Manganese-(II)-hexacyanomanganate (II) sodium salts

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Between 2020-03-12 and 2020-06-15

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosomal Aberration Test)

Version / remarks:

2016-07-29

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Version / remarks:

21 December 2015

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Test material

Test material information

Constituent 1

Reference substance name:

Manganese-(II)-hexacyanomanganate (II) sodium salts

EC Number:

847-971-4

Cas Number:

2363126-59-2

Molecular formula:

NaxMny[Mn(CN)6]z X = 1.2-1.4; Y = 1.0; Z = 0.8-0.85

IUPAC Name:

Manganese-(II)-hexacyanomanganate (II) sodium salts

impurity 1

Reference substance name:

Water

EC Number:

231-791-2

EC Name:

Water

Cas Number:

7732-18-5

Molecular formula:

H2O

IUPAC Name:

Oxidane

Test material form:

solid: particulate/powder

Method

Species / strain

Species / strain / cell type:

lymphocytes: see details on mammalian cells

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: male, aged 27 years and female, aged 27 years
- Suitability of cells: yes
- Normal cell cycle time (negative control):

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9 : Moltox (Lot no, 4127), expiry date 25 July 2021
- method of preparation of S9 mix : S9-mix was prepared prior to the dosing of the test cultures and contained the S9 fraction (20% (v/v)), MgCl2 (8mM), KCl (33mM), sodium orthophosphate buffer pH 7.4 (100mM), glucose-6-phosphate (5mM) and NADP (5mM).
- concentration or volume of S9 mix and S9 in the final culture medium : 2%
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): S9 was pre-tested for acceptability by the supplier prior to purchase and was supplied with a relevant “Quality Control & Production Certificate”

Test concentrations with justification for top dose:

4 hrs exposure without S9 followed by 20-hour recovery period in treatment-free media prior to cell harvest:
0, 2, 4, 8, 16, 24, 32 to 64 μg/mL

4 hrs exposure with S9-mix (2%), followed by 20-hour recovery period in treatment-free media prior to cell harvest:
0, 2, 4, 8, 16, 24, 32 to 64 μg/mL

24-hour continuous exposure to the test item without S9-mix prior to cell harvest:
0, 0.5, 1, 2, 4, 8, 12, to 16 μg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Minimal Essential Medium (MEM)

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration : 2
- Number of independent experiments : 2 (Preliminary Toxicity Test and Main Experiment)

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable):
- Test substance added in medium; in agar (plate incorporation); preincubation; in suspension; as impregnation on paper disk

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: 48 h (24 h w/o S9)

- 4-Hour Exposure With Metabolic Activation (S9)
4-Hour Exposure Without Metabolic Activation (S9)
24-Hour Exposure Without Metabolic Activation (S9)

FOR CHROMOSOME ABERRATION AND MICRONUCLEUS:
- Mitosis was arrested by addition of demecolcine (Colcemid 0.1 μg/mL) two hours before the required harvest time.
- After incubation with demecolcine, the cells were centrifuged, the culture medium was drawn off and discarded, and the cells re-suspended in 0.075M hypotonic KCl.
- After approximately fourteen minutes (including centrifugation), most of the hypotonic solution was drawn off and discarded, cells were re-suspended and then fixed by dropping the KCl cell suspension into fresh methanol/glacial acetic acid (3:1 v/v). The fixative was changed at least three times and the cells stored at approximately 4 ºC to ensure complete fixation prior to slide preparation.
- The lymphocytes were re-suspended in several mL of fresh fixative before centrifugation and re-suspension in a small amount of fixative.
- Several drops of this suspension were dropped onto clean, wet microscope slides and left to air dry.
- The slides were dry they were stained in 5% Giemsa for 5 minutes, rinsed, dried and a cover slip applied using mounting medium

- A total of 1000 lymphocyte cell nuclei were counted and the number of cells in metaphase recorded and expressed as the mitotic index and as a percentage of the vehicle control value.

- Where possible, 300 consecutive well-spread metaphases from each concentration were counted (150 per duplicate)

- Where at least 15 cells with aberrations (excluding gaps), slide evaluation was terminated.

- If the cell had 44-48 chromosomes, any gaps, breaks or rearrangements were noted according to the simplified system of Savage (1976) recommended in the 1983 UKEMS guidelines for mutagenicity testing and the ISCN (1985).
- Cells with 69 chromosomes or more were scored as polyploid cells and the incidence of polyploid cells (%) (including the incidence of cells with endoreduplicated chromosomes) was also reported.

Evaluation criteria:

The frequency of cells with structural chromosome aberrations (excluding gaps) in the vehicle control cultures was within the laboratory historical control data range.
• All the positive control chemicals induced a positive response (p≤0.01) and demonstrated the validity of the experiment and the integrity of the S9-mix.
• The study was performed using all three exposure conditions using a top concentration which meets the requirements of the current testing guideline.
• The required number of cells and concentrations were analyzed.

Statistics:

Fisher's Exact test

Results and discussion

Test results

Key result

Species / strain:

lymphocytes:

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

not determinable

Applicant's summary and conclusion

Conclusions:

The test item was considered to be non-clastogenic to human lymphocytes in vitro.

Executive summary:

The study research the structural chromosomal aberrations in cultured mammalian cells (in-vitro).

All vehicle (MEM) controls had frequencies of cells with aberrations within the range expected for normal human lymphocytes.

All the positive control items induced statistically significant increases in the frequency of cells with aberrations. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

The test item did not induce any toxicologically relevant increases in the frequency of cells with aberrations, using a dose range that included a dose level that was either the lowest precipitating dose level in the short-term exposures or induced 55±5% mitotic inhibition in the 24 hour exposure group.