PROTOCOL PRINCIPLE (SkinEthicTM RHE model)
The purpose of this study is to determine the protective effect of the test product (formulation), after topical application on the SkinEthicTM RHE model and UVB irradiation.
Four in vitro reconstructed human epidermal tissues are dosed after topical application with the test agent. Duplicate tissues are consequently irradiated with 300 mJ/cm² UVB, and duplicate non-irradiated control tissues are stored in the dark. Immediately after irradiation, cultures are assessed for histology (H&E staining) and pyrimidine dimer detection.
DETAILED ASSAY PROCEDURE
2 µL of the test product and of the positive control (Minesol (Roc) SPF 60) are applied onto the surface of the stratum corneum of four SkinEthicTM RHE tissues (size 0.5 cm²). Untreated negative control are run in parallel. Duplicate tissues are irradiated with 300 mJ/cm² UVB (Solar Simulator BIOSUN). Duplicate non-irradiated tissues are stored in the dark at room temperature. Cultures are fixed in formaline immediately after irradiation.
Histology (H&E staining), and pyrimidine dimer detection are performed.
For each tested product, triplicate tissues will be fixed in a balanced 10% formalin solution and embedded in paraffin. Four micron vertical sections will be stained with hematoxylin/eosin and photographed under a microscope.
Immunohistochemistry is also performed to detect pyrimidine dimers (H3 antibody).
Assessment of P53, P21 protein expression and cyclobutane pyrimidine dimer formation in a human skin culture model following UVB exposure. Robert C., Doucet O., Bonnet M., Bertino B., Zastrow L. ATLA special issue, p. 304, 1999.