Skin Irritation

ecvam 7

  1. ECVAM validated test methods
  2. Test methods under validation by ECVAM

  3. Development and optimisation of alternative methods

 

 

Background 

 

Topical exposure to chemicals can lead to adverse skin effects. According to the severity and reversibility of effects one distinguishes skin corrosion (=skin burns) from skin irritation. Corrosive substances irreversibly damage the skin beyond repair, while irritant substances lead to a reversible local inflammatory reaction caused by the innate (non-specific) immune system of the affected tissue: in response to chemically-induced tissue trauma and cell damage, skin cells release inflammatory mediators (chimokines and cytokines) which increase the diameter and permeability of blood vessels, attract immune cells (e.g. mast cells, neutrophiles) to the site of injury and trigger the migration of immune cells through the endothelium into the tissue where they participate in antigen clearance and tissue repair. Moreover inflammatory mediators stimulate nerve endings leading to itching and stinging sensations.

 

While some chemicals will only trigger an irritant response after repeated exposure of the same skin area (=cumulative irritants), other chemicals will even after a one-time exposure cause irritation (=acute irritants). Current regulatory requirements focus on the assessment of the acute irritation potential of chemicals in order to support the risk management associated with the handling and transport of chemicals, but also possible exposure to acutely irritant substances contained in various products. Data on skin irritation effects are required by several pieces of legislations, notably

  • the Cosmetics Directive (76/768/EEC, which will be repealed, from July 2013 onwards, by the EU regulation on cosmetics products (EC 1223/2009)
  • the Classification, Labelling and Packaging (CLP) Regulation (1272/2008)
  • the REACH Regulation (1907/2006)

 

Currently, internationally accepted test methods for skin irritation testing include the traditional in vivo animal test (Draize rabbit test) as well as in vitro test methods (Table 1). All accepted in vitro test methods are based on the RhE technology (Reconstructed human Epidermis) validated by ECVAM. RhE models use normal human keratinocytes that, during culturing, form a multi-layered epidermis including a stratum corneum at the top, functioning as a barrier.

 

Table 1: EU test methods and OECD test guidelines for skin irritation testing

Generic description of test method Standardised description of test method in EU legislation and OECD test guidelines
  EU Test Method* number and title OECD Test Guideline (TG) number and title

In vivo Draize rabbit test for skin corrosion/irritation testing

B.4 pdf icon

Acute toxicity: dermal irritation/corrosion

TG 404 pdf icon

Acute Dermal Irritation/Corrosion

In vitro skin irritation testing using RhE:

EpiskinTM , EpidermTM and SkinEthicTM test methods

B.46 pdf icon

In Vitro Skin Irritation: Reconstructed Human Epidermis (RHE) Model Test

TG 439 pdf icon

In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method

*) Test Method Regulation 440/2008


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Alternative test methods and approaches

 

1. ECVAM validated test methods


Up to date, ECVAM has validated three in vitro test methods based on Reconstructed human Epidermis (RhE) summarised in table 2:

  • EpiSkin™ test method
  • (modified) EpiDerm™ SIT test method
  • SkinEthic™ RHE test method

 

The predictive capacity of these methods was originally validated in reference to the cut-off value for hazard categorisation as used in the EU Dangerous Substance Directive, EU DSD (ESAC statements 2007 and 2008). As a result of the implementation, from 2008 on, of the UN Globally Harmonised System (GHS) for Classification and Labelling in the EU through the EU Classification, Labelling and Packaging Directive (1272/2008), the cut-off value for distinguishing irritants from non-irritants shifted from an in vivo score of 2.0 to 2.3. This required a re-evaluation of the original results on predictive capacity as well as an update of the ECVAM performance standards.

 

More details on the validation studies and downloads of relevant study documents can be found in the sections (a) and (b) below. Table 2 to 4 show the relevant ESAC statements of scientific validity of all ECVAM-validated in vitro skin irritation methods (table 2), the current ECVAM performance standards for in vitro skin irritation testing as well as background information (table 3), the current SOP versions of the ECVAM validated in vitro skin irritation test methods.

 

Table 2: ECVAM-validated in vitro test methods for skin irritation testing

Nr.

Test method name

Validation study type

Download documents

1

EpiSkin™

Full prospective validation study

ESAC statement 2007 (validation study) pdf icon

ESAC statement 2009 (performance under EU CLP) pdf icon

2

EpiDerm™ SIT (EPI-200)

 

Full prospective validation study.

A modification of the EpiDerm was validated based on the ECVAM PS (Nr.4) in 2008.

2a

Modified EpiDerm (EPI-200)

 

 

Validation studies based on the original ECVAM Performance Standards for Skin Irritation Testing

ESAC statement 2008 (validation studies) pdf icon

ESAC statement 2009 (performance under EU CLPpdf icon

3

SkinEthic™ RHE

 

 

Table 3: Current ECVAM performance standards and background documents relating to the validation study

Nr. Document description Download documents
 1  ECVAM performance standards for in vitro skin irritation testing (updated in 2009 in view of the new classification rules under UN GHS/EU CLP) Updated ECVAM PS pdf icon
 2  ESAC statement on the updated ECVAM performance standards for in vitro skin irritation testing ESAC statement 2009 on updated PS pdf icon
 3  ECVAM background document on in vitro skin irritation testing* ECVAM background document on in vitro skin irritation testing using RhE pdf icon

*) This document has also been published by the OECD Test Guidelines Programme in its series on assessment and testing (Nr. 137).

 


 

Table 4: Current SOPs of ECVAM-validated in vitro skin irritation methods

Nr. Document description Download documents
 1  

SOP of EpiSkin™ using MTT reduction as parameter to make prediction on the skin irritancy of substances

Scientifically validated as replacement to the Draize skin irritation test on the basis of prospective ECVAM validation study


SOP EpiSkin Revised MTTpdf icon
 2

SOP of EpiSkin™ using IL-1alpha release as an adjunct to the EpiSkin MTT assay (Nr. 1) to increase the sensitivity of the assay

 SOP EpiSkin IL1apdf icon
 3  

SOP of modified EpiDerm™ SIT using MTT reduction as parameter to make prediction on the skin irritancy of substances

Scientifically validated as replacement to the Draize skin irritation test on the basis of the ECVAM performance standards

 SOP Modified EpiDerm MTT pdf icon
 4 SOP of SkinEthic™ RHE test method using MTT reduction as parameter to make prediction on the skin irritancy of substances

Scientifically validated as replacement to the Draize skin irritation test on the basis of the ECVAM performance standards

 SOP SkinEthic MTT pdf icon

 

 

1.1 ECVAM-coordinated and sponsored full prospective validation study (2005-2007)

 

Following a Prevalidation study, ECVAM conducted and sponsored, from 2003-2007, a full prospective validation study (ECVAM skin irritation validation study = SIVS) of two in vitro skin irritation test methods (EpiSkin™ and EpiDerm™) based on the test system of Reconstructed human Epidermis (RhE). As a result of the validation study, ECVAM developed Performance Standards for the assessment and validation of test methods found sufficiently similar with regard to the essential test method components of the validated reference methods. These performance standards were used for the validation of two test methods in 2008 (see section b). All relevant validation study documents of the full study are summarised in Table 5.

 

The EpiSkin™ test method was validated as a potential stand-alone method, capable of reliably distinguishing non-irritant (non-classified) from irritant chemicals according to EU DSD categorisation. In the EU and other global areas, The EpiSkin™ test method can thus replace the traditional in vivo Draize skin irritation test introduced into safety assessment regimes for drugs and chemicals over 60 years ago.

 

The EpiDerm™ test method did not qualify as a stand-alone replacement but was recommended for the identification of irritant chemicals, and thus to be used as an element of a testing strategy. A modification of the EpidermTM's Standard Operating Procedure (SOP) and/or Prediction Model was recommended by ESAC. In 2008, the modified EpiDermTM test method was validated according to the ECVAM performance standards (see section b). The modified test method was found to be scientifically valid as a full replacement alternative to the Draize rabbit test.

 

In addition to the cell viability endpoint as main parameter for making extrapolative predictions on irritancy classes, both test methods were also evaluated with regard to the predictive capacity as a result of the measurement of an inflammatory mediator (Interleukin 1 alpha). This endpoint was found a useful adjunct in case of the EpiSkin™ test method, potentially increasing the sensitivity of the assay.

 

Table 5: Relevant documents of the ECVAM full prospective validation study of the EpiSkin and EpiDerm test methods, conducted from 2005-2007.

Nr. Document description Download documents
 1  Skin Irritation Validation Study Phase II: Analysis of the primary endpoint MTT and the secondary endpoint IL-1  Analysis MTT.IL1a pdf icon
 2  ESAC Peer Review Panel Consensus Report: Skin Irritation Validation Study  ESAC Report SIVS pdf icon
 3  Report from the Chemicals Selection Sub-Committee to the Management Team on Potential Reasons for the Misclassification of Chemicals in the EpiSkin™ and EpiDerm™ Assays  SIVS Chemical   Selection report pdf icon
 4  Application of the BfR Rulebase for Skin Irritation to Test Chemicals in the ECVAM Skin Irritation Validation Study  BfR Rulebase application pdf icon
 5  

Original ECVAM Performance Standards (PS) for applying human skin models to in vitro skin irritation testing

IMPORTANT NOTE: These PS should not be used any longer and have been replaced by the updated ECVAM PS taking the provisions of UN GHS/EU CLP into account (table 2)
 Original ECVAM PS pdf icon
 6  Original SOP of the EpiSkin test method as used during the ECVAM SIVS  SOP original EpiSkin MTT pdf icon
 7  Original SOP of EpiDerm™ test method as used during the ECVAM SIVS  SOP Original EpiDerm MTT pdf icon

 

1.2 Validation studies based on the ECVAM performance standards (2008) 


In 2008 ECVAM evaluated two submissions of validation studies performed on the basis of the original ECVAM performance standards (PS: see document 5, table 4). These were

  • the modified EpiDermSIT test method
  • the SkinEthic™ test method

The relevant documents relating to these PS-based validations are listed in table 6.

 

The SkinEthic™ RHE, a similar/me-too method, underwent a non-ECVAM coordinated catch-up study based on the ECVAM PS and was submitted to ECVAM for evaluation. The SkinEthic™ test method also underwent experimental evaluation at ECVAM in order to assess its transferability and robustness. The SkinEthic™ RHE was confirmed by ECVAM as sufficiently similar with regard to its structural and functional characteristics and its performance in reference to the PS and the test method was therefore forwarded to ESAC for peer review. It was endorsed as scientifically valid by ESAC in November 2008.

 

The EpiDerm™ model, a modification of the previously validated EpiDerm test method underwent a non-ECVAM coordinated update validation study in reference to the ECVAM PS and was submitted to ECVAM for evaluation. The main modification performed is the prolongation of the exposure time to the test substances from 15 ('common protocol', ECVAM SIVS) to 60 minutes, while all other essential model parameters remained unchanged. The test method was confirmed by ECVAM to qualify for a PS-based validation study and forwarded to ESAC for scientific peer review. ESAC endorsed the test method as scientifically valid in November 2008.

 

Table 6: Study documents relating to the PS-based validation studies of the modified EpiDerm™ and the SkinEthic™ test methods

Nr. Document description Download documents
 1  ESAC peer review panel report of the PS-based validation study of the SkinEthic test method  Peer Review SkinEthic pdf icon
 2  ESAC peer review panel report of the PS-based validation study on the modified EpiDerm test method  Peer Review EpiDerm pdf icon
 3  Result of the experimental evaluation of the SkinEthic test method at ECVAM (May 2008)  ECVAM evaluation SkinEthic pdf icon

 

 

2. Test Methods under validation by ECVAM


There are currently no test methods for in vitro skin irritation testing under validation at ECVAM.

However, ECVAM continues to receive submissions of external test method validation studies performed in reference to the ECVAM performance standards. These submissions are evaluated by ECVAM and, if found compliant with the PS, will be forwarded for ESAC peer review.


  

3. Development and optimisation of alternative methods


Having been the lead organisation for the drafting and regulatory acceptance of TG 439 on in vitro skin irritation, EURL ECVAM is continuing activities in collaboration with the OECD test guidelines programme in view of improving the scientific use of in vitro skin irritation test methods, their standardisation but also their evaluation and validation. The latter concerns at present predominantly so-called me-too methods i.e. test methods that are similar to the methods validated by ECVAM in 2007 and which were used to define the Performance Standards of in vitro skin irritation testing. ECVAM is regularly collaborating with OECD on updates of TG 439 in view of integrating recently validated and accepted test methods for in vitro skin irritation test method into this test guideline. In addition, ECVAM experts are participating in the OECD expert group on skin corrosion / irritation and have recently (2012-2013) been involved in the development of guidance on Integrated Approaches to Testing and Assessment (IATA) for skin irritation and skin corrosion testing. The IATA builds on the Integrated Testing Strategy for skin corrosion/irritation testing originally developed for the EU REACH legislation under the coordination of ECVAM (2006-2007), but provides more prescriptive guidance based on additional data and experiences that became available in the past seven years.