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

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2020-03-24 til 2020-03-27

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 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.40 (In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test (TER))

Version / remarks:

"bis"

Deviations:

no

GLP compliance:

yes (incl. QA statement)

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

Specific details on test material used for the study:

See section "Test material information"

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

other: target cells are epithelial, derived from human skin, and formed into a stratified, cornified epithelium.

Cell source:

other: EpiDerm™ Reconstructed Human Epidermis Model Kit

Vehicle:

other: Dullbeccos Phosphate Buffered Saline

Details on test system:

SKIN DISC PREPARATION
- Procedure used: not applicable
- Quality control for skin discs: Electrical resistance obtained with two of the isolated skin discs was [complete, e.g. 10 kΩ] not applicable

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 °C
- Temperature of post-treatment incubation (if applicable): not applicable

REMOVAL OF TEST MATERIAL AND CONTROLS
- Number of washing steps: Dulbecco’s Phosphate Buffered Saline (DPBS) (without Ca++ Mg++) for approximately 40 seconds
- Observable damage in the tissue due to washing: not reported
- Modifications to validated SOP: no

DYE BINDING METHOD
- Dye used in the dye-binding assay: MTT
- Spectrophotometer: Labtech LT-4500 microplate reader and LT-com analysis software
- Wavelength: 570 nm
- Filter:
- Filter bandwidth: 10 nm

NUMBER OF INDEPENDENT TESTING RUNS / EXPERIMENTS TO DERIVE FINAL PREDICTION:
PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be corrosive to skin if [complete, e.g. if the mean TER value is less than or equal to 5 kΩ and the skin disk is obviously damaged, or if the mean TER value is less than or equal to 5 kΩ, and the skin disc is showing no obvious damage, but the mean disc dye content is greater than or equal to the mean disc dye content of the 10M HCl positive control obtained concurrently.]

- The test substance is considered to be non-corrosive to skin if [complete, e.g. if the mean TER value obtained for the test substance is greater than 5 kΩ, or if the mean TER value is less than or equal to 5 kΩ, and the skin disc is showing no obvious damage, and the mean disc dye content is well below the mean disc dye content of the 10M HCl positive control obtained concurrently.]

- Justification for the selection of the cut-off point(s) if different than recommended in TG 430: not applicable

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit):
- Concentration (if solution): 25 mg + 25 µL H2O per plate

NEGATIVE CONTROL
- Concentration (if solution): 50 µL H2O per plate

POSITIVE CONTROL
- Concentration (if solution): 50 µL 8 N KOH per plate

Duration of treatment / exposure:

3 minutes and 60 minutes

Duration of post-treatment incubation (if applicable):

3 hrs MTT incubation

Number of replicates:

2

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 minutes

Value:

96.6

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

set as 100 %

Positive controls validity:

valid

Remarks:

4.5%

Remarks on result:

no indication of irritation

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

60 minutes

Value:

92

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

set as 100%

Positive controls validity:

valid

Remarks:

3.9 %

Remarks on result:

no indication of irritation

Any other information on results incl. tables

Tissue	Exposure Period	Mean OD570of individual tissues	Mean OD570of duplicate tissues	Standard Deviation	Coefficient of Variation (%)	Relative Mean Viability (%)
Negative Control⊕	3 Minutes	1,804	1.885	0.115	6.1	100*
		1,966
	60 Minutes	1,882	1.869	0.019	1.0
		1,855
Positive Control⊕	3 Minutes	0,096	0.085	0.016	na	4.5
		0,074
	60 Minutes	0,083	0.073	0.015	na	3.9
		0,062
Test Item	3 Minutes	1,747	1.820	0.103	5.7	96.6
		1,893
	60 Minutes	1,670	1.719	0.069	4.0	92.0
		1,719

negative control set as 100%

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The test item was considered to be non-corrosive to the skin.

Executive summary:

Purpose

The purpose of this test was to evaluate the corrosivity potential of the test item using the EpiDerm™ Human Skin Model after treatment periods of 3 and 60 minutes.

Corrosion is directly related to cytotoxicity in the EpiDerm™ tissue. Cytotoxicity is determined by the reduction of MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to formazan by viable cells in the test item treated tissues relative to the corresponding negative control. Viable cells are able to reduce MTT to formazan whereas non-viable cells cannot. The results are used to make a prediction of the corrosivity potential of the test item (increased cytotoxicity is indicative of corrosion potential).

Methods

Duplicate tissues were treated with the test item for exposure periods of 3 and 60 minutes. Negative and positive control groups were treated for each exposure period. The test item was found to have the potential to cause color interference and therefore additional tissues were incorporated for color correction purposes. An assessment of the test item’s capability to directly reduce MTT was inconclusive due to the intrinsic color of the test item and therefore, an additional procedure using freeze killed tissues was performed. A third set of controls was included, comprising freeze killed tissues, in order to prevent a double correction from a colored test item that also reduces MTT. At the end of the exposure period the test item was rinsed from each tissue before each tissue was taken for MTT loading. After MTT-loading each tissue was placed in 2 mL of isopropanol for MTT extraction.

At the end of the formazan extraction period each well was mixed thoroughly and triplicate 200 μL samples were transferred to the appropriate wells of a pre-labeled 96-well plate. The optical density (OD) was measured at 570 nm (OD570).

Data are presented in the form of percentage viability (MTT reduction in the test item treated tissues relative to negative control tissues).