Registration Dossier

Administrative data

skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

Data source

Reference Type:
study report
Report Date:

Materials and methods

Test guidelineopen allclose all
according to
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
according to
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
GLP compliance:

Test material

Test material form:
other: liquid

Test animals

other: human skin model
Details on test animals and environmental conditions:
EPISKIN Small ModelTM (EPISKIN-SMTM, 0.38 cm2, Batch No.: 12-EKIN-028, See APPENDIX 4). This model is a three-dimensional human epidermis
model, which consists of adult human-derived epidermal keratinocytes which have been seeded on a dermal substitute consisting of a collagen type
I matrix coated with type IV collagen. The keratinocytes were cultured for 13 days, which results in a highly differentiated and stratified epidermis
model comprising the main basal, supra basal, spinous and granular layers and a functional stratum corneum.

Test system

unchanged (no vehicle)
Amount / concentration applied:
25 µl
Duration of treatment / exposure:
15 minutes
Observation period:
44 hours
Details on study design:
The test was performed on a total of 3 tissues per test substance together with negative and positive controls. Twenty-five µl of the undiluted test
substance was added (with a pipette) into 12-well plates on top of the skin tissues. Three tissues were treated with 25 µl PBS (negative control) and 3
tissues with 25 µl 5% SDS (positive control) respectively. The positive control was re-spread after 7 minutes contact time. After the exposure period of15 minutes at room temperature, the tissues were washed with phosphate buffered saline. After rinsing the cell culture inserts were each dried carefully and moved to a new well on 2 ml pre-warmed maintenance medium until all tissues were dosed and rinsed.
Subsequently the skin tissues were incubated for approximately 44 hours at 37°C.
After incubation, cell culture inserts were dried and transferred into a 12-wells plate prefilled with 2 ml MTT-medium. The tissues were incubated for 3 h at 37°C. After incubation the tissues were placed on blotting paper. Total biopsy was made by using a biopsy punch. Epidermis was separated from the collagen matrix and both parts were placed in prelabeled microtubes and extracted with 500 µl isopropanol. Tubes were stored refrigerated and protected from light for approximately 69 hours. The amount of extracted formazan was determined spectrophotometrically at 570 nm in duplicate. Skin irritation is expressed as the remaining cell viability after exposure to the test substance.

Results and discussion

In vivo

Irritant / corrosive response data:
The relative mean tissue viability obtained after 15 minutes treatment with the test compared to the negative control tissues was 113%. Since the mean relative tissue viability for the test item was above 50%, the test item is considered to be non-irritant.
The positive control had a mean cell viability after 15 minutes exposure of 8%.

Any other information on results incl. tables

It is concluded that this test is valid.

Applicant's summary and conclusion

Interpretation of results:
study cannot be used for classification
Migrated information
It is concluded that the substance is non-irritant in the in vitro skin irritation test under the experimental conditions described.