505 A new tool for modelling stinging test in vitro: A comparative evaluation with in vivo results using a bacterial polysaccharide

Abstract

Stinging test is an in vivo protocol that evaluates sensitive skin and tests formula or ingredients on soothing sensation. Stinging test is made using lactic acid (LA) at 10% placed on nasolabial fold right and left of the face of volunteers before addition of a product. To predict the soothing sensation of a product and to modelize effect on the skin, we developed culture models based on LA test and substance P (SP) release using human keratinocytes and PC12 cells. In order to predict a calming effect, we used a bacterial polysaccharide (bPS) present in Fucogel® in in vivo stinging test and our in vitro model. Cells were placed on a 96-wells plate. PC12 cells were differentiated using NGF for 3 days and keratinocytes were maintained one day in proliferation medium before application of LA or bPS or both. Firstly, the ideal concentration of LA (10%, 5%, 1%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.07%, 0.04% or 0%) was determined to release SP without cytotoxicity. Excluding toxic concentrations, the release of SP was significant from 0.2% of lactic acid for PC12 (pH6.2) cells and from 0.1% of lactic acid for keratinocytes (pH6.6). When pH was adjusted around 7.2, LA did not provoke SP release. In keratinocytes, 0.1% LA showed a cytosolic calcium entry. At these concentrations of LA, 0.1% of bPS showed a significant decrease of SP release on two cellular types and in co-culture without modified the pH of the medium. In vivo, stinging test using Fucogel® showed a decrease by 30% of prikling intensity versus placebo on 19 women from 21 to 69 years old. Our in vitro model is ethically interesting and is adapted for cosmetic ingredients screening because it does not use animal experimentation. Moreover, Fucogel® has an attractive soothing activity revealed by in vivo stinging test and our new in vitro stinging test based on SP release