In vitro permeation kinetics, skin layers distribution and emission in the surrounding environment of biologically active Melaleuca alternifolia essential oil (Tea Tree oil) components from topic formulations

Melaleuca alternifolia essential oil (Tea Tree Oil, TTO) is widely employed both as such and as ingredient in skin care products for its several well-known biological activities. The European Scientific Committee on Consumer Products constantly promotes the collection of information on both skin distribution and systemic exposure to tea tree oil components after the application of topic formulations. In this context, our study aimed to obtain quantitative data on the percutaneous absorption and skin layers distribution of seven TTO major components (i.e. alpha-pinene, beta-pinene, alpha-terpinene, 1,8-cineole, gamma-terpinene, 4-terpineol, alpha-terpineol) when applying a 5% TTO cream. The study also focused on the evaluation of the TTO major components release in the surrounding environment due to their high volatility. The permeation study was performed in vitro on full thickness pig ear skin samples using both conventional and ad hoc modified static glass Franz diffusion cells to monitor the compounds permeation and environmental release respectively. The amount of formulation to apply was chosen to be representative for the in-use condition (finite dose regime). The applied formulation (before and after the study), the collected receptor fluid and the individual skin layers (stratum corneum, epidermis and dermis)- previously separated- were quantitatively analyzed by a fully-automatized and solvent-free analytical method employing Headspace Solid Phase Microextraction (HS-SPME) in combination with Gas Chromatography coupled with Mass Spectrometry (GC–MS). Method validation showed satisfying values of sensitivity, repeatability and intermediate precision with percent relative standard deviation (%RSD) below 15%. External calibration curves were built through both simple HS-SPME-GC-MS and HS-SPME-GC-MS in Multiple Head Space Extraction mode (MHE) to quantify TTO markers in the receptor fluid and in skin layers and formulation respectively. At the end of the permeation study, the applied formulation was almost free from TTO components. Skin layers overall contained less than 1% of each TTO component. Only the oxygenated terpenes: 4-terpineol, -terpineol and 1,8 cineole, significantly permeated through the skin membrane, (around 50% and 12% of the total for 4-terpineol/ -terpineol and 1,8-cineole respectively) while hydrocarbons were found at trace level in the receptor fluid. As expected from the TTO markers volatile nature, a substantial fraction of total amount of each TTO component applied on the skin at the beginning of the study was released in the surrounding environment. To the best of the authors’ knowledge, in this work for the first time 1) an ad hoc modified static Franz cells was used to quantify the loss of TTO components by evaporation and 2) a complete solvent-free method was employed to quantify and extract seven major volatile TTO components from skin layers.