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Toxicological information
Skin irritation / corrosion
Currently viewing:
Administrative data
- Endpoint:
- skin irritation: in vitro / ex vivo
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 014
Materials and methods
Test guidelineopen allclose all
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
- Version / remarks:
- Reconstructed Human Epidermis Test Method of 2013
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- other: L’Oreal. In Vitro Skin Irritation Test: Human Epidermis Model EPISKIN, EPISKIN Skin Irritation Test 15min - 42 hours, Standard Operating Procedure: February 2009 Version 1.8.
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
Test material
- Test material form:
- solid: particulate/powder
- Remarks:
- migrated information: powder
Constituent 1
Test system
- Vehicle:
- unchanged (no vehicle)
- Amount / concentration applied:
- Types of Treatment in the Main Test
- Negative control: Dulbecco's Phosphate Buffered Saline (DPBS), dose volume 10 µl/tissue sample
- Test Substance: WS405777, dose 10 mg/tissue sample after wetting with 5 µl purified water
- Positive control: 5% Sodium Dodecyl Sulphate (SDS) in purified water, dose volume 10 µl/tissue sample - Duration of treatment / exposure:
- 15 ± 0.5 minutes with the test substance, negative or positive control at room temperature, followed by thorough rinsing of each epidermis unit with Dulbecco's phosphate buffered saline
- Details on study design:
- Test System
EPISKIN human epidermis skin constructs consisting of normal, human-derived epidermal keratinocytes and forming a multilayered, highly differentiated model of the human epidermis with a functional multilayered stratum corneum (matrix: collagen type 1 coated with type IV collagen).
Principle of the Test – Main Test
Irritant substances are sufficiently cytotoxic to cause cell deaths in the cell layers. Therefore, cell viability of the multilayers was determined by measurement of mitochondrial dehydrogenase activity assessed by reduction of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) to a soluble, coloured, formazan salt. The degree of formazan salt formation (positively correlated with the degree of cell viability) was measured photometrically (i.e. determination of the optical density of formazan extracts from tissue at 540 nm).
Depending on the percentage of tissue viability attained (compared to negative control viability) a test substance is classified as skin irritating or not skin irritating.
Pre-Tests – Checking for Interference of the Test Substance with the Assay
It was demonstrated, that the test material WS405777 itself did not interact with MTT. Also the test substance WS405777 did not show any potential for colouring water.
Main Test
Each treatment group (test substance, negative/positive controls) comprised 3 live (viable) tissue samples placed into wells of 12 well plates.
Incubation of these tissues before treatment in maintenance medium: ≥ 24 h at 37°C, 5% CO2 in air, in humidified atmosphere , in wells
each containing 2 mL fresh pre-warmed maintenance medium.
Test material administration: Spreading of thin even layer over the epidermal surface.
Termination of 15 ± 0.5 minute exposure period: Removal of residual test material or positive control substance by thorough
rinsing of each epidermis unit with Dulbecco's PBS;
removal of remaining PBS by blotting on absorbent paper.
Posttreatment incubation (all tissue samples) : 42 ± 1 h at 37°C , 5% CO2 in humidified atmosphere, in wells each containing
2 mL fresh pre-warmed maintenance medium
Then MTT incubation: 3 hours (± 5 minutes) at 37°C , 5% CO2 in humidified atmosphere, in wells each containing 2 mL of 0.3 mg/mL MTT.
Formazan extraction (all tissue samples) : Further processing of tissue samples & formazan extraction by vortexing in
acidic isopropanol, 500 µL/sample. The vortexed samples were stored at 2-8°C
protected from light for 48 - 70 hours.
Qantitative determination of optical density: At 540 nm with acidified isopropanol (0.04 N HCl final concentration,
6 x 200 µL) as blanks.
Results and discussion
In vitro
Resultsopen allclose all
- Irritation / corrosion parameter:
- % tissue viability
- Run / experiment:
- replicate a
- Value:
- 107.2
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Irritation / corrosion parameter:
- % tissue viability
- Run / experiment:
- replicate b
- Value:
- 95.7
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Irritation / corrosion parameter:
- % tissue viability
- Run / experiment:
- replicate c
- Value:
- 93.9
- Negative controls validity:
- valid
- Positive controls validity:
- valid
Any other information on results incl. tables
Applicant's summary and conclusion
- Interpretation of results:
- GHS criteria not met
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