Life Power® L1

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation / corrosion

Remarks:

in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study with GLP.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Human Skin Model Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Principles of method if other than guideline:

The reconstructed human epidermis model EPISKIN-SM is designed to predict and classify the corrosive potential of chemicals. The present test is based on the experience that corrosive chemicals show cytotoxic effects following short-term exposure of the stratum corneum of the epidermis.
EPISKIN-SM is a three-dimensional human skin model comprising a reconstructed epidermis with a functional stratum corneum. Its use for skin corrosivity testing involves the topical application of test materials to the surface of the skin, and the subsequent assessment of their effects on cell viability. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT.

GLP compliance:

yes (incl. QA statement)

Test material

Reference

Name:

Unnamed

Type:

Constituent

Test material form:

other: fine dust

Test animals

Species:

other: No animal. A human skin was used.

Details on test animals or test system and environmental conditions:

EPISKIN-SM (Manufacturer: SkinEthic, France) is a three-dimensional human epidermis model. Adult human-derived epidermal keratinocytes are seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen. A highly differentiated and stratified epidermis model is obtained after 13-day culture period comprising the main basal, supra basal, spinous and granular layers and a functional stratum corneum. Its use for skin irritation testing involves topical application of test materials to the surface of the epidermis, and the subsequent assessment of their effects on cell viability.
Kit Contents:
Units: EPISKIN-SM plate containing up to 12 reconstructed epidermis units (area: 0.38 cm2) each reconstructed epidermis is attached to the base of a tissue culture vessel with an O-ring set and maintained on nutritive agar for transport.
Plate: 12-well assay plate.
Punch: EPISKIN-SM biopsy punch for easy sampling of epidermis.
Medium: A flask of sterile “Assay Medium” for use in assays was provided as part of the kit.

Test system

Number of animals:

Replicate wells:
In this assay 3 replicates per test item and 3 negative controls + 3 positive controls were used. As the test item was coloured, in addition to the normal procedure, additional test item-treated tissue was used for the non specific OD evaluation.

Details on study design:

Negative Control: NaCl (9 g/L saline).
Positive Control: Glacial acetic acid.

INDICATOR FOR POTENTIAL FALSE VIABILITY
- Check-method for possible direct MTT reduction with test substance
- Check-method to detect the colouring potential of test-substances
- Additional control(s) for dyes and chemicals able to colour the tissue

APPLICATION
The appropriate number of an assay plate wells were filled with the pre-warmed medium (2.2 mL per well, 37°C). The epidermis units were placed with the media below them, in contact with the epidermis into each prepared well. Three skin units were used for each test or control materials.
- 20 mg of test item was applied evenly to the epidermal surface of each of the three test skin units, then 20 μl NaCl (9 g/L saline) was added to the test item to ensure good contact with the epidermis.
- 50 μl NaCl (9 g/l saline) was added to each of the three negative control skin units.
- 50 μl glacial acetic acid was added to each of the three positive control skin units.
- For additional control for staining effects of the test item, 20 mg of test item was applied evenly to the epidermal surface, then 20 μl NaCl (9 g/L saline) was added to the test item to ensure good contact with the epidermis.
The plates with the treated epidermis units were incubated for the exposure time of 4 hours at room temperature (18-28°C) covered with the plate lids.

RINSING
After the incubation time (4 hours), each skin unit was removed and rinsed thoroughly with PBS 1x solution (0.9 %) to remove all of the test material from the epidermal surface. The rest of the PBS was removed from the epidermal surface using a Pasteur pipette linked to a vacuum source (without touching the epidermis) and the culture medium was removed from the assay plate wells.

MTT TEST
MTT solution (2.2 mL of 0.3 mg/mL MTT) was added to each well below the skin units (except the additional chemical-treated tissue). The lid was replaced and the plate incubated at room temperature (18-28°C) for 3 hours, protected from light.

FORMAZAN EXTRACTION
At the end of incubation with MTT a formazan extraction was undertaken

CELL VIABILITY MEASUREMENTS
Following the formazan extraction, 3×200 μL sample from each tube were placed into the wells of a 96-well plate (labelled appropriately) and read the OD (Absorbance / Optical Density) of the samples in a 96-well plate spectrophotometer using wavelength filter of 540 nm and acidified isopropanol solution as blank (6×200 μL).

Results and discussion

In vivo

Results

Irritation parameter:

other: not relevant

Basis:

mean

Time point:

other: Not relevant for in vitro studies.

Score:

0

Remarks on result:

other: Not relevant for in vitro studies.

Irritant / corrosive response data:

CELL VIABILITY
The results of the optical density (OD) measured at 540 nm of each extract and the calculated % viability of the cells is presented in the attachment.

Other effects:

VALIDITY OF THE TEST
The mean OD value of the three negative control tissues was 0.239. The positive control result showed 18 % viability. All validity criteria were within acceptable limits and therefore the study can be considered as valid.

INDICATOR FOR POTENTIAL FALSE VIABILITY
- Possible direct MTT reduction with Test Substance
No colour change was observed after three hours of incubation of the test item in MTT solution. The test material did not interact with the MTT, therefore additional controls and data calculations were not necessary. A false estimation of viability can be precluded.
- Colouring potential of test substances
As the test item is coloured, one additional test item-treated tissue was used for the non specific OD evaluation. Optical density (measured at 540 nm) of this tissue was determined as 0.024, Non Specific Colour % was calculated as 10%. Therefore additional data calculation was necessary.

Applicant's summary and conclusion

Interpretation of results:

not classified

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

In this in vitro EPISKIN model test with the test substance, the results indicate that the test item is not a skin corrosive.

Executive summary:

The reconstructed human epidermis model EPISKIN-SM is designed to predict and classify the corrosive potential of chemicals, by measuring its cytotoxic effect, as reflected in the MTT assay, on the EPISKIN reconstituted human epidermis.

Disks of EPISKIN (three units / chemical) were treated with test item and incubated for 4 hours at room temperature. Exposure of test material was terminated by rinsing with PBS 1x solution (0.9 %). The viability of each disk was assessed by incubating the tissues for 3 hours with MTT solution. The formazan precipitated was then extracted using acidified isopropanol and quantified spectrophotometrically.

NaCl (9 g/l saline) and glacial acetic acid treated epidermis were used as negative and positive controls. An additional disk was used to provide an estimate of colour contribution from the test item. For each treated tissue viability was expressed as a % relative to negative control. If the mean relative viability after 4 hours of exposure is below 35% of the negative control, the test substance is considered to be corrosive to skin.

Following exposure with the test substance, the mean cell viability (after adjustment for colour) was 101% compared to the negative control and therefore non-corrosive. All validity criteria were within acceptable limits and therefore the study can be considered as valid.

In this in vitro EPISKIN model test with the test substance, the results indicate that the test item is not a skin corrosive.