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Toxicological information

Skin irritation / corrosion

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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:
other: EU method B.46 (in vitro skin irritation)
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
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 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:
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™.