Registration Dossier

Administrative data

skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
22.6.2010 - 14.8.2010
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guideline
according to guideline
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Instead of lefting the 24-well-plat to stand over night in order to produce formazan, the plat was shaked for two hours at room temperature. This deviation can be seen as uncritical since the protocol of MatTek Corporation describes this.
GLP compliance:
yes (incl. QA statement)

Test material


In vitro test system

Test system:
human skin model
Source species:
Cell type:
non-transformed keratinocytes
Cell source:
other: The EpiDermTM tissue consists of normal, human-derived epidermal keratinocytes which have been cultured to form a multi-layered, highly differentiated model of the human epidermis.
Source strain:
not specified
Details on test system:
Commercially available EpiDermTM-Kit.
The EpiDermTM tissue consists of human-derived epidermal keratinocytes which have been cultured to form a multi-layered, highly differentiated model of the human epidermis. It consists of organized basal, spinous and granular layers, and a multi-layered stratum corneum containing intercellular lamellar lipid layers arranged in patterns analogous to those found in vivo. The EpiDermTM tissues are cultured on specially prepared cell cultures inserts.
Control samples:
yes, concurrent MTT non-specific colour control
Amount/concentration applied:
30 µl of the liquid test item
Duration of treatment / exposure:
One plate (three tissues) was used as negative control; each tissue was treated with 30 µl DPBS buffer. One plate was used as positive control; each tissue was treated with 30 µl SDA-solution. One plate was used for treatment with the test item. 30 µl test item were applied, and a nylon mesh was added in order ot ensure sufficient contact with the tissue surface.
Tissues were dosed on one minute intervals. After dosing the last tissue, al plates are transferred into the incubator for 35 min.. 60 min after the first application, the inserts were removed from the plates using sterile forceps and rinsed immediately in one-minute-intervals.
After rinsing, each tissue was dried with a sterile cotton tip and then transferred into a new 6-well-plate with fresh assay medium (0.9 mL). The tissue surfaces were evaluated visually under the stereo microscop, excess test item was removed, where necessary.
Then the tissues were set in the incubator for 24 hours.
Duration of post-treatment incubation (if applicable):
For three incubated tissues, a new 6-well-plate with 0.9 mL assay medium in the upper row was prepared. The tissues were removed from the incubator and shaken for ten min (500 rpm). Then the inserts were transferred into the new 6-well-plate and set into the incubator for 18+/-2 hours for post-incubation.
The medium from the "old" 6-well-plates was collected in the labelled 24-well-plate. It can be stored for 12 monthes at -20 °C for possible interleukin alalysis.
MTT Assay: After a total icubation time of 42 hours, a 24-well-plate was prepared with 300 µL freshly prepared MTT-reagent in each well. The tissues were blotted on the bottom and then transferred into the 24-well-plate. Then the 24-well-plate was set into the incubator for 3 hours.
After this time, the MTT reagent was aspirated and replaced by PBS buffer. This was the aspirated, too, and replaced several times. At last, each insert was thoroughly dried and set into the empty, pre-warmed 24-well-plate. Into each well, 2 mL isopropanol were pipetted, taking care to reach the upper rim of the insert. The plate was then shaked for two hours at room temperature.
After two hours, the inserts in which formazan had been produced in two hours wer pierced with an injection needle, taking care that all colour was extracted. The inserts were then discarded and the content of each well was thoroughly mixed in order to achieve homgenisation.
From each well two replicates with 200 µl solution (each) were pipetted into a 96-well-plate which was read in a plate spectral photometer at 570 nm.
Number of replicates:
From each well two replicates with 200 µl solution (each) were pipetted into a 96-well-plate which was read in a plate spectral photometer at 570 nm.

Results and discussion

In vitro

Irritation / corrosion parameter:
% tissue viability
Percentage values of formazan production were calculated in comparison to the negative control
Run / experiment:
Cell viability is measured by dehydrogenase conversion of MTT 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide, present in cell mitochondria, into a blue formazan salt that is quantitatively measured after extraction from tissues.
Vehicle controls validity:
not applicable
Negative controls validity:
Positive controls validity:
Remarks on result:
positive indication of irritation

Applicant's summary and conclusion

Interpretation of results:
Category 2 (irritant) based on GHS criteria
After the treatment, the relative absorbance values were decreased to 13 %. This value is well below the threshold for irritation (50%).
The optical density of the negative control was well within the required acceptability criterion of 1.0 < mein OD < 2.5. The positive control induced a decrease in the relative absorbance as compared to the negative control to 10.9% (required: <20%) for thus ensuring the validity of the test system. Variation within replicates was within the accepted range for negative control, positive control and test item. For these reasons, the result of the test is considered valid.
The test item is considered irritant.