After exposure for 6 or 24 h the compound was washed off with cotton swabs and washing fluid. During the experimental period, samples were taken from the stirred (magnetic stirrers, Variomag Telemodul 20C/40C, H + P Labortechnik, Germany) receptor fluid at distinct time points and replaced with fresh receptor fluid by a fraction collector (222 L, Abimed, Germany) and a multi-channel peristaltic

pump (MC 360, Ismatec, Germany). At the end of the run each diffusion cell was dismantled and all parts were processed for balancing. Two to six tape strips (Crystal Clear Tape 600, Scotch, France) were used to remove the upper stratum corneum from the skin samples. The tapes with stratum corneum and the remaining skin were digested with Soluene 350®, lasting a minimum of 24 h; cotton swabs as well as the class devices were extracted with ethanol or water – depending on the solubility of the test www.selleckchem.com/products/LDE225(NVP-LDE225).html compounds. All samples were diluted with LSC-Cocktail Nutlin-3a datasheet and measured by Liquid Scintillation Counting (LSC; TriCab 2800TR, Perkin-Elmer, USA; linear range up to 1,000,000 dpm). Absolute and percentage

amounts in receptor fluid, skin, tape strips and washing fluids were calculated as well as the total recovery. Only a recovery of 100 ± 10% was assumed to be valid for mean calculations. The sum of content in receptor fluid (including receptor chamber washings) and skin was defined as the potentially absorbable dose (AD); if applicable also the amount recovered from the underlying membrane of the reconstructed human skin was assigned to AD. The cumulative absorbed amount was plotted against time. The steepest slope – the maximal absorption rate in μg cm−2 h−1 Bcl-w – divided by the applied concentration in μg cm−3 provides the maximal permeability constant maxKp in cm h−1. The intercept of the elongated steepest slope line with the x-axis represents the lag time (h). Test compound dependent experimental conditions as well as logP and molecular weight are listed in Table 1. All four test compounds were applied to full-thickness and split-thickness human skin, 14C-testosterone, 14C-caffeine and 14C-MCPA

were also applied to rat skin and to reconstructed human skin. Unintentionally damaged skin samples were left in the set up and examined along with the intact samples. Intentionally impaired rat skin samples were used for 14C-MCPA experiments. Besides a visual check at least two of the five following integrity tests were conducted in each experiment, the skin being mounted on the Franz cell. TEER, TEWL and TWF were performed in advance, ISTD concurrently and BLUE at the end of the run. To measure the transepidermal electrical resistance to an alternating current (impedance), the receptor and donor compartment of the diffusion cell were filled with physiological saline (0.9% aqueous NaCl solution). Electrodes were immersed in each compartment and the impedance was measured via a LCR bridge (LCR400, Thurbly Thandar Instruments, Great Britain) at a frequency of 1 kHz.