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: Well documented and reliable GLP guideline study

Data source

Reference Type:
study report
Report Date:

Materials and methods

Test guideline
according to
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
GLP compliance:
yes (incl. certificate)
BASF SE, Ludwigshafen, Germany

Test material

Test material form:
other: solid
Details on test material:
Please refer to confidential details on test material.

Test animals

other: human-derived epidermal keratinocytes
other: not applicable
Details on test animals and environmental conditions:
Three dimensional human epidermal model

The EpiDerm™ model consists of normal, 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 (surface 0.6 cm²) are cultured on specially prepared cell culture inserts and commercially available as kits, containing 24 tissues on shipping agarose.

Test system

Type of coverage:
other: not applicable
Preparation of test site:
other: not rapplicable
other: PBS
other: not applicable
Amount / concentration applied:
25 µl of solid test item
Duration of treatment / exposure:
1 hour
Observation period:
about 42 hours post-incubation
Number of animals:
not applicable
Details on study design:

Direct MTT reduction
To assess the ability of the test material to directly reduce MTT a pretest was performed. The test substance was added to 0.9 mL of a MTT solution. The mixture was incubated in the dark at about 37 °C for 55 to 65 minutes. A negative control (de-ionized water) was tested
concurrently. If the MTT solution color or, in case of water-insoluble test substances the border to the water-phase, turned blue / purple, the test substance was presumed to directly reduce MTT. The direct reduction of MTT by a test substance interferes with the color density produced by metabolic capacity of the tissue and would falsify the test results.

Basic procedure
On the day of arrival in the laboratory, the tissues were transferred to sterile 6-well plates with 0.9 mL assay medium and preconditioned in the incubator at 37°C. After 1 hour the preincubation medium was replaced with fresh medium and preconditioning continued for 18 ± 3 hours. Three tissues were treated with the test substance, the positive control (PC) and negative control (NC), respectively. A volume of 25 μL sterile PBS was applied first. Thereafter, a bulk volume of 25 μL of the solid test material was applied with a sharp spoon and homogeneously distributed together with the fluid. Control tissues were concurrently applied with 30 μL of sterile PBS (NC) or with 30 μL of 5% SDS (PC). A nylon mesh was placed carefully onto the tissue surface afterwards. The tissues were kept under the laminar flow hood at room temperature for 25 minutes overall and for 35 minutes in the incubator. The tissues were washed with sterile PBS to remove residual test material 1 hour after start of application. Rinsed tissues were blotted on sterile absorbent paper and transferred into new 6-well plates, pre-filled with 0.9 mL fresh medium. When all tissues were rinsed, the surface of each tissue was carefully dried with a sterile cotton swab. Subsequently, the tissues were incubated in the incubator at 37°C for 24 ± 2 hours. After 24 ± 2 hours the tissues were transferred into new 6-well plates pre-filled with 0.9 mL of fresh medium and placed into the incubator for additional 18 ± 2 hours post-incubation period. After the postincubation period, the assay medium was replaced by 0.3 mL MTT solution and the tissues were incubated in the incubator for 3 hours. After incubation, the tissues were washed with PBS to stop the MTT-incubation. The formazan that was metabolically produced by the tissues was extracted by incubation of the tissues in isopropanol. The optical density at a wavelength of 570 nm (OD570) of the extracts was determined spectrophotometrically. Blank values were established of 6 microtiter wells filled with isopropanol for each microtiter plate.

Data evaluation
The OD570 values determined for the various tissues are measures of their viability. The quotient of the OD570 of tissues treated with the test material and the mean OD570 values of the NC (percent of control) is used for evaluating whether or not a test material is an irritant.

Calculation of individual and mean optical densities
The individual tissue OD570 is calculated by subtracting the mean blank value of the respective microtiter plate from the respective individual tissue OD570 value. The mean OD570 for a test group of three tissues (irritation test) treated in the same way is calculated.

Tissue viability
The quantification of tissue viability is presented as the quotient of the mean OD570 divided by the respective OD570 NC value in percent.

Assay acceptance criterion for the negative control (NC)
The absolute OD570 of the tissues in the MTT test is an indicator of tissue viability obtained under specific conditions of the assay. Tissue viability is acceptable if the mean OD570 of the NC is ≥ 1.0. The mean OD570 of the NC should not exceed 2.5

Assay acceptance criterion for the positive control (PC)
5% SDS is used as PC and reflects the sensitivity of the tissues used in the test conditions. A viability of ≤ 20% is acceptable.

Assay acceptance criterion for tissue variability
For every treatment, 3 tissues are treated in parallel. The inter-tissue variability is considered to be acceptable if the SD of %-viability is less or equal to 20.


Irritant potential of the test materials is predicted from the mean relative tissue viabilities compared to the negative control tissues concurrently treated with sterile PBS. A chemical is considered as "irritant", if the mean relative tissue viability with a test material is less than or equal to 50%.


Historical control values of negative and positive controls, gathered over an appropriate time period, are used to derive suitable acceptance criteria for the test system.

Results and discussion

In vivo

Irritant / corrosive response data:
The mean relative reliable viability of the test substance-treated tissues determined after an exposure period of 1 hour with about 42 hours post-incubation was 101%. The PC item demonstrated appropriate sensitivity of the tissues used under test conditions.
Other effects:
The test substance was unable to reduce MTT directly.
Yellow discoloration of the tissues was observed after washing.

Any other information on results incl. tables

Table 1: Relative viability of EpiDermTM tissues samples





Mean OD570



Viability [% of NC]



Test substance

Mean OD570



Viability [% of NC]




Mean OD570



Viability [% of NC]



NC: negative control

OD: optical density

PC: positve control

SD: standard deviation

The SD of %-viability of the test substance was out of the acceptance range. Since all other quality criteria of the test were met and the viability values of the tissues are well above the cut off for skin irritation, this deviation was not considered to adversely affect the result of this study.

Applicant's summary and conclusion