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The EpiSkin human epidermis model can be used to assess skin irritation potential of chemicals.

The method has previously been validated as part of the ECVAM international validation study on in vitro tests for acute skin irritation. Furthermore, the method has been peer-reviewed by ESAC and was recommended for consideration for regulatory approval for skin irritation assessments.

Subsequently, the test has been adopted as OECD test guideline (No 439).

The results of the validation study, the ESAC statement and our recommendation will be soon available on TSAR, the Tracking System for Alternative methods towards Regulatory acceptance.

Skin irritation

[collapsed]Acute skin irritation is a local, reversible inflammatory response of normal skin when it is exposed to a chemical with irritant properties. This is in contrast to skin corrosion, which is normally considered to be an injury to skin that is non-reversible.

The potential to cause irritation is an important consideration for developing procedures for the safe-handling, packing and transportation of chemicals.[/collapse]

EpiSkin Skin Irritation Test (SIT)

[collapsed]The EpiSkin model is a three dimensional reconstructed human epidermis that, once established, comprises of a main basal, supra basal, spinous and granular layers and a functional stratum corneum (the outer barrier layer of skin).

The use of the model specifically for skin irritation testing involves the topical application of a test chemical followed by an assessment of cell viability, and specifically cytotoxicity (as measured via the MTT assay). Reductions in cell viability are then expressed as a % of the negative control. The % reduction is then used to predict the irritation potential of a chemical.

A substance is considered to be irritating to skin (i.e. categorised as R38) if tissue viability after exposure and post incubation (see protocol, below) is less than or equal to 50%. Cell viability above 50% is considered a marker of a chemical being a non-irritant.[/collapse]

Animal testing replacement

[collapsed]The method has been scientifically validated and has received regulatory approval as a replacement for in vivo (rabbit) skin testing.

An OECD test guideline (OECD TG 439) has previously been adopted that includes this method as validated for replacing the in vivo equivalent.[/collapse]

Validation study

[collapsed]The ECVAM international validation study on in vitro tests for acute skin irritation was a prospective two-phase validation study in which the EpiSkin method was assessed.

The two phases of the study included prevalidation and formal validation stages.

In phase one of the study, 20 chemicals (nine irritants and 11 non-irritants) were assessed in a blinded manner and outcomes compared with those expected in the in vivo version of the test. In phase two of the study, 58 chemicals (25 irritants and 33 non-irritants) were blindly assessed but this time in three separate laboratories to assess both within and between lab validity.

In both phases the main outcome was the change in cell viability following exposure, as measured via the MTT assay.[/collapse]

Validation study outcomes

[collapsed]The outcome of phase 1 of the study indicated that the method could correctly predict 91% of chemicals that are known non-irritants. In comparison it could also correctly predict 67% of known R38 irritants.

While the second outcome was considered by the study's management team to be low, reassessment of the data indicated that almost all mis-classified substances were lying at the predetermined threshold between what is considered an irritant or not.

On that basis, they judged that the method should nevertheless proceed to phase 2 of the study.

Following phase 1 of the study, some protocol optimisation was made to improve predictive outcomes.

This included the addition of a secondary outcome that measured IL-1α release (reportedly, this additional outcome appeared to improve overall sensitivity of the assay).

Following the testing of the 58 chemicals in the three laboratories over three separate experiments, within laboratory reproducibility was 94% and between laboratory reproducibility was 90%. Sensitivity, specificity and concordance/accuracy measures were equally high.[/collapse]

EURL ECVAM recommendations

[collapsed]As a result of the outcomes of the study, ESAC and EURL ECVAM recommended that the EpiSkin method was scientifically validated as a stand-alone method to replace the equivalent in vivo Draize skin irritation test.

Subsequently, EpiSkin was included in the relevant EU Test Method Regulation (440/2008/EC) and also as OECD Test Guideline No 439.

Further, as a consequence of the outcomes of this study, performance standards have also been developed on which subsequent methods can/should be benchmarked (more details below). As well as regulatory documents being available, an SOP and DB-ALM protocol are also available.[/collapse]