What is the official description of skin irritation?
Skin irritation refers to the production of reversible damage to the skin following the application of a test chemical for up to 4 hours [as defined by the United Nations (UN) Globally Harmonized System of Classification and Labelling of Chemicals (GHS)] (from OECD TG 439)
What is the current OECD Guideline for skin irritation testing?
The current OECD Test Guideline / OECD Ligne Directrice 439 "OECD GUIDELINES FOR THE TESTING OF CHEMICALS; In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method" (OECD TG 439).
Here, we provide an extract of this Test Guideline. If you need more detailed information, please consult the full guideline as PDF.
[...] This Test Guideline (TG) provides an in vitro procedure that may be used for
the hazard identification of irritant chemicals (substances and mixtures) in
accordance with UN GHS Category 2. In member countries or regions that do not
adopt the optional UN GHS Category 3 (mild irritants), this Test Guideline can
also be used to identify non-classified chemicals. Therefore, depending on the
regulatory framework and the classification system in use, this Test Guideline
may be used to determine the skin irritancy of chemicals either as a
stand-alone replacement test for in vivo
skin irritation testing or as a partial replacement test within a tiered
testing strategy. [...]
[...] This Test Guideline addresses the human health endpoint skin irritation. It is based on the in vitro test system of reconstructed human epidermis (RhE), which closely mimics the biochemical and physiological properties of the epidermis. The RhE test system uses human derived non-transformed keratinocytes as cell source to reconstruct an epidermal model with representative histology and cyto-architecture. [...]
[...] Pre-validation, optimization and validation studies have been completed for four commercially available in vitro test methods based on the RhE test system. These four test methods are included in this TG and are listed in Annex 2, which also provides information on the type of validation study used to validate the respective test methods. [...]
[...] While this Test Guideline 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. [...]
[...] This Test Guideline is based on RhE-models using human keratinocytes, which represent in vitro the target organ of the species of interest. Moreover, it directly covers the initial step of the inflammatory cascade/mechanism of action (cell and tissue damage resulting in localized trauma) that occurs during irritation in vivo. [...]
[...] The Test Guideline is applicable to solids,
liquids, semi-solids and waxes. [...]
How does the test work?
The test chemical is applied topically to
a three-dimensional RhE model, comprised of non-transformed human-derived
epidermal keratinocytes, which have been cultured to form a multilayered,
highly differentiated model of the human epidermis...
... the RhE-based test methods (...) measure the initiating events in the cascade, e.g. cell / tissue damage, using cell viability as readout.
Cell viability in RhE models is measured by enzymatic conversion of the vital dye (...) 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). Depending on the regulatory framework and applicability of the Test Guideline, chemicals that produce cell viabilities above the defined threshold level, may be considered non-irritants (i.e. > 50%, No Category).
According to the test procedure, it is essential that the viability measurement is not performed immediately after exposure to the test chemical, but after a sufficiently long post-treatment incubation period of the rinsed tissue in fresh medium. This period allows both for recovery from weak cytotoxic effects and for appearance of clear cytotoxic effects. A 42 hours post-treatment incubation period was found optimal during test optimization of two of the RhE-based test methods underlying this TG.
How can I translate viability data into substance classification?
Viability measured after 20 min exposure
and 42 h post incubation
|Prediction to be considered|
Category 2 (Cat. 2)
Non - irritant
Non - classified
How does this work in practice?
This case study illustrates skin irritation testing results and the classification of test chemicals for two test substances as an example.
Case study skin irritation
Substance A is a solid substance, which is applied as a powder (after grinding) topically onto the epidermis.
|Exposure time||20 min|
|Classification||No Category (non-irritant)|
Substance B is a creamy substance, which is applied with a brush topically onto the epidermis.
|Exposure time||20 min|
|Classification||Category 2; irritant|
Which methods are validated and accepted?
Six methods are validated: EpiSkinTM, EpidermTM, SkinEthicTM RHE and LabCyteTM,, Skin +® and epiCS®
The validation study of the epiCS® Skin Irritation Test (SIT) based on the EURL ECVAM/ OECD Performance Standards for in vitro skin irritation testing using Reconstructed human Epidermis (RhE) [EURL ECVAM Scientific Advisory Committee (2016) (ESAC OPINION 2016)] and was performed in a ring trial with three participating laboratories. Advanced CellSystems GmbH (ACS), Harlan Cytotest Cell Research GmbH (HCCR) and the Institute for In Vitro Sciences, Inc. (IIVS).) A set of 20 reference test chemicals as specified of the OECD Test Guideline (TG) 439 and EC Testing Method B.46. (In vitro Skin Irritation: reconstructed human Epidermis model test. Council Regulation (EC) No 761/2009) was tested in each laboratory (OECD 2013) (B 46. (EC) No 761/2009) The Evaluation of the catch-up Validation study is based and guided by the Performance Standards (PS) of the OECD TG 439 (OECD 2015), following ESAC opinion in (ESAC Opinion Number 2016-5) and independent peer review in 2018 (Peer Review Report of the Validation of the SkinIrritation Tests 2018).
|epiCS method||Other validated test methods|
|Sensitivity||100 %||70 - 90 %|
|Specificity||80 %||73,3 - 80 %|
|Overall Accuracy||90 %||75 - 85 %|
The epiCS method showed a "Within Laboratory Reproducibility" of 96,58 % and "Between Laboratory Reproducibility" of 95 %.
In November 2018 the non-Commission members of the ECVAM Scientific Advisory Committee (ESAC) accepted epiCS test method for in vitro irritations test and adopted the epiCS test method in the TG439 (OECD; 2019)
What do I need for skin irritation testing?
We have developed a specific kit that you can use for in vitro skin irritation testing. In addition, you need laboratory material which are standards in cell culture laboratories.
- epiCS (tissues)
- epiCS Culture Medium - to culture the tissues - for three medium changes (sufficient for skin irritation testing)
- epiCS MTT-Assay Medium - for MTT-Assay
- 6-well plates
- Lot specific epiCS certificate of analysis
For further Information, please check the epiCS SOP and manual:
We provide competent swift technical and scientific support by telephone or e-mail.
How can I compare test performances in different labs?
We have designed a validation kit for skin irritation to conduct a multicenter study to validate between laboratory reliability.
Two (blinded) test substances are tested at your site(s) and at our ISO9001:2015 certified laboratories. Testing is carried out at the same time on the same lot of tissues to minimize material variations between the sites. Test results are compared and in case of concordant results a certificate is issued by SkinInVitro GmbH (formerly known as " Cells´Systems Biotechnologie Vertrieb GmbH") to the qualified user. This study can be carried out with one or more external laboratories at the same time.
Who should participate?
a) Beginners - to prove that the test
method is carried out correctly following the SOP.
b) Routine users - to prove on a routine basis the reproducible performance of the technicians.
For further information, please download the PDF below
- UN (2009), United Nations Globally Harmonized System of Classification and Labeling of Chemicals (GHS), Third revised edition, UN New York and Geneva.
- EC-ECVAM (2009), Statement on the “Performance under UN GHS of three in vitro assays for skin irritation testing and the adaptation of the Reference Chemicals and Defined Accuracy Values of the ECVAM skin irritation Performance Standards”, issued by the ECVAM Scientific Advisory Committee (ESAC30), 9 April 2009.
- EC-ECVAM (2009), ESAC Statement on the Performance Standards (PS) for in vitro skin irritation testing using Reconstructed human Epidermis, issued by the ECVAM Scientific Advisory Committee (ESAC31), 8 July 2009.
- OECD (2010), Explanatory background document to the OECD draft Test Guideline on in vitro skin irritation testing. Published in OECD Series on Testing and Assessment, No. 137, OECD, Paris.
- OECD (2015), OECD guidelines for the testing of chemicals; in vitro skin Irritation: reconstructed human Epidermis test method.
- OECD, 2019. OECD Guideline for Testing of Chemical, No. 439: In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method
- Peer Review Report of the Validation of the Skin Irritation Tests Using Skin+ ® and Using epiCS® (2018) 2. EC Testing Method B.46. In vitro skin irritation: reconstructed human epidermis model test. Council Regulation (EC) No 761/2009
- EC Testing Method B.46. In vitro skin irritation: reconstructed human epidermis model test. Council Regulation (EC) No 761/2009
- EURL ECVAM DataBase service on Alternative Methods to animal experimentation (DB-ALM) Protocol n° 212: epiCS® Skin Irritation Test (epiCS® SIT)
- ESAC Opinion on the Validation Study of the epiCS® Test Method based on the EURL ECVAM/OECD Performance Standards for in vitro Skin Irritation Testing using Reconstructed human Epidermis (RhE), ESAC Opinion Number 2016-5