The EpiDerm Skin Irritation Test (SIT) method can be used to predict the skin irritation potential of chemicals/substances.
The method has previously been validated in a series of validation studies and has now received regulatory approval as a full replacement method for equivalent in vivo test methods for skin irritation potential.
The results of the validation study, the peer review of ESAC and our recommendation can be found on TSAR, the Tracking System for Alternative methods towards Regulatory acceptance.
Read more about the EpiDerm Skin Irritation Test (SIT) on TSARSkin irritation
[collapsed]Skin irritation is a local inflammatory response of normal skin that occurs upon exposure to a chemical or substance that has irritant properties.
Potential irritant chemicals penetrate the barrier that is the outer most layer of cells of the skin (the stratum corneum) and disrupt cell integrity.
Damage may be caused directly through the physicochemical properties of the chemical. In this case, the irritant may be a surfactant that can destabilise lipid bilayers to create pores and disrupt proteins in skin cells.
Alternatively, damage might occur indirectly where the chemical induces release of reactive oxygen species which in turn disrupt membranes and proteins.
In either case the outcome is eventually cell death and an inflammatory response in exposed tissues.[/collapse]
EpiDerm Skin Irritation Test (SIT)
[collapsed]The EpiDerm Skin Irritation Test (SIT) utilises the cellular outcomes of skin irritation (cell death/inflammation) as biomarkers for the potential of chemicals/ substances to cause skin irritation.
Specifically, the test method is based on a one hour topical exposure of a chemical to the reconstructed human epidermis model EpiDerm EPI-200 followed by a test for cell viability via MTT reduction.
A secondary outcome that can also be measured is the release of inflammatory cytokines as represented by the cytokine IL-1α.
Based on the cell viability outcome, a chemical is classed as an irritant if ≤50% are viable after the exposure time in comparison to a negative control (exposure to water). Conversely a chemical is classed as a non-irritant when >50% are viable after exposure.
The method can be used to test a wide range of chemicals, chemical classes and substances. This includes solids, liquids, semi-solids and waxes.
The approach has not been evaluated for testing gases, aerosols or highly volatile compounds. Certain chemicals that are either highly coloured or directly reduce MTT may interfere with cell viability measurements.
In these cases, specific steps can be taken to improve accuracy of the measurements.
A video demonstration of the method is available via the Journal of Visualized Experiments.[/collapse]
Animal testing replacement?
[collapsed]The EpiDerm skin irritation test was granted regulatory approval as a full replacement method for the in vitro Draize rabbit skin irritation test under the EC test method regulation (method B.46) and OECD test guideline No.439.
For full characterisation of irritation/corrosion potential, the method should be used as part of a sequential dermal irritation/corrosion testing strategy.[/collapse]
Validation studies
[collapsed]Validation of the EpiDerm skin irritation test is the result of a series of formal validation studies involving both EURL ECVAM and a number of other organisations.
Initially the EpiDerm method was evaluated alongside the EpiSkin skin irritation test method in the ECVAM Skin Irritation Validation Study (2003-2007).
In the case of EpiDerm both the management team and ESAC concluded that the method could identify irritants but that, due to a series of false negatives, it could not at this stage be used as a standalone method to correctly identify irritants and non-irritants - other approaches (such as in vivo skin irritation tests) would still be needed to verify the outcomes.
ESAC went further and recommended that the method should be optimised to increase sensitivity.
In 2006 and 2007, MatTek Corporation (the manufacturer) did further optimise the method and concluded that a longer incubation time (increased to 1 hour) and some other minor modifications improved the sensitivity.
Following this, a further validation study was carried out by MatTek and another four laboratories. EURL ECVAM Performance Standards were used to evaluate the method.[/collapse]
Validation study outcomes
[collapsed]The initial analysis from the ECVAM Skin Irritation Validation Study indicated that the within- and between-laboratory reproducibility over three experiments was high.
However, while specificity of the method was high (83.3%), sensitivity was much lower (57.3%) indicating that the method was likely sufficient for predicting irritants but not non-irritants.
For this reason, a recommendation was made to optimise the method for subsequent validation studies were considered.
After modifications were made (see above), the method was re-evaluated in a further validation study and in this case the method met all pre-defined performance standards with regards to the predictive capacity of the method.[/collapse]
EURL EVCAM recommendations
[collapsed]As a result of the validation studies, ESAC and EURL ECVAM recommended that the EpiDerm Skin Irritation Test (SIT) method was scientifically validated as a stand-alone method to replace the equivalent in vivo Draize skin irritation test.
Subsequently, EpiDerm Skin Irritation Test (SIT) was included in the relevant EU Test Method Regulation (440/2008/EC) and also as OECD Test Guideline No 439. As well as regulatory documents being available, an SOP and DB-ALM protocol are also available.[/collapse]