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Skin irritation / corrosion

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skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
24 Jan 2012 - 08 Feb 2012
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guidelineopen allclose all
according to guideline
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
according to guideline
other: Commission Regulation (EU) No. 761/2009 of 23 July 2009 amending, for the purpose of its adaptation to technical progress, Regulation (EC) No 440/2008 laying down test methods pursuant to Regulation (EC) No 1907/2006.
GLP compliance:
yes (incl. QA statement)
Experimental Toxicology and Ecology, BASF SE, 67056 Ludwigshafen, Germany

Test material

Constituent 1
Chemical structure
Reference substance name:
EC Number:
EC Name:
Cas Number:
Molecular formula:
Test material form:
Details on test material:
- Physical state: solid/white

In vitro test system

Test system:
human skin model
Reconstructed human epidermis model EpiDermTM
Source species:
Details on animal used as source of test system:
Tissue model: Epi-200
Origin of tissue: MatTek Corporation, Ashland MA, USA
Details on test system:
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.
The test substance was applied minimally moistened with PBS.
Control samples:
other: Negative control (NC): PBS, sterile. Positive control (PC): 5% (w/v) sodium dodecyl sulfate (SDS, Sigma, Germany) in deionized water, sterile.
Amount/concentration applied:
25 μL of the solid ground test material (about 20 mg) was applied with a sharp spoon.
Duration of treatment / exposure:
The tissues were kept under the laminar flow hood at room temperature for 25 minutes overall and for 35 minutes in the incubator.
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.
Duration of post-treatment incubation (if applicable):
The tissues were washed with sterile PBS to remove residual test material 1 hour after start of application.

The present test is based on the experience that irritant chemicals produce cytotoxicity in human reconstructed epidermis. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity. The mitochondrial dehydrogenase reduces the yellow colored water-soluble 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) to the insoluble blue colored formazan.
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.
Number of replicates:
Three tissues were treated with the test substance, the PC and NC, respectively.

Test system

unchanged (no vehicle)

Results and discussion

In vitro

Irritation / corrosion parameter:
% tissue viability

Any other information on results incl. tables


tissue 1 tissue 2 tissue 3 mean SD
negative control mean OD570 1.932 2.087 1.96 1.993


[% of NC]

96.9 104.7 98.3 100 4.15
test material mean OD570 1.958 2.046 1.959 1.988


[% of NC]

98.2 102.7 98.3 100 2.54
positive control mean OD570 0.13 0.126 0.13 0.129


[% of NC]

6.5 6.3 6.5 6 0.11

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Based on the observed results and applying the evaluation criteria cited it was concluded, that the test substance does not show a skin irritation potential in the EpiDermTM skin irritation test under the test conditions chosen.
Executive summary:

The potential of the test substance to cause dermal irritation was assessed by a single topical application of 25 μL bulk volume (about 20 mg) of the test substance to a reconstructed three dimensional human epidermis model (EpiDerm™). Three EpiDerm™ tissue samples were incubated with the test substance for 1 hour followed by an about 42-hours post-incubation period. Tissue destruction was determined by measuring the metabolic activity of the tissue after exposure/ post-incubation using a colorimetric test. The reduction of mitochondrial dehydrogenase activity, measured by reduced formazan production after incubation with a tetrazolium salt (MTT) was chosen as endpoint. The formazan production of the testsubstance treated epidermal tissues is compared to that of negative control tissues. The quotient of both values indicates the relative tissue viability. The EpiDerm skin irritation test showed the following results: The test substance is not able to reduce MTT directly. The mean viability of the test-substance treated tissues determined after an exposure period of 1 hour with about 42 hours post-incubation was 100%. Based on the observed results and applying the evaluation criteria cited it was concluded, that the test article does not show a skin irritation potential in the EpiDerm™ skin irritation test under the test conditions chosen.