In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method

Commonly used acronym: RHE irritation test method, OECD TG 439

Scope of the method

The Method relates to
  • Human health
The Method is situated in
  • Regulatory use - Routine production
Type of method
  • In vitro - Ex vivo
This method makes use of
  • Human derived cells / tissues / organs
Specify the type of cells/tissues/organs
Reconstructed human Epidermis (RhE) obtained from human derived non-transformed keratinocytes


Method keywords
  • 3D Reconstructed Human Tissue model
  • Human epidermis
  • cytotoxicity
  • Cell viability
  • MTT
  • Skin irritation
Scientific area keywords
  • Hazard identification
  • Irritating chemicals
  • GHS
  • Local toxicity of substances and mixtures
  • Classification
  • Labelling
Method description

The method is described in detail in OECD TG 439 as SkinEthicTM RHE and provides an in vitro procedure that may be used for the hazard identification of irritant chemicals (substances and mixtures) in accordance with the United Nations Globally Harmonized System of Classification and Labelling (UN GHS) Category 2. This Test Guideline (TG) can also be used to identify non-classified chemicals. Therefore, depending on the regulatory framework and the classification system in use, this procedure may be used to determine the skin irritancy of test substances as a stand-alone replacement test for in vivo skin irritation testing, or as a partial replacement test, within a tiered testing strategy. It is based on reconstructed human epidermis (RhE), which in its overall design closely mimics the biochemical and physiological properties of the upper parts of the human skin, i.e. the epidermis. The test chemical is applied topically to a three-dimensional RhE model. The method measures the initiating events in the cascade, e.g. cell/tissue damage, using cell viability as readout. Cell viability is measured by enzymatic conversion of the vital dye MTT into a blue formazan salt that is quantitatively measured after extraction from tissues. Irritant chemicals are identified by their ability to decrease cell viability below defined threshold levels (i.e. ≤ 50%, for UN GHS Category 2). Test chemicals that produce cell viabilities above the defined threshold level, may be considered non-irritants (i.e. > 50%, No Category).

Lab equipment
  • - Standard equipment for working with cell cultures;
  • - Microplate reader (OD) or HPLC/UPLC-spectrophotometer.
Method status
  • Validated by an external party (e.g. OECD, EURL ECVAM,…)

Pros, cons & Future potential

  • - It is based on an in vitro test system of reconstructed human epidermis (RhE), which closely mimics the biochemical and physiological properties of the upper parts of the human skin, i.e. the epidermis.
  • - The RhE test system uses human-derived non-transformed keratinocytes as cell source to reconstruct an epidermal model with representative histology and cytoarchitecture.
  • - Performance Standards (PS) are available to facilitate the validation and assessment of similar and modified RhE-based test methods.
  • - The method is applicable to solids, liquids, semi-solids and waxes. The liquids may be aqueous or non-aqueous; solids may be soluble or insoluble in water. Whenever possible, solids should be ground to a fine powder before application; no other pre-treatment of the sample is required.

For more information: see OECD TG 439

  • - It does not allow the classification of chemicals to the optional UN GHS Category 3 (mild irritants).
  • - Gases and aerosols have not been assessed yet in a validation study.
  • - A lack of applicability of the RhE based in vitro skin irritation test for agrochemical formulations.
  • - Test chemicals absorbing light in the same range as MTT formazan and test chemicals able to directly reduce the vital dye MTT (to MTT formazan) may interfere with the cell viability measurements and need the use of adapted controls for corrections.
  • - This method does not provide adequate information on skin corrosion. It should be noted that OECD TG 431 on skin corrosion is based on the same RhE test system, though using another protocol.

For more information: see OECD TG 439

References, associated documents and other information

Associated documents
OECD TG 439.pdf
OECD Test Guideline No.439: In Vitro Skin Irritation: Reconstructed Human Epide…

Contact person

Sandra Verstraelen


Vlaamse Instelling voor Technologisch Onderzoek (VITO)
Flemish Region