Extrapolation of systemic bioavailability assessing skin absorption and epidermal and hepatic metabolism of aromatic amine hair dyes in vitro.

Approaches to assess the role of absorption, metabolism and excretion of cosmetic ingredients that are based on the integration of different in vitro data are important for their safety assessment, specifically as it offers an opportunity to refine that safety assessment. In order to estimate systemic exposure (AUC) to aromatic amine hair dyes following typical product application conditions, skin penetration and epidermal and systemic metabolic conversion of the parent compound was assessed in human skin explants and human keratinocyte (HaCaT) and hepatocyte cultures. To estimate the amount of the aromatic amine that can reach the general circulation unchanged after passage through the skin the following toxicokinetically relevant parameters were applied: a) Michaelis-Menten kinetics to quantify the epidermal metabolism; b) the estimated keratinocyte cell abundance in the viable epidermis; c) the skin penetration rate; d) the calculated Mean Residence Time in the viable epidermis; e) the viable epidermis thickness and f) the skin permeability coefficient. In a next step, in vitro hepatocyte Km and Vmax values and whole liver mass and cell abundance were used to calculate the scaled intrinsic clearance, which was combined with liver blood flow and fraction of compound unbound in the blood to give hepatic clearance. The systemic exposure in the general circulation (AUC) was extrapolated using internal dose and hepatic clearance, and Cmax was extrapolated (conservative overestimation) using internal dose and volume of distribution, indicating that appropriate toxicokinetic information can be generated based solely on in vitro data. For the hair dye, p-phenylenediamine, these data were found to be in the same order of magnitude as those published for human volunteers.

Narrow-bore sample trapping and chromatography combined with quadrupole/time-of-flight mass spectrometry for ultra-sensitive identification of in vivo and in vitro metabolites.

The identification of in vitro and in vivo metabolites is vital to the discovery and development of new pharmaceutical therapies. Analytical strategies to identify metabolites at different stages of this process vary, but all involve the use of liquid chromatography separations combined with detection via mass spectrometry (HPLC/MS). Reported here is the use of narrow-bore column (0.5-1.0 mm i.d.) trapping of metabolites, followed by back-flushing onto a matching analytical column. Separated metabolites were then identified using quadrupole time-of-flight mass spectrometry (MS) and tandem MS. Metabolites in human plasma and from low-level in vitro incubations, that were not identified using standard HPLC/MS approaches, were characterized using the instrumental configuration described here.