Potassium dicyanoargentate

Genetic toxicity: in vitro

Administrative data

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.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

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

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: crystalline

Details on test material:

- Physical state: White crystals
- Expiration date of the lot/batch: 31 December 2015
- Storage condition of test material: Stored at room temperature.

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).

Method

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

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

Controls

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

Results and discussion

Test results

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

Applicant's summary and conclusion

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.