Manganese dinitrate

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2010-01-19 to 2010-01-25

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study follows GLP and internationally accepted guideline

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2010

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

other: EU method B.46 (in vitro skin irritation)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

manganese dinitrate tetrahydrate

IUPAC Name:

manganese dinitrate tetrahydrate

Details on test material:

- Name of test material: manganese nitrate
- Molecular formula : Mn(NO3)2.4H2O
- Substance type: Pink solid
- Physical state: solid
- Storage condition of test material: Approximately 4°C in dark over silica gel

Test animals

Species:

other: EPISKIN™ Reconstituted Human Epidermis model

Strain:

other: not applicable

Details on test animals or test system and environmental conditions:

Not applicable

Test system

Type of coverage:

other: not applicable

Preparation of test site:

other: not applicable

Vehicle:

unchanged (no vehicle)

Controls:

not required

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 10 ± 2 mg


VEHICLE
Test material was used as supplied

Duration of treatment / exposure:

15 minutes

Observation period:

42 hours

Number of animals:

Not applicable

Details on study design:

APPLICATION OF TEST MATERIAL
- Area of exposure: The test material was applied topically to the reconstituted epidermis ensuring uniform coverage. The epidermis surface had been moistened with 5 µL of sterile distilled water to improve contact between the solid test material and the epidermis.


REMOVAL OF TEST SUBSTANCE
- Washing (if done): At the end of the exposure period each tissue was rinsed with a Phosphate Buffered Saline solution containing Ca2+ and Mg2+ before incubating for approximately 42 hours at 37 °C in 5% CO2 air
- Time after start of exposure: 15 minutes


SCORING SYSTEM:
At the end of the post-exposure incubation period each tissue was taken for MTT-loading. The maintenance medium from beneath each tissue was transferred to pre-labelled micro tubes and stored in a freezer for possible inflammatory mediator determination. After MTT loading a total biopsy of each epidermis was made and placed into micro tubes containing acidified isopropanol for extraction of formazan crystals out of the MTT-loaded tissues.
At the end of the formazan extraction period each tube was mixed thoroughly and duplicate 200 µL samples were transferred to the appropriate wells of a pre-labelled 96-well plate. The optical density was measured at 540 nm.

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

other: other: Relative tissue viability %

Value:

>= 107 - <= 110.6

Remarks on result:

other:

Remarks:

Basis: mean. Time point: 15 minutes. Max. score: 100.0. Reversibility: other: not applicable. Remarks: Please refer to table 1 for tabulated results including control.

Other effects / acceptance of results:

The relative mean viability of the test material treated tissues was 108.2 % after a 15-minute exposure.

QUANTITATIVE MTT ASSESSMENT (percentage tissue viability):For the test material, the relative mean tissue viabilities were compared to the mean of the negative control treated tissues (n = 3). The relative mean viabilities were calculated in the following way:

% Relative mean viability = (mean OD540 of test material/mean OD540 of negative control) x 100
The test material was found not to directly reduce MTT.

Any other information on results incl. tables

Table 1: Mean OD₅₄₀ values and % viabilities for the negative control material, positive control material and test material

Material	OD540of tissues	Mean OD540of triplicate tissues	± SD of OD540	Relative individual tissue viability %	Relative mean % viability	± SD of % viability
Negative control material	0.827	0.833	0.040	99.3	100*	4.7
	0.776			95.6
	0.875			105.0
Positive control material	0.065	0.054	0.010	7.8	6.5	1.2
	0.052			6.2
	0.045			5.4
Test material	0.611	0.505	0.092	73.3	60.6	11.1
	0.458			55.0
	0.445			53.4

SD = Standard deviation * = The mean viability of the negative control tissues is set at 100%.

Table 2: Qualitative evaluation of tissue viability (MTT uptake visual evaluation)

Material	Tissue 1	Tissue 2	Tissue 3
Negative control Material	-	-	-
Positive Control Material	++	++	++
Test Material	-	-	-

MTT visual scoring scheme:

-         - = blue tissue (viable)

-         + = blue/white tissue (semi-viable)

-         ++ = tissue is completely white (dead)

Quality criteria

The quality criteria required for acceptance of results in the test were satisfied, i.e. positive control and negative control acceptance criteria.

Applicant's summary and conclusion

Interpretation of results:

not irritating

Remarks:

Criteria used for interpretation of results: other: Expert judgement based on the criteria set out in the study protocol

Conclusions:

The test material was considered to be a non-irritant to the reconstituted human epidermis model EPISKIN™.