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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:
June-July 2009
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:
OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)
Version / remarks:
Study was performed before publication of the corresponding OECD- and EU-testing methods
Deviations:
no
Principles of method if other than guideline:
The purpose of this study was to determine the skin irritation potential of the test material using the SkinEthic Reconstituted Human Epidermal model (RHE, SkinEthic Laboratories, Nice, France) following exposure periods of 4 and 24 hours. The SkinEthic RHE model incorporates several features which make it advantageous in the study of skin irritancy potential. The model consists of an airlifted, living, multi-layered epidermal tissue construct, produced in polycarbonate inserts in serum-free and chemically defined medium, featuring normal ultra-structure and functionally equivalent to human epidermis in vivo. Test materials are applied directly to the culture surface, at air interface, so that undiluted and/or end use dilutions can be tested directly. The test is based on the hypothesis that irritant chemicals are able to penetrate the stratum corneum of the Skin Ethic RHE model and are sufficiently cytotoxic to cause cell death in the underlying cell layers.
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: crystalline
Details on test material:
- Colour: colourless
- Odour: odourless
- CAS-Number: 89-32-7
- Molecular formula: C10 H2 O6
- Molecular weight: 218.12
Specific details on test material used for the study:
- Sponsor's identification: LZ 6119
- Description: white powder
- Batch number: CSA0901038
- Purity: >= 99.5% (w/w)
- Date received: 01 May 2009
- Storage conditions: at room temperature in the dark

In vitro test system

Test system:
human skin model
Remarks:
SkinEthicTM RHE is an in vitro reconstructed human epidermis from normal human keratinocytes cultured on an inert polycarbonate filter at the air-liquid interface, in a chemically defined medium.
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: not specified (Skin Ethic Laboratories, Nice, France)
Details on animal used as source of test system:
no animals tested in this study
Justification for test system used:
as the test item is know to be a severe skin irritant (listed in Annex I), it was decided to run an in-vitro test instead of an in-vivo study
Vehicle:
unchanged (no vehicle)
Details on test system:
- Supplier: Skin Ethic Laboratories, Nice, France
- Date received: 03 June 2009
- SkinEthic Tissues (0.5cm2): Lot Number 09 022A0506
- Maintenance Medium: Lot Number 0905 011J 398

Receipt of Tissues:
On arrival, the SkinEthic RHE tissues (Day 19), were stored at room temperature prior to transferring into 6-well plates designated 'arrival plates' containing 1 ml of maintenance
medium. It was important to ensure that there were no air bubbles present under the tissue inserts. The tissues were incubated overnight at 37"C, 5% COz in air.

Preparation of Tissues:
Using sterile techniques, 800 ul of maintenance medium at room temperature, was dispensed into the appropriate number of wells of 6-well plates designated 'treatment plates'. Each well was labelled with details of the treatment and the appropriate exposure time. Separate treatment plates were used for each substance and negative control to avoid the possibility of cross contamination. Before treatment, the Day 20 tissues were transferred from the 'arrival plates' into the wells of the 'treatment plates' containing the maintenance medium.
Control samples:
other: Triton X-100
Amount/concentration applied:
The positive control material, Triton X-100, was prepared as a 0.1% w/v solution in sterile distilled water.
Duration of treatment / exposure:
tissues were treated with the test material for exposure periods of 4 and 24 hours
Duration of post-treatment incubation (if applicable):
three hours incubation at 37"C, 5% CO2 in air.
Number of replicates:
triplicate tissues were treated with the test material

Results and discussion

In vitro

Resultsopen allclose all
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
at 4h exposure time
Value:
108.1
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
at 24h exposure time
Value:
96.7
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
Assessment of Direct Test Material Reduction of MTT:
The solution did not turn blue/purple. This was taken to indicate that the test material was not able to directly reduce MTT.

Assessment of Skin Irritation Potential:
The relative mean viability of the test material treated tissues was 108.1% after 4 hours exposure and 96.7% after 24 hours exposure. It was considered unnecessary to proceed with tissue histology or analysis of inflammatory mediator levels.

Qualitative Evaluation of Tissue Viability (MTT Uptake Visual Assessment):
Following the 4 and 24 hour exposures the test material treated tissues appeared blue which was considered to be indicative of viable tissue.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
The test material was considered to be a non-irritant under the conditions of this test.
Executive summary:

The purpose of this study was to determine the skin irritation potential of the test material using the SkinEthic Reconstituted Human Epidermal model (RHE, SkinEthic Laboratories, Nice, France) following exposure periods of 4 and 24 hours. The experimental design of the study consists of a test for Direct Reduction of MTT by the test material, followed by the main test. For the main test, triplicate SkinEthic tissues were treated with approximately 25 mg of test material and exposed for 4 hours and 24 hours. The tissues were incubated at 37°C in a humidified atmosphere of 5% C02 in air for the appropriate exposure times. Duplicate untreated tissues were used for each exposure period to serve as negative controls. Duplicate tissues treated with 50 ul of Triton X-100 0.1% w/v were used for the 24 hour exposure period to serve as a positive control. The relative mean viability of the test material treated tissues was 108.1% after 4 hours exposure and 96.7% after 24 hours exposure. It was considered unnecessary to proceed with tissue histology or analysis of inflammatory mediators. In conclusion, the test material was considered to be a non-irritant in this in-vitro study.