Skin irritation: '42 bis' validated protocol

Predicting the cutaneous irritant potential of chemical products represents an important component of the overall safety evaluation programme for new and existing chemicals.

The SkinEthic Skin Irritation Test assay (42 minutes application + 42 hours post-incubation, “42 bis” protocol) is designed for the prediction of acute skin irritation of chemicals.

At its 29th meeting, held on 4-5th November, 2008 the non-Commission members of the ECVAM Scientific Advisory Committee (ESAC) endorsed that SkinEthic RHE assay “which measures or predicts the same biological or toxic effect as the fully validated and accepted reference method” (see ESAC statement, 2007 on EpiSkin Draize full replacement method). This statement was based on the evaluation of multicentric validation study results performed in 3 independent laboratories using the “42 bis” protocol. A statistical analysis showed an excellent intra- and inter-laboratory reproducibility. Moreover, this protocol provided 90% sensitivity, 80% sensitivity, false positive and false negative (ie. 85% overall accuracy) for the 20 ECVAM reference chemicals. This unique performance profile set up the “42 bis” protocol as the most relevant and reproducible skin irritation protocol commercially available for regulatory and/or research testing of chemicals.


  • An ethical method

Carried out in vitro, the SkinEthic reconstructed Epidermis method makes it possible to evaluate the irritant potential of chemical products, without using animal testing.

  • The use of a suitable standardized model

The validated method uses a model equivalent to the human epidermis (3). The reproducibility of the model is checked by histological and function tests (4). Compared to in vivo study, the SkinEthic reconstructed epidermis  allow use of measurement systems that produced quantitative, reproducible results.

  • A validated method

The ECVAM validation of the method has shown its reproducibility within and between laboratories, and that it is rapid and economical.


    Skin irritation protocole
    Prediction model : Irritant if < 50% viability

    Each test substance (chemical tested, negative and positive controls) is topically applied concurrently on three tissues replicates (Figures 1 and 2) for 42 minutes at room temperature (RT), comprised between 18°C to 24°C). For each test substance, three independent experiments are performed.

    Figure 1: Liquid test substance application according to “42 bis” protocolFigure 2: Solid test substance application according to “42 bis” protocol

    Exposure to the test substance was followed by rinsing with phosphate buffer saline (PBS) and mechanically dried. RHE were then transferred to fresh medium and incubated at 37°C for 42 additional hours. Cell viability is assessed by incubating the tissues for 3 hours with 0.3 mL MTT solution (1 mg/mL). The formazan crystals are extracted using 1.5 mL isopropanol for 2 hours at RT and quantified by spectrophotometry at 570 nm wavelength. Sodium Dodecyl Sulphate (SDS 5%), and PBS treated epidermis are used as positive and negative controls, respectively. For each treated tissue, the cell viability is expressed as the percentage of the mean negative control tissues.
    The mean relative tissue cell viability above 50 % predicts a non irritancy potential of the test substance. The prediction model is defined as described in Table 1:

    In vitro results

    In vivo classification
    Mean tissue cell viability ≤50%

    R38, Irritant (I)
    Mean tissue cell viability >50%

    Non-irritant (NI)

    Table 1:
    “42 bis” protocol prediction model

    4) RESULTS

    MTTLab 180809090
    Lab 280809090
    Lab 380809090
    MTT + IL-1alphaIn the 3 labs (median classification)80809090

    Table 1:
    Predictive capacity (specificity and sensitivity) in the three laboratories considering either all chemical runs or three valid runs for MTT alone and for the combined prediction based on MTT and the total amount of IL-1alpha, IL-1alpha NC corrected or IL-1alpha fold increase released. Compared to the predictive capacity of the MTT alone, no increase in sensitivity and specificity were achieved using the three MTT + IL-1alpha combined prediction models.

     EpiSkin (Validated Reference Method)SkinEthic RHE
    MTTMTT + IL1-alphaMTT
    False positive rate202020
    False negative rate301010

    Table 2: Predictive values (in %) of the three validated in vitro tests for skin irritation testing. Similar predictive performance of SkinEthic RHE compared to EpiSkinTM model with 2 endpoints (MTT & IL-1alpha)


    The excellent performances of the “42 bis” protocol are mainly due to 2 parameters:
    -         standardized quality of the RHE model
    -         relevant, reliable, robust  and user friendly design of the  “42 bis” protocol
    The participation of laboratory 1, as naïve concerning the manipulation of RHE tissues, in the multicentric validation study  was possible and successful  during each steps of the validation process due to a protocol transfer effective after just one-day training, an adaptation of the protocol using a nylon mesh. for applying all liquid and viscous tested substances in order to ensure their homogeneous spreading, a high performance customer focused 42 bis  protocol designed to allow the evaluation of series of 20 chemicals  using only 2 sets  which the cell viability (MTT) can be  measured by one technician  in  the same day.

    6) References

    Statement on the validity of in vitro tests for skin irritation.
    ESAC, ECVAM. November, 5th, 2008.