Reverse Iontophoresis: Noninvasive Assessment of Topical Drug Bioavailability.

Assessing drug disposition in the skin after the application of a topical formulation is difficult. It is hypothesized that reverse iontophoresis (RI), which can extract charged/polar molecules for monitoring purposes, may provide a noninvasive approach for the assessment of local drug bioavailability. The passive and RI extraction of salicylic acid (SA) and nicotine (NIC) from porcine skin in vitro was assessed after a simple solution of the former and a transdermal patch of the latter had been applied for 24 and 8 h, respectively. Immediately after this "passive skin loading", the amount of drug in the stratum corneum (SC) and "viable" tissue (VT) was measured either (a) after tape-stripping and subsequent solvent extraction of both skin layers or (b) following RI extraction over 4 h. Parallel experiments were then performed in vivo in healthy volunteers; in this case, the VT was not sampled and the skin loading period for NIC was only 4 h. RI extraction of both drugs was significantly higher (in vitro and in vivo) than that achieved passively, and the cumulative RI extraction profiles as a function of time were mathematically analyzed using a straightforward compartmental model. Best-fit estimates of drug amounts in the SC and VT (ASC,0 and AVT,0, respectively) at the end of "loading" and two first-order rate constants describing transfer between the model compartments were then determined. The in vitro predictions of ASC,0 and AVT,0 were in excellent agreement with the experimental results, as was the value of the former in vivo. The rate constants derived from the in vitro and in vivo results were also similar. In summary, the results provide proof-of-concept that the RI method has the potential to noninvasively assess relevant metrics of drug bioavailability in the skin.

A new 3D model for genotoxicity assessment: EpiSkin™ Micronucleus Assay.

The European Regulation on Cosmetics (no. 1223/2009) has prohibited the use of animals in safety testing since March 2009 for ingredients used in cosmetics. Irreversible events at the chromosome level (clastogenesis and aneugenesis) are commonly evaluated by scoring either micronuclei or chromosome aberrations using cell-based genotoxicity assays. Like most in vitro genotoxicity assays, the 2D in vitro micronucleus assay exhibits a poor specificity and does not mimic the dermal route. To address these limitations, the current project aims to develop and validate a 3D micronucleus assay using the EpiSkin™ model. This project is scientifically supported by the Cosmetics Europe Genotoxicity Task Force. In a first step, two key criteria for the development of micronucleus assay, namely, the sufficient yield of cells from the EpiSkin™ model and an acceptable proliferation rate of the basal layer, were assessed and demonstrated. Subsequently, six chemicals (vinblastine, n-ethylnitrosourea, ß-butyrolactone, 2-acetylaminofluorene, 2,4-dichlorophenoland d-limonene) were evaluated in the EpiSkin™ Micronucleus Assay. At least two independent experiments using 48- and 72-h incubations were performed for each chemical. Results showed good inter-experimental reproducibility, as well as the correct identification of all six tested chemicals. The metabolism of 2-acetylaminofluorene on the EpiSkin™ model was also investigated and confirmed by the formation of an intermediate metabolite (2-aminofluorene). These preliminary results from the EpiSkin™ Micronucleus Assay indicate that it is a promising in vitro assay for assessing genotoxicity. The availability and suitability of this test method contribute significantly to the development of non-animal testing methods in China and its impact on the worldwide field.

Assessment of a defined approach based on a stacking prediction model to identify skin sensitization hazard.

Skin sensitization is an important toxicological endpoint in the safety assessment of chemicals and cosmetic ingredients. Driven by ethical considerations and European Union (EU) legislation, its assessment has progressed from the reliance on traditional animal models to the use of non-animal test methods. It is generally accepted that the assessment of skin sensitization requires the integration of various non-animal test methods in defined approaches (DAs), to cover the mechanistic key events of the adverse outcomes pathway (AOP) (OECD, 2014). Several case studies for DAs predicting skin sensitization hazard or potency have been submitted to the OECD, including a stacking meta-model developed by L'Oréal Research & Innovation (OECD, 2017b; Del Bufalo et al., 2018; Noçairi et al., 2016). The present study evaluated the predictive performance of the defined approach integrating a stacking meta-model incorporating in silico, in chemico and in vitro assays, using the Cosmetics Europe (CE) skin sensitization database. Based on the optimized prediction cut-offs, the defined approach provided a hazard prediction for 97 chemicals with a sensitivity of 91%, a specificity of 76% and accuracy of 86% (kappa of 0.67) against human skin sensitization hazard data and a sensitivity of 85%, specificity of 91% and accuracy of 87% (kappa of 0.67) against Local Lymph Node Assay (LLNA) hazard data. A comparison of the in vivo LLNA with human hazard data for the same 97 chemicals showed a sensitivity of 92%, specificity of 51% and accuracy of 78% (kappa of 0.48). Thus, the defined approach showed a higher degree of concordance, as compared to the LLNA for predicting human skin sensitization hazard. Moreover, a comparison with the six DAs selected for evaluation of their predictivity in the study by Kleinstreuer et al. (2018) showed a similar high accuracy of 86% for 97 overlapping chemicals. The next step will be an independent evaluation of the DA for its integration in the performances based test guidelines (PBTG) for skin sensitization.

A strategy for systemic toxicity assessment based on non-animal approaches: The Cosmetics Europe Long Range Science Strategy programme.

When performing safety assessment of chemicals, the evaluation of their systemic toxicity based only on non-animal approaches is a challenging objective. The Safety Evaluation Ultimately Replacing Animal Test programme (SEURAT-1) addressed this question from 2011 to 2015 and showed that further research and development of adequate tools in toxicokinetic and toxicodynamic are required for performing non-animal safety assessments. It also showed how to implement tools like thresholds of toxicological concern (TTCs) and read-across in this context. This paper shows a tiered scientific workflow and how each tier addresses the four steps of the risk assessment paradigm. Cosmetics Europe established its Long Range Science Strategy (LRSS) programme, running from 2016 to 2020, based on the outcomes of SEURAT-1 to implement this workflow. Dedicated specific projects address each step of this workflow, which is introduced here. It tackles the question of evaluating the internal dose when systemic exposure happens. The applicability of the workflow will be shown through a series of case studies, which will be published separately. Even if the LRSS puts the emphasis on safety assessment of cosmetic relevant chemicals, it remains applicable to any type of chemical.

Use of human in vitro skin models for accurate and ethical risk assessment: metabolic considerations.

Several human skin models employing primary cells and immortalized cell lines used as monocultures or combined to produce reconstituted 3D skin constructs have been developed. Furthermore, these models have been included in European genotoxicity and sensitization/irritation assay validation projects. In order to help interpret data, Cosmetics Europe (formerly COLIPA) facilitated research projects that measured a variety of defined phase I and II enzyme activities and created a complete proteomic profile of xenobiotic metabolizing enzymes (XMEs) in native human skin and compared them with data obtained from a number of in vitro models of human skin. Here, we have summarized our findings on the current knowledge of the metabolic capacity of native human skin and in vitro models and made an overall assessment of the metabolic capacity from gene expression, proteomic expression, and substrate metabolism data. The known low expression and function of phase I enzymes in native whole skin were reflected in the in vitro models. Some XMEs in whole skin were not detected in in vitro models and vice versa, and some major hepatic XMEs such as cytochrome P450-monooxygenases were absent or measured only at very low levels in the skin. Conversely, despite varying mRNA and protein levels of phase II enzymes, functional activity of glutathione S-transferases, N-acetyltransferase 1, and UDP-glucuronosyltransferases were all readily measurable in whole skin and in vitro skin models at activity levels similar to those measured in the liver. These projects have enabled a better understanding of the contribution of XMEs to toxicity endpoints.