Manganese ores, reduced

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2009-07-07 to 2009-07-13

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2009

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

Details on test material:

- Name of test material: Sinter ore
- Physical state: Solid
- Substance type: Dark grey rocks
- Composition of test material, percentage of components: Certificate of analysis included within report
- Lot/batch No.: not reported
- Storage condition of test material: room temperature in the dark

Test animals

Species:

other: EPISKIIN™ 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:

other: Positive and negative controls were included in study

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:

The test was performed in triplicate

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:

>= 92.39 - <= 101.3

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 97.3% 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^b 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	0.826	0.756	0.061	109.3	100^a	8.15
	0.710			93.9
	0.733			97.0
Positive control material	0.069	0.074	0.012	9.1	9.7	1.55
	0.065			8.6
	0.087			11.5
Test material	0.744	0.736	0.035	98.4	97.3	4.59
	0.698			92.3
	0.766			101.3

^aThe mean viability of the negative control tissues is set at 100%;^bData are presented in the form of % viability (MTT reduction in the test material treated tissues relative to negative control tissues).

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:

other: Not classified in accordance with EU criteria.

Conclusions:

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

Executive summary:

The skin irritation potential of the test material was assessed in vitro using a method equivalent to EU Method B.46 and in compliance with GLP using an EPISKIIN™ Reconstituted Human Epidermis model.

The relative mean viability of the test material treated tissues was 97.3 % after a 15-minute exposure and was therefore considered to be a non-irritant.