) x 100
The Cloning Efficiency (CE) = (Number of empty wells / Total number of wells) / cells per well
The Relative cloning efficiency (RCE) = (CE test / CE controls) x 100
The Relative Total Growth (RTG) = (RSG / RCE) x 100

CALCULATION OF MUTATION FREQUENCY
The mutation frequency (MF) = CE / CE day2 x 10^6

DATA EVALUATION
Any increase of the mutation frequency was evaluated for its biological relevance, in consideration of the historical control data range. A test item is considered positive (mutagenic) in the mutation assay if it induces a MF of more than MF(controls) + 126 in a dose-dependent manner. An observed increase should be biologically relevant and will be compared with the historical control data range. A test item is considered equivocal (questionable) in the mutation assay if no clear conclusion for positive or negative result can be made after an additional confirmation study. A test item is considered negative (not mutagenic) in the mutation assay if: none of the tested concentrations reaches a mutation frequency of MF(controls) + 126.

Statistics:

No formal hypothesis testing was done.

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Conclusions:

The in vitro L5178Y mouse lymphoma gene mutation assay (OECD 490) detects forward mutations at the thymidine kinase (TK) locus in L5178Y cells, via base pair mutation, frame shift mutations and/or small deletions. Cells deficient in the thymidine kinase (TK), due to the forward mutation (TK+/- to TK-/-) are resistant to the cytotoxic effects of the pyrimidine analogue trifluorothymidine (TFT). The in vitro mammalian cell gene mutation test is required to fulfil Annex VIII information requirements. It is concluded that Potassium dicyanoargentate is mutagenic in the mouse lymphoma L5178Y test system. Conducted according to the aforementioned guidelines and GLP, the study passed all validity criteria and was considered to be reliable without restriction (Klimisch 1).

Executive summary:

Mutagenicity refers to the induction of permanent transmissible changes in the amount or structure of the genetic material of cells or organisms. These changes may involve a single gene or a gene segment, a block of genes or chromosomes (i.e. cytogenicity). The in vitro L5178Y mouse lymphoma gene mutation assay (OECD 490) detects forward mutations at the thymidine kinase (TK) locus in L5178Y cells. The TK mutational system detects base pair mutations, frame shift mutations and small deletions. Cells deficient in the thymidine kinase (TK), due to the forward mutation (TK+/- to TK-/-) are resistant to the cytotoxic effects of the pyrimidine analogue trifluorothymidine (TFT).

In the mutation experiment, Potassium dicyanoargentate was tested up to concentrations of 1 and 7 μg/ml in the absence and presence of S9-mix, respectively. The incubation time was 3 hours. Relative total growth (RTG) was 9 and 30% in the absence and presence of S9-mix, respectively. The mutation frequency found in the solvent control cultures was within the acceptability criteria of this assay and within the 95% control limits of the distribution of the historical negative control database. Positive control chemicals, methyl methanesulfonate and cyclophosphamide, both produced significant increases in the mutation frequency. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly. In the absence of S9-mix, the test item induced an up to 10-fold dose related increase in the mutation frequency. The increase was above the 95% control limits of the distribution of the historical negative control database and also above the GEF + MF(controls) (189 per 10^6 survivors).

In the absence of S9-mix, the test item showed 11- and 6.0-fold increases in the mutant frequency of the small colonies and large colonies respectively, compared to the mean mutant frequency of the small and large colonies of the solvent controls. In the presence of S9-mix, the test item showed 6.1- and 5.5-fold increases in the mutant frequency of the small colonies and large colonies respectively, compared to the mean mutant frequency of the small and large colonies of the solvent controls. The increases in small and large colonies indicate that the test item has the potential to induce both chromosomal aberrations and single cell gene mutations. It is concluded that Potassium dicyanoargentate is mutagenic in the mouse lymphoma L5178Y test system.

The in vitro gene mutation assay in mammalian cells (OECD 490) is an intentionally accepted in vitro test method to detect genotoxicity, as described in the Annex to the EU Test Methods (TM) Regulation (Council Regulation (EC) No 440/2008). Conducted according to the aforementioned guidelines and GLP, the test passed all validity criteria and was considered to be reliable without restriction (Klimisch 1).

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

10 March 2016 - 04 April 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Mutagenicity refers to the induction of permanent transmissible changes in the amount or structure of the genetic material of cells or organisms. These changes may involve a single gene or a gene segment, a block of genes or chromosomes (i.e. cytogenicity). Genetic toxicity is a broader term and refers to processes which alter the structure, information content or segregation of DNA and are not necessarily associated with mutagenicity. Column 1 of REACH Annex VII and VIII inform on the standard mutagenicity information requirements, for substances produced or imported in quantities >1 tpa and >10 tpa, respectively. The Bacterial Reverse Mutation Test (OECD 471, EU B.13/14) detects gain of function point mutations and frameshifts in vitro, and is required to fulfil both Annex VII and VIII information requirements.

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

Adopted July 21, 1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Version / remarks:

Official Journal of the European Union No. L142, 31 May 2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Identification: Potassium dicyanoargentate
- Source and lot/batch No. of test material: Batch 112415

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: No correction was made for the purity/composition of the test item. A solubility test was performed, consequently, Potassium dicyanoargentate was dissolved in dimethyl sulfoxide.

Target gene:

The Salmonella typhimurium and Escherichia coli histidine (his) reversion system is a microbial assay which measures his-to his+ reversion induced by chemicals which cause base changes or frameshift mutations in the genome of this organism. S. typhimurium strains TA98 and TA1537 primarily respond to frameshift mutations at the histidine gene locus his D3052 and his C3076, respectively. S. typhimurium trains TA100 and TA1535 respond to base-pair substitutions in the his G46 locus. The Escherichia coli WP2uvrA strain detects base-pair substitutions. In addition to the mutation in the histidine operon, these strains contain several other mutations that increase their ability to detect mutagens, such as rfa, which refers to partial loss of lipopolysaccharide (LPS) barriers, increasing permeability to macromolecules. All strains presented a loss of DNA excision repair systems (uvrB). Each test train contained the following additional mutations:
rfa: deep rough (defective lipopolysaccharide cellcoat)
gal: mutation in the galactose metabolism
chl: mutation in nitrate reductase
bio: defective biotin synthesis
ubrB: loss of the excision repair system (deletion of the ultraviolet-repair B gene)
A pKM101 plasmid was also used to increase error-prone DNA repair and tetracycline resistance.

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Additional strain / cell type characteristics:

nitroreductase deficient

Metabolic activation:

with and without

Metabolic activation system:

Rat liver microsomal enzymes (S9 homogenate) from Trinova Biochem GmbH

Test concentrations with justification for top dose:

In the dose range finding test, the test item was tested up to concentrations of 5000 µg/plate in the absence and presence of S9-mix in the strains TA100 and WP2uvrA. Potassium dicyanoargentate did not precipitate on the plates at this dose level. Severe toxicity was observed in both tester strains in the absence and presence of S9-mix. Based on the results of the dose range finding test, Potassium dicyanoargentate was tested in the first mutation assay at concentrations of 1.7, 5.4, 17. 52, 164, 512, 1600 and 5000 µg/plate in the presence of 5% (v/v) S9-mix. A second dose range finding test was conducted to clarify the toxicity at lower test item concentrations.
The following dose ranges were selected for the second dose range finding test:
TA100 (-S9 mix): 0.006, 0.018, 0.056, 0.18, 0.55 and 1.7 µg/plate
TA100 (+S9 mix): 0.18, 0.55, 1.7, 5.4, 17 and 52 µg/plate
WP2uvrA (-S9 mix): 0.056, 0.18, 0.55, 1.7, 5.4 and 17 µg/plate
WP2uvrA (+S9 mix): 0.18, 0.55, 1.7, 5.4, 17 and 52 µg/plate
Based on the results of the dose range finding test, the following dose range was selected for the first mutation assay with the tester strains TA1535, TA1357 and TA98:
Absence of S9 (-S9 mix): 0.018, 0.056, 0.18, 0.55, 1.0, and 1.7 µg/plate
Presence of S9 (+S9 mix): 0.55, 1.7, 5.4, 17, 28 and 52 µg/plate
The highest concentration of Potassium dicyanoargentate used in the subsequent mutation assay was the level at which the test item inhibited bacterial growth.

Vehicle / solvent:

Dimethyl sulfoxide (DMSO, SeccoSolv, Merck, Darmstadt, Germany)

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

2-nitrofluorene

sodium azide

methylmethanesulfonate

other: ICR-191 (Sigma)

Details on test system and experimental conditions:

METHOD
- Method of application: in agar (plate incorporation)
- Method: To 3 ml molten top agar, 0.1 ml of a fresh bacterial culture of each of the tester strains and 0.1 ml of the test item with or without 0.5 ml of S9-mix or 0.5 ml 0.1M phosphate buffer, was added. Ingredients were mixed on a Vortex and the content was poured onto a selective agar plate. After solidification, the plates were inverted and incubated at 37 ± 1.0 °C for 48 ± 4 hours. After this the revertant colonies (histidine independent (His+) for salmonella typhimurium bacteria and tryptophan independent (Trp+) for Escherichia coli, were counted.
- Cell density at seeding (if applicable): 10^9 cells/ml
- Colony counting: Sorcerer colony counter

NUMBER OF REPLICATIONS: 3

Rationale for test conditions:

Test conditions were consistent with OECD TG 471

Evaluation criteria:

In addition to the criteria stated below, any increase in the total number of revertants was evaluated for its biological relevance including a comparison of the results with the historical control data range.
A test item is considered negative (not mutagenic) in the test if:
- The total number of revertants in the tester strain TA100 or WP2uvrA is not greater than two (2) times the concurrent control, and the total number of revertants in tester strains TA1535, TA1537 or TA98 is not greater than three (3) times the concurrent vehicle control.
- The negative response should be reproducible in at least one follow-up experiment.

A test item is considered positive (mutagenic) in the test if:
- The total number of revertants in the tester strain TA100 or WP2uvrA is greater than two (2) times the concurrent control, or the total number of revertants in tester strains TA1535, TA1537, TA98 is greater than three (3) times the concurrent vehicle control.
- In case a follow up experiment is performed when a positive response is observed in one of the tester strains, the positive response should be reproducible in at least one follow up experiment.

Statistics:

No formal hypothesis testing was done

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:

not specified

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:

not specified

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:

not specified

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:

not specified

Positive controls validity:

valid

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Additional information on results:

CYTOTOXICITY
Extreme toxicity, as observed by a decrease in the bacterial background lawn, was observed in all Salmonella typhimurium strains (TA1535, TA1537, TA100 and TA98) and Escherichia coli strain (WP2uvrA).

MUTAGENICITY
No statistically significant increase in revertant colony numbers relative to control counts was reported for Potassium dicyanoargentate in any of the five test strains, in two independent experiments, in the presence or absence of S9 metabolic activation. The positive control items showed a significant increase in the number of revertant colonies of the respective test strain and confirmed the validity of the test conditions and sensitivity of the test system.

Conclusions:

The Bacterial Reverse Mutation Test (OECD 471, EU B.13/14) detects gain of function point mutations and frameshifts in vitro. Under the test conditions, Potassium dicyanoargentate did not present any mutagenic properties in the Bacterial Reverse Mutation Test. Conducted according to the aforementioned guidelines and GLP, the Ames Test passed all validity criteria and was considered to be reliable without restriction (Klimisch 1).

Executive summary:

Mutagenicity refers to the induction of permanent transmissible changes in the amount or structure of the genetic material of cells or organisms. The Bacterial Reverse Mutation Test detects gain of function point mutations and frameshifts in vitro. A trace of histidine allows the logarithmic division of the histidine-requiring bacteria in the presence of the test item. This period of auxotrophic cell division forms a lawn of histidine-requiring bacteria, whose growth is prevented by exhaustion of histidine. Only the small fraction of bacteria which has reverted to histidine- independence (either spontaneously or by the action of the test chemical) will continue to divide to form discrete, randomly distributed visible colonies, each one of which consists of the progeny of a single mutant bacterium. The assay determines whether the addition of graded concentrations of the test item to a series of such plates induces a concentration-related increase in mutant colonies compared with plates treated only with the appropriate solvent.

Potassium dicyanoargentate (0.018 to 52.0 µg/plate) was examined in four Salmonella typhimurium strains, TA98, TA100, TA1535 and TA1537, in addition to the Escherichia coli WP2uvrA strain in plate incorporation experiments conducted in the presence and absence of S9 metabolic activation. Cytotoxicity was observed in all test strains. No statistically significant increase in revertant colony numbers relative to control counts was reported for Potassium dicyanoargentate in any of the five test strains, in two independent experiments, in the presence or absence of S9 metabolic activation. The positive control items showed the expected increases in revertant colonies and confirmed the validity of the test.

The Ames Test (OECD 471, EU B.13/14) is an intentionally accepted in vitro test method to detect mutagenicity, as described in the Annex to the EU Test Methods (TM) Regulation (Council Regulation (EC) No 440/2008). Conducted according to the aforementioned guidelines and GLP, the Ames Test passed all validity criteria and was considered to be reliable without restriction (Klimisch 1).

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

OECD 474 - in-vivo mammalian erythrocyte micronucleus test 

OECD 489 - in-vivo mammalian alkalin comet assay

Link to relevant study records

Reference

Reference 1

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

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

Specific details on test material used for the study:

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

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)

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

Dose / conc.:

18 mg/kg bw/day

Dose / conc.:

9 mg/kg bw/day

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.

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.

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

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.

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.

Reference 2

Endpoint:

in vivo mammalian germ cell study: cytogenicity / chromosome aberration

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian comet assay

Specific details on test material used for the study:

- Expiry data: 17 December 2026 (5 year)
- Storage condition: at room temperature

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)

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: milli-Q water
- Concentration of test material in vehicle: 450 mg/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 Milli-Q water is 1.0 g/mL.

Test material concentrations were vortexed until the test material is completely dissolved.
This resulted in colourless solutions for all formulations. Test materials 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 consecutives days

Frequency of treatment:

Once per day

Dose / conc.:

18 mg/kg bw/day

Dose / conc.:

9 mg/kg bw/day

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 Comet Assay test was Ethyl Methanesulfate (EMS; CAS n°62-50-0), Sigma Aldrich, Steinheim, Germany) at 200 mg/kg bw/day dissolved in physiological saline.

- 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 10 mL/kg bw.
- Doses / concentration: 200 mg/kg bw/day dissolved in 10 mL of physiological saline

Tissues and cell types examined:

Liver, glandular stomach and duodenum cells

Details of tissue and slide preparation:

1. Isolation of liver:
The isolation method was based on the publication of Hu et al. 2022. A portion of 0.6-0.7 gram from the liver was removed and minced thoroughly on aluminium foil in ice. The minced liver tissues were added to 10 mL of collagenase (20 Units/mL) dissolved in HBBS (Ca2+ and Mg2+ - free) and incubated in a shaking water bath at 37°C for 20 minutes. Thereafter, a low centrifugation force was applied two times to remove large undigested liver debris (40g for 5 min). The supernatant was collected and centrifuged to precipitate the cells (359g for 10min). The supernatant was removed, and the cell pellet was resuspended in ice cold HBSS (Ca2+ and Mg2+ free) and kept on ice.

2. Isolation of glandular stomach cells:
This isolation method for glandular stomach is based on the JACVAM Comet validation study.
The stomach was cut open and washed free from food using cold Hank's Balanced Salt Solution (HBSS; Ca+2 and Mg2+ free). The force-stomach was removed and discarded. the glandular stomach was stored on ice in mincing buffer incomplete (HBSS containing 20 mM EDTA).

The glandular stomach was then transferred to a petri-dish on ice containing 10mL mincing buffer incomplete. The surface epithelia of the glandular epithelia were gently scraped 3-4 times with a cell scraper. This layer was discarded since the lifetime of these cells is very short in the body with a maximum of 3 days. Therefore, this layer contains a high amount of apoptotic cells which disturb the interpretation in the Comet Assay. Moreover, since the lifetime of these cells is very short it is unlikely that these cells play a role in carcinogenesis.

The glandular stomach was then rinsed with mincing buffer incomplete and transferred to a petri-dish containing 10 mL mincing buffer. The glandular stomach was then scraped multiple times with a cell scraper and the cells were collected in the mincing buffer present in the petri-dish. The mincing buffer consists of 20 mM EDTA (disodium) and 10% DMSO in Hank's Balanced Salt Solution (HBSS), pH 7.5 (DMSO) was added immediately before use).

The cells suspension was filtered through a 100 µm Cell Strainer to purify the cell suspension and collected in a tube and stored on ice.

3. Isolation of duodenum:

This isolation method for duodenum is based on the JACVAM Comet validation study.
The duodenum was then transferred to a petri-dish on ice containing 10 mL mincing buffer incomplete. The duodenum was cut open and the surface epithelia of the glandular epithelia were gently scraped 3-4 times with a cell scraper to remove apoptotic cells in the upper cell layer. This layer was discarded.

The duodenum was then rinsed with mincing buffer incomplete and transferred to a petri-dish containing 10 mL mincing buffer. the duodenum was then scraped multiple times with a cell scraper and the cells are collected in the mincing buffer present in the petri-dish.

The mincing buffer consists of 20 mM EDTA (disodium) and 10% DMSO in Hank's Balanced Salt Solution (HBSS) (Ca2+, Mg 2+ free, and phenol red free if available), pH 7.5 (DMSO was added immediately before use).
The cell suspension was filtered through a 100 µm cell strainer to purify the cell suspension and collected in a tube and stored on ice.

4. Sampling, fixation and storage of tissue for histotechnology and histopathology:
Part of the liver, glandular stomach and duodenum from the animals (with exception of the positive control) used (after isolation of a part of the comet assay) was collected and fixed and stored in 10% buffered formalin (neutral phosphate buffered 4% formaldehyde solution). No histotechnology and histopathology was needed.

5. Preparation of Comet slides:
To the cell suspension, melted low melting point agarose was added (ratio 10:140). The cells were mixed with the LMAgarose and 50 µL was layered on a pre-coated Comet slide in duplicate. Three slides per tissue were prepared. The slides were incubated with the study identification number, animal number and group number. The slides were incubated for 11 to 24 minutes in the refrigerator in the dark until clear appears at the edge of the Comet slide area.

6. Lysis, Electrophoresis and staining of the slides:
The cells on the slides were overnight (approximately 16-18h) immersed in pre-chilled lysis solution in the refrigerator. After this period the slides were immersed/rinsed in neutralization buffer (0.4 M Tris-HCl pH 7.4). The slides were then placed in freshly prepared alkaline solution for 20 minutes (or 30 minutes for liver) at room temperature in the dark. The slides were placed in the electrophoresis unit just beneath the alkaline buffer solution and the voltage was set to 0.7 - 1 Volt/cm. The electrophoresis was performed for 20 to 30 minutes under constant cooling (actual temperature 4.0 to 4.5°C). After electrophoresis the slides were immersed/rinsed in neutralized buffer for 5 minutes. The slides were subsequently immersed for 5 minutes in Absolut ethanol (>99.6% pure) and allowed to dry at room temperature. The slides were stained for approximately 5 to 6 minutes with the fluorescent dye in the refrigerator. Thereafter, the slides were washed with Milli-Q water and allowed to dry at room temperature in the dark and fixed with a coverslip.

7. Comet Scoring:
To prevent bias, slides were randomly coded (per tissue) before examination of the Comets. An adhesive label with study identification and code were placed over the marked slide. The slides were examined with a fluorescence microscope connected to a Comet Assay IV image analysis system. One hundred fifty comets (150) (50 comets of each replicate LMAgarose circle) were examined per sample.

Evaluation criteria:

The following criteria for scoring Comet were used:
- Only horizontal orientation Comets were scored, with the head on the left and the tail on the right.
- Cells that showed overlap or were not sharp were not scored.

The frequency of hedgehogs was determined and documented based on the visual scoring of at least 150 cells per tissues per animal in the repeat experiment. The occurrence of hedgehogs was scored in all treatment groups and the control.

Acceptance criteria:
The in vivo comet is considered acceptable if it meets the following criteria:
1. 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.
2. The positive control EMS should produce at least a statistically significant increase in the percentage. Tail Intensity compared to the vehicle treated animals. The response should be compatible with the data in the historical control databased.
3. Adequate number of cells and dose have been analysed
4. The highest test dose is the MTD or 2000 mg/kg bw/day

Statistics:

ToxRat Professional v 3.3.0 (ToxRat Solutions) was used for statistical analysis of the data.

A test material is considered positive in the Comet Assay if all the following criteria are met:
1. At least one of the treatment group exhibits a statistically significant (one-sided, p< 0.05) increase in percentage Tail Intensity is detected compared with the concurrent negative control.
2. The increase is dose related when evaluated with a trend test
3. Any of the results are outside the 95% control limits of the historical control data range.

A test material is considered negative in the Comet Assay if:
1. None of the treatment groups exhibits a statistically significant (one-side p<0.05) increase in percentage Tail Intensity is detected compared with the concurrent negative control.
2. There is a no concentration-related increase when evaluated with a trend test
3. All results are within the 95% control limits of the negative historical control range.

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Positive controls validity:

valid

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

Bioanalysis

Blood was sampled 0, 1, 2, 4, 6 and 24 hour after 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 blood. All test material dosed animals showed increased levels of the otal silver in blood, confirming systemic exposure.

Main study Comet slide analysis:

Comet slides were prepared and analyzed. 

No statistically significant increase in the mean Tail Intensity (%) was observed in liver, duodenum and glandular stomach cells of test materials treated male animals compared to the vehicle treated animals. In addition, there were no Hedgehogs observed in vehicle and test material treated groups.

The mean Tail Intensity in liver, duodenum and glandular stomach cells of vehicle-treated rats was 1.77+/-0.40% (mean +/- SD), 2.86 +/- 0.26% (mean +/- SD) and 5.08 +/- 1.84% (mean +/- SD) in male animals, respectively, which is within the 95% control limits of the distributions of the historical control data for the vehicle control. The positive control EMS induced a significant increase and showed a mean Tail Intensity of 80.46 +/- 7.69% (mean +/- SD), 44.64 +/- 6.06% (mean +/-SD) and 53.54 +/- 0.20% (mean +/- SD) in male animals in liver, duodenum and glandular stomach cells, respectively. The mean positive control Tail Intensity was within the 95% control limits of the distribution of the historical positive control database.

Adequate numbers of cells (150 cells per animal) and doses were analyzed and the highest test dose was the MTD. Hence, all criteria for an acceptable assay were met.

	Main Tail Intensity in liver cells	Mean Tail Intensity in Duodenum Cells	Mean Tail Intensity in Glandular Stomach Cells
Vehicle controls
Group	Tail Intensity (%)
Rat 1	2,09	2,92	5,53
Rat 2	1,74	2,84	4,59
Rat 3	2,19	2,55	2,14
Rat 4	1,66	3,26	6,71
Rat 5	1,17	2,74	6,43
Mean	1,77	2,86	5,08
SD	0,4	0,26	1,84
Group 2: 4.5 mg/kg
Group	Tail Intensity (%)
Rat 6	1,83	3,75	3,8
Rat 7	2,09	3,63	6,2
Rat 8	1,33	3,6	4,03
Rat 9	2,07	3,63	3,07
Rat 10	3,31	2,27	3,87
Mean	2,13	3,38	4,19
SD	0,73	0,62	1,18
Group 3: 9 mg/kg
Group	Tail Intensity (%)
Rat 11	2,53	3,77	6,25
Rat 12	2,53	1,89	4,46
Rat 13	1,16	2,59	3,39
Rat 14	3,41	2,57	6,21
Rat 15	1,71	2,33	4,02
Mean	2,27	2,63	4,87
SD	0,86	0,7	1,3
Group 4: 18 mg/kg
Group	Tail Intensity (%)
Rat 16	2,44	3,18	2,95
Rat 17	1,56	4,09	2,87
Rat 18	1,38	4,18	4,99
Rat 19	1,41	3,96	2,72
Rat 20	1,44	2,03	4,58
Mean	1,65	3,49	3,62
SD	0,45	0,91	1,07
EMS 200 mg/kg
Group	Tail Intensity (%)
Rat 24	84,47	48,03	53,56
Rat 25	85,31	48,24	53,33
Rat 26	71,59	37,65	53,72
Mean	80,46	44,64	53,54
SD	7,69	6,06	0,2

	Historical Positive Control Data for Comet Assay
	Duodenum Tail Intensity (%) Males and Females	Liver Tail Intensity (%) Males and Females	Glandular Stomach Tail Intensity (%) Males and Females
Mean	50,7	81,6	56,5
SD	9,6	7,6	8,7
n	34	44	33
Lower control limit (95% control limit)	31,9	66,6	39,5
Upper control limit (95% control limit)	69,5	96,6	73,5
SD: standard deviation
n= number of observations
HCD from experiments in May 2019 to May 2022

Conclusions:

The Comet Assay is valid and Potassium dicyanoargentate is not genotoxic in the Comet Assay in liver, duodenum and glandular stomach cells when sampled approximately 3-4 hours post dosing, of males rats that were dosed via oral gavage for three consecutive days up to a dose of 18 mg/kg (MTD) under the experimental conditions described in this report.

Executive summary:

The objective of this study was to obtain information on the potential genotoxicity of Potassium Dicyanoargentate when administered to rats at the maximum recommended dose in accordance with current regulatory guidelines by measuring the increase in DNA strand breaks in liver, duodenum and glandular stomach.

The Wistar Han rat (only males) was the species and strain of choice because it is a 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.
The study has been performed according to the OECD TG 489 and followed the GLP principles.
The test material was dissolved in Milli-Q water.
Based on the results of the dose-range finding study test concentrations of 18 mg/kg/day for male animals was selected as maximum dose for the main test (maximum tolerated dose). Since there were no substantial differences in toxicity between sexes only males were used in the main study.
A small peak 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 concentrations 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%).

The dose formulation samples were homogeneous (i.e. coefficient of variation ≤ 10%). In the main study male animals were dosed three times by oral gavage with vehicle or with 4.5, 9 or 18 mg test material per kg body weight for three consecutive days. A positive control group was dosed twice by oral gavage with 200 mg Ethyl Methane Sulfonate (EMS) per kg
body weight. 

In total 5 treatment groups were used, each consisting of 5 animals, with exception of the positive control and TK animals (3 animals per group) and the highest dose group (8 animals).
Clinical signs of toxicity were limited to the high dose group and included lethargy. Only one animal showed signs of toxicity and included convulsions, tremors, lethargy, no reaction to stimulus and pale skin.
In addition, blood for bioanalysis of total Silver was collected from satellite animals for the 18 mg/kg group (highest dose group) and from satellite animals for the vehicle control group.
Blood was sampled 0, 1, 2, 4, 6 and 24 h after the second dose of satellite animals dosed with the vehicle or the highest concentration of the test material. Vehicle dosed animals showed silver levels below the lower limit of quantification in the blood. All test material dosed animals showed increased levels of silver (as indicator of the test material) in the blood,
confirming systemic exposure.

The results show no statistically significant increase in the mean Tail Intensity (%) was observed in liver, duodenum and glandular stomach cells of test material treated male animals compared to the vehicle treated animals.
The mean Tail Intensity in liver, duodenum and glandular stomach cells of vehicle-treated rats was 1.77 ± 0.40% (mean ± SD), 2.86 ± 0.26% (mean ± SD) and 5.08 ± 1.84% (mean ± SD) in male animals, respectively, which is within the 95% control limits of the distribution of the historical control data for the vehicle control. The positive control EMS induced a
significant increase and showed a mean Tail Intensity of 80.46 ± 7.69% (mean ± SD), 44.64 ± 6.06% (mean ± SD) and 53.54 ± 0.20% (mean ± SD) in male animals in liver, duodenum and glandular stomach cells, respectively. The mean positive control Tail Intensity was within the 95% control limits of the distribution of the historical positive control database. Adequate numbers of cells and doses were analysed and the highest test dose was the MTD. Hence, all criteria for an acceptable assay were met.

In conclusion, the comet assay is valid and Potassium Dicyanoargentate is not genotoxic in the Comet assay in liver, duodenum and glandular stomach cells up to a dose of 18 mg/kg (the maximum tolerated dose) under the experimental conditions described in this report.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Potassium dicyanoargentate was tested for its ability to induce reverse mutations in a screening and definitive Ames assay (Verbaan, 2016) in four strains of Salmonella typhimurium (TA98, TA100, TA1535 and TA1537) and one strain of Escherichia coli (WP2uvrA), following OECD guideline 471. Two experiments were carried out with up to 8 test concentrations. There was no mutation induced at concentrations up to toxic concentrations in the presence and absence of rat liver S9 metabolic activation. Cytotoxicity was observed in various strains at various concentrations with and without metabolic activation.

In an in vitro experiment for chromosomal damage, human lymphocytes were exposed to potassium dicyanoargentate in two independent experiments. Concentrations up to 8 µg/mL for 3 hours in the first cytogenetic assay, and up to 5 µg/mL for 24 hours in the second (Verbaan, 2016). Potassium dicyanoargentate did not induce a statistically significant or biologically relevant increase in the number of mono- and binucleated cells with micronuclei in the absence and presence of S9-mix, in either of the two experiments.

In the mammalian cell gene mutation experiment, potassium dicyanoargentate was tested in the mouse lymphoma L5178Y test system up to concentrations of 1 and 7 μg/mL in the absence and presence of S9-mix, respectively. In the absence of S9-mix, the test item showed 11- and 6.0-fold increases in the mutant frequency of the small colonies and large colonies respectively, compared to the mean mutant frequency of the small and large colonies of the solvent controls. In the presence of S9-mix, the test item showed 6.1- and 5.5-fold increases in the mutant frequency of the small colonies and large colonies respectively, compared to the mean mutant frequency of the small and large colonies of the solvent controls. The increases in small and large colonies indicate that the test item has the potential to induce both chromosomal aberrations and single cell gene mutations. It is concluded that potassium dicyanoargentate is mutagenic in the mouse lymphoma L5178Y test system.

 

Following the positive results of the in-vitro mammalian cell gene mutation test (OECD 490) and in accordance with Regulation 1907/2006 (REACH Regulation) further investigations were performed and an combined in-vivo micronucleus (OECD 474) and comet assay (OECD 489) was performed.

The in-vivo micronucleus test - concluded that 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. 

The in-vivo comet assay test - concluded that potassium dicyanoargentate is not genotoxic in liver, duodenum and glandular stomach cells when sampled approximately 3-4 hours post dosing, of males rats that were dosed via oral gavage for three consecutive days up to a dose of 18 mg/kg bw (MTD) under the experimental conditions.

 

 

Justification for classification or non-classification

The mammalian cell gene mutation assay (OECD490) represents the most relevant genetic toxicity study using mammalian cells and was a guideline study conducted in accordance with GLP. However, this study produced a positive result indicating a potential for the test substance to cause chromosome aberrations or single cell gene mutations. Therefore, a testing proposal for the conduct of a suitable in vivo assay has been made in accordance with Annex IX Section 8.4 Column 2 rules.

Following the positive in-vitro mammalian cell gene mutation assay (OECD 490) - according to Annex VIII of Regulation 1907/2006, further investigations were performed to assess the genotoxicity of potassium dicyanoargentate. A combined Micronucleus and Alkaline Comet Test in the Rat (OECD 474 and OECD 489) was performed. Both test concluded on the non-genotoxicity/mutagenicity of potassium dicyanoargentate. Therefore, it is concluded that potassium dicyanoargenate does not need to be classified for that endpoint.