Cutaneous phototoxicity assay


Protocol principle

The purpose of this study is to determine the relative phototoxic potential of the test product after topical application on the SkinEthic in vitro reconstructed human epidermis model for 24 hours.

Detailed assay procedure

Test method

The test product, and the positive control are deposited onto the surface of the stratum corneum of six reconstructed epidermal tissues (size 0.5 cm²). All cultures (including six untreated tissues) are incubated at 37°C, 5% CO2 for 24 hours. At the end of the test period, triplicate tissues are irradiated with 6J/cm² UVA (Solar Simulator BIOSUN, Vilber Lourmat, France). Triplicate non-irradiated control tissues are stored in the dark at room temperature. After the treatment, the test material is removed by a washing step, and the cultures are incubated for an additional 24 hours at 37° C, 5% CO2 . After incubation, duplicate irratiated and non-irradiated tissues are assessed for tissue viability using the MTT method. The remaining tissues are assessed for histology (H&E staining).

Quantity applied and positive controls are differents if the test product is a cream or a liquid:
-         For a cream: 2.5 µl of the test product is applied; positive control: Phenergan (Rhone Poulenc)
-         For a liquid: 80 µl of the test product is applied; positive control: chlorpromazine 0.005%.

Evaluation of cell viability (MTT test)

For each test condition, duplicate cultures are placed on 300 µl of 0,5 mg/ml MTT and incubated 3 hours at 37° C 5% CO². Extraction is performed in 1.5 ml of isopropanol at room temperature, for a minimum of 2 hours. Optical density is measured at 570 nm. Results are expressed as percentage of viability compared to negative control: % Viability = [OD (570nm) test compound / OD (570nm ) negative control)] x 100.


For each test condition, one tissue is fixed in a balanced 10% formalin solution and embedded in paraffin. Four micron vertical sections are stained with hematoxylin/eosin and photographed under a microscope.


Assessment of the phototoxic potential of compounds and finished topical products using a human reconstructed epidermis.
J. Medina, C. Elsaesser, V. Picarles, O. Grenet, M. Kolopp, S Chibout, and A. de Fraissinette. In Vitro & Molecular Toxicology, Vol. 14, n°3, p.157-168, 2001.
Development of a highly sensitive in vitro phototoxicity assay using the SkinEthic reconstructed human epidermis. F-X. Bernard, C. Barrault, A. Deguercy, B. De Wever, M. Rosdy. (1) BIOalternatives, Genēay, France. (2) SkinEthic Laboratories, Nice, France. Cell Biology and Toxicology, Vol. 16,6, p.391-400, 2000.
UVA effects on reconstituted human epidermis (SkinEthic®): new developments in the determination of the phototoxic potential of cosmetic raw materials. De Wever B., Cappadoro M., Deguercy A., and Bernard F-X., SkinEthic, Nice France; BIOalternatives, Gencay, France. Cosmetics & Toiletries Manufacture Worldwide, 221-227, 1999.