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.

In vitro to in vivo extrapolation to derive a metabolism factor for estimating the aggregate exposure to salicylic acid after dermal exposure of its esters.

As part of the safety assessment of salicylate esters in cosmetics, we developed a metabolism factor based on in vitro to in vivo extrapolation (IVIVE) to provide a better estimation of the aggregate internal exposure to the common metabolite, salicylic acid. Optimal incubation conditions using human liver S9 were identified before measuring salicylic acid formation from 31 substances. Four control substances, not defined as salicylic esters but which could be mistaken as such due to their nomenclature, did not form salicylic acid. For the remaining substances, higher in vitro intrinsic clearance (CLint, in vitro) values generally correlated with lower LogP values. A "High-Throughput Pharmacokinetic" (HTPK) model was used to extrapolate CLint, in vitro values to human in vivo clearance and half-lives. The latter were used to calculate the percentage of substance metabolised to salicylic acid in 24 h in vivo following human exposure to the ester, i.e. the "metabolism factor". The IVIVE model correctly reproduced the observed elimination rate of 3 substances using in silico or in vitro input parameters. For other substances, in silico only-based predictions generally resulted in lower metabolism factors than when in vitro values for plasma binding and liver S9 CLint, in vitro were used. Therefore, in vitro data input provides the more conservative metabolism factors compared to those derived using on in silico input. In conclusion, these results indicate that not all substances contribute equally (or at all) to the systemic exposure to salicylic acid. Therefore, we propose a realistic metabolism correction factor by which the potential contribution of salicylate esters to the aggregate consumer exposure to salicylic acid from cosmetic use can be estimated.

Use of in vitro ADME methods to identify suitable analogs of homosalate and octisalate for use in a read-across safety assessment.

Case studies are needed to demonstrate the use of human-relevant New Approach Methodologies in cosmetics ingredient safety assessments. For read-across assessments, it is crucial to compare the target chemical with the most appropriate analog; therefore, reliable analog selection should consider physicochemical properties, bioavailability, metabolism, as well as the bioactivity of potential analogs. To complement in vitro bioactivity assays, we evaluated the suitability of three potential analogs for the UV filters, homosalate and octisalate, according to their in vitro ADME properties. We describe how technical aspects of conducting assays for these highly lipophilic chemicals were addressed and interpreted. There were several properties that were common to all five chemicals: they all had similar stability in gastrointestinal fluids (in which no hydrolysis to salicylic occurred); were not substrates of the P-glycoprotein efflux transporter; were highly protein bound; and were hydrolyzed to salicylic acid (which was also a major metabolite). The main properties differentiating the chemicals were their permeability in Caco-2 cells, plasma stability, clearance in hepatic models, and the extent of hydrolysis to salicylic acid. Cyclohexyl salicylate, octisalate, and homosalate were identified suitable analogs for each other, whereas butyloctyl salicylate exhibited ADME properties that were markedly different, indicating it is unsuitable. Isoamyl salicylate can be a suitable analog with interpretation for octisalate. In conclusion, in vitro ADME properties of five chemicals were measured and used to pair target and potential analogs. This study demonstrates the importance of robust ADME data for the selection of analogs in a read-across safety assessment.

Solubility of Foreign Molecules in Stratum Corneum Brick and Mortar Structure.

The barrier function of the skin is mainly assured by its outermost layer, stratum corneum (SC). One key aspect in predicting dermal drug delivery and in safety assessment of skin exposure to chemicals is the need to determine the amount of chemical that is taken up into the SC. We here present a strategy that allows for direct measures of the amount of various solid chemicals that can be dissolved in the SC in any environmental relative humidity (RH). A main advantage of the presented method is that it distinguishes between molecules that are dissolved within the SC and molecules that are not dissolved but might be present at, for example, the skin surface. In addition, the method allows for studies of uptake of hydrophobic chemicals without the need to use organic solvents. The strategy relies on the differences in the molecular properties of the added molecules in the dissolved and the excess states, employing detection methods that act as a dynamic filter to spot only one of the fractions, either the dissolved molecules or the excess solid molecules. By measuring the solubility in SC and delipidized SC at the same RHs, the same method can be used to estimate the distribution of the added chemical between the extracellular lipids and corneocytes at different hydration conditions. The solubility in porcine SC is shown to vary with hydration, which has implications for the molecular uptake and transport across the skin. The findings highlight the importance of assessing the chemical uptake at hydration conditions relevant to the specific applications. The methodology presented in this study can also be generalized to study the solubility and partitioning of chemicals in other heterogeneous materials with complex composition and structure.

Practical application of the interim internal threshold of toxicological concern (iTTC): a case study based on clinical data.

We present a case study that provides a practical step-by-step example of how the internal Threshold of Toxicological Concern (iTTC) can be used as a tool to refine a TTC-based assessment for dermal exposures to consumer products. The case study uses a theoretical scenario where there are no systemic toxicity data for the case study chemicals (avobenzone, oxybenzone, octocrylene, homosalate, octisalate, octinoxate, and ecamsule). Human dermal pharmacokinetic data following single and repeat dermal exposure to products containing the case study chemicals were obtained from data published by the US FDA. The clinical studies utilized an application procedure that followed maximal use conditions (product applied as 2 mg/cm2 to 75% of the body surface area, 4 times a day). The case study chemicals were first reviewed to determine if they were in the applicability domain of the iTTC, and then, the human plasma concentrations were compared to an iTTC limit of 1 µM. When assessed under maximum usage, the external exposure of all chemicals exceeded the external dose TTC limits. By contrast, the internal exposure to all chemicals, except oxybenzone, was an order of magnitude lower than the 1 µM interim iTTC threshold. This work highlights the importance of understanding internal exposure relative to external dose and how the iTTC can be a valuable tool for assessing low-level internal exposures; additionally, the work demonstrates how to use an iTTC, and highlights considerations and refinement opportunities for the approach.

Non-destructive, reverse iontophoretic extraction of phytochemicals from Mangifera indica, Centella asiatica, Punica granatum, and Citrus sinensis.

For the commercial-scale isolation of phytochemicals, a suitable plant biomass source (including species, origin, growing season, etc.) must be identified, and frequent analytical verification is required to ensure that the phytochemicals are present at predefined minimum threshold concentrations. While the latter are typically assessed in the laboratory, a more efficient and less resource-intensive approach would involve non-destructive and environmentally friendly measurements in situ. Reverse iontophoretic (RI) sampling offers a potential solution to this challenge. OBJECTIVE: We aimed to demonstrate the non-destructive, RI sampling of phytochemicals of interest from biomass from four different sources. MATERIALS AND METHODS: RI experiments were performed in side-by-side diffusion cells using a current density of 0.5 mA/cm2 , for a predetermined time in a defined pH environment, using (1) fresh leaves from Mangifera indica and Centella asiatica and (2) isolated peel from Punica granatum and Citrus sinensis. RESULTS: Mangiferin, madecassoside, punicalagin, ellagic acid, and hesperidin were extracted from the different biomasses by RI. The amounts extracted ranged from 0.03 mg/100 mg of biomass for the cathodal extraction of madecassoside to 0.63 mg/100 mg of biomass for the anodal extraction of punicalagin. A linear relationship (r2  = 0.73) between the RI-extracted quantities of punicalagin and those determined using conventional methods was demonstrated. CONCLUSION: The non-destructive, in situ measurement of phytochemical levels by RI represents a feasible approach for timing the harvesting process.

An eco-friendly system for stabilization of retinol: A step towards attending performance with improved environmental respect.

OBJECTIVE: Retinol (Vitamin A) is one of the most effective molecules for the treatment of skin aging. However, it degrades rapidly under the influence of light, oxygen, metal ions, and oxidizing agents. To prevent this, stabilizing systems are used commonly. Notably, butylated hydroxytoluene (2,6-di-tert-butyl-p-cresol) (BHT) and ethylenediaminetetraacetic acid (EDTA) salts exhibit excellent antioxidant and metal-chelating properties but are not eco-friendly. In this study, our goal was to develop a new eco-friendly stabilization system for retinol-based formulations such that the system does not interfere with retinol skin absorption, nor its clinical efficacy. METHODS: An evaluation tool called the Sustainable Product Optimization Tool (SPOT) was used to assess the environmental performance of formulations containing retinol and the various stabilizers investigated. Accelerated stability tests were performed on formulations stored for 2 months at 4 and 45°C (ISO/TR Standard 18811 2018 directives). Long-term stability evaluation was done on formulations stored for 24-months at room temperature. Retinol skin absorption was assessed by the Franz cell method using human skin explants (OECD guideline 428). Finally, a clinical study was performed to evaluate the cosmetic performance of a 0.3% stabilized retinol formulation. RESULTS: N,N'-ethylenediamine disuccinic acid ([S,S]-EDDS isomer) and pentaerythritol tetrakis(3,5-di-tert-butyl-4-hydroxyhydrocinnamate) (PBHC) showed higher biodegradability and a reduced water footprint compared with those of BHT and EDTA. The SPOT simulation gave [S,S]-EDDS + PBHC a score of 10 versus 8.84 for EDTA + BHT. Moreover, [S,S]-EDDS + PBHC better controlled the chemical degradation of retinol compared with EDTA + BHT. Retinol skin absorption was also achieved in the case of a formulation containing [S,S]-EDDS + PBHC, and several skin attributes improved significantly after 12 weeks of product use, with over 75% of the panel perceiving benefits. CONCLUSION: Regarding retinol stabilization, the PBHC + [S,S]-EDDS combination is an eco-friendlier and more effective alternative to BHT + EDTA.

OBJECTIF: Le rétinol est l'une des molécules les plus efficaces sur les signes du vieillissement. Cependant, il se dégrade rapidement, notamment sous l'influence de la lumière, de l'oxygène, des ions métalliques et des oxydants. Il existe des systèmes de stabilisation notamment à base d'hydroxytoluène butylé (2,6-di-tert-butyl-p-crésol) (BHT) et de sels d'acide éthylènediamine tétra acétique (EDTA) qui possèdent respectivement d'excellentes propriétés antioxydantes et chélatrices des métaux bien que certaines études aient montré que les deux présentaient un profil environnemental moins que parfait. Notre objectif était de développer un nouveau système de stabilisation ayant un meilleur respect environnemental. Ce nouveau système plus durable ne doit pas impacter l'absorption cutanée du rétinol, ni son efficacité clinique. MÉTHODES: Un outil d'évaluation appelé SPOT (Sustainable Product Optimization Tool) a été utilisé pour évaluer la performance environnementale des formules contenant du rétinol et des stabilisants. Des tests de stabilité accélérés ont été réalisés 2 mois après la formulation avec des produits gardés à 4°C et 45°C (norme ISO/TR 18811 directives 2018). Les stabilités de longue durée ont été évalués sur des produits gardés 24 mois à la température ambiante. L'absorption cutanée du rétinol a été réalisée par la méthode des cellules de Franz avec des explants de peau humaine (directive OCDE 428). Enfin, une étude clinique a prouvé la performance cosmétique d'une formule de rétinol stabilisé à 0,3%. RÉSULTATS: L'association de l'acide N,N'-éthylènediamine disuccinique (isomère [S,S]-EDDS) et du pentaérythritol tétrakis (3,5-di-tert-butyl-4-hydroxyhydrocinnamate) (PBHC) présente une biodégradabilité plus élevée et une empreinte eau réduite par rapport à BHT et EDTA. La simulation SPOT a donné au [S,S]-EDDS et au PBHC un score de 10 contre 8,84 pour l'EDTA et le BHT. De plus, le [S,S]-EDDS et le PBHC contrôlent mieux la dégradation chimique du rétinol par rapport à l'association BHT plus EDTA. L'absorption cutanée du rétinol a également été obtenue dans une formule contenant du [S,S]-EDDS et du PBHC et plusieurs attributs cutanés ont été significativement améliorés après 12 semaines d'utilisation du produit. Les bénéfices ont été perçus par plus de 75% du panel testé CONCLUSION: Concernant la stabilisation du rétinol, l'association PBHC et [S,S]-EDDS est une alternative plus écologique et plus performante que le BHT et l'EDTA.

In-Vivo Tape Stripping Study with Caffeine for Comparisons on Body Sites, Age and Washing.

PURPOSE: Assessing the percutaneous absorption of cosmetic ingredients using in-vitro human skin reveals certain limitations, such as restricted anatomical sites and repeated exposure, and to overcome these issues, in-vivo studies are required. The aim of the study is to develop a robust non-invasive in-vivo protocol that should be applicable to a wide range of application. METHODS: A robust tape stripping protocol was therefore designed according to recent recommendations, and the impact of two different washing procedures on caffeine distribution in tape strips was investigated to optimise the protocol. The optimised protocol was then used to study the effect of age and anatomical area on the percutaneous absorption of caffeine, including facial areas which are not readily available for in-vitro studies. RESULTS: With tape stripping, a difference between the percutaneous absorption on the face (forehead, cheek) and the volar forearm was observed. No obvious difference was observed between percutaneous absorption in young and post-menopausal women, but this could be due to the limited number of subjects. CONCLUSION: This tape stripping protocol is now to be deployed to address many other factors, such as percutaneous absorption in other anatomical areas (e.g. abdomen, axilla, etc.), impact of repeated applications and effect of formulation.

3D Molecular Imaging of Stratum Corneum by Mass Spectrometry Suggests Distinct Distribution of Cholesteryl Esters Compared to Other Skin Lipids.

The crucial barrier properties of the stratum corneum (SC) depend critically on the design and integrity of its layered molecular structure. However, analysis methods capable of spatially resolved molecular characterization of the SC are scarce and fraught with severe limitations, e.g., regarding molecular specificity or spatial resolution. Here, we used 3D time-of-flight secondary ion mass spectrometry to characterize the spatial distribution of skin lipids in corneocyte multilayer squams obtained by tape stripping. Depth profiles of specific skin lipids display an oscillatory behavior that is consistent with successive monitoring of individual lipid and corneocyte layers of the SC structure. Whereas the most common skin lipids, i.e., ceramides, C24:0 and C26:0 fatty acids and cholesteryl sulfate, are similarly organized, a distinct 3D distribution was observed for cholesteryl oleate, suggesting a different localization of cholesteryl esters compared to the lipid matrix separating the corneocyte layers. The possibility to monitor the composition and spatial distribution of endogenous lipids as well as active drug and cosmetic substances in individual lipid and corneocyte layers has the potential to provide important contributions to the basic understanding of barrier function and penetration in the SC.

Elongated microparticles tuned for targeting hyaluronic acid delivery to specific skin strata.

OBJECTIVE: Microneedle or fractional laser applications are the most common topical delivery enhancement platforms. However, these methods of drug delivery are not skin strata specific. Drug delivery approaches which could target specific stratum of the skin remains a challenge. Elongated microparticles (EMPs) have been used in enhancing drug delivery into the skin. The aim of this study was to evaluate, for the first time, elongated silica microparticles with two different length profiles to enhance delivery of hyaluronic acid into different strata of human skin. METHODS: Two types of EMPs-long (milled EMPs) or short (etched EMPs) length ranges were characterized. A prototypical liquid formulation (Fluorescent hyaluronic acid) with and without EMP enhancement were evaluated for hyaluronic acid delivery in ex-vivo human skin. High performance liquid chromatography, Typhoon fluorescence scanning system, laser scanning confocal microscopy and reflectance confocal microscopy (RCM) were used to validate F-HA stability, visualize fluorescein in the skin, image the depth of F-HA delivery in the skin and define EMP penetration in skin strata, respectively. Statistical analysis was conducted using GraphPad Prism 6 software (GraphPad Software Inc, USA). RESULTS: Fluorescein-hyaluronic acid was stable and EMP enhanced skin penetration. RCM revealed that 'etched EMP' penetrated the skin to the stratum spinosum level. The vast majority (97.8%; p < 0.001) of the etched EMP did not penetrate completely through the viable epidermis and no obvious penetration into the dermis. In contrast, milled EMP showed 41-fold increase in penetration compared to the etched EMP but penetrated beyond the dermoepidermal junction. CONCLUSION: EMPs can enhance delivery of hyaluronic acid. Using EMPs with defined length distributions, which can be tuned for a specific stratum of the skin, can achieve targeted hyaluronic acid delivery.

OBJECTIF: Les microaiguilles ou le laser fractionné sont couramment utilisés pour augmenter l'absorption d'actif appliqué par voie topique. Toutefois, ces approches ne permettent de cibler une strate spécifique de la peau. Ainsi les méthodes permettant de cibler spécifiquement l'épiderme reste un défi. Les microparticules allongées (EMP) ont déjà été utilisé pour augmenter l'absorption cutanée. L'objectif de l'étude est d'évaluer pour la première fois, la capacité de microparticules allongées de silice (de deux longueurs différentes) à délivrer l'acide hyaluronique dans les différentes couches de la peau. MÉTHODES: Deux types d'EMP, longues (EMP broyé) ou courtes (EMP gravé), ont été caractérisées. Une formulation liquide contenant de l'acide hyaluronique marquée avec une sonde fluorescente (F-HA) a été évaluée avec et sans EMP sur peau humaine ex vivo. La chromatographie liquide haute performance, le scanner à fluorescence Typhoon, la microscopie laser confocal à balayage et la microscopie confocale par réflectance (RCM) ont été utilisées respectivement pour contrôler la stabilité de la F-HA, visualiser le signal de la fluorescéine dans la peau, imager l'absorption du F-HA dans la peau en fonction de la profondeur et caractériser la pénétration des EMP. Les analyses statistiques ont été réalisées avec le logiciel GraphPad Prims 6 (GraphPad Software Inc, USA). RÉSULTATS: L'acide hyaluronique marquée avec la fluorescéine est stable et les EMP permettent d'augmenter son absorption cutanée. Le RCM a montré que les EMP gravées pénètrent dans la peau jusqu'au niveau du stratum spinosum. La grande majorité des EMP gravés (97.8% ; p < 0,001) ne pénètre pas complétement dans l'épiderme viable et aucune pénétration mesurable dans le derme. Au contraire, les EMP broyées ont montrées une pénétration 41 fois plus importantes que les EMP gravées et peuvent aller au-delà de la jonction derme-épiderme. CONCLUSION: Les EMP peuvent augmenter l'absorption cutanée de l'acide hyaluronique. En utilisant des EMP de longueur définie et en ajustant celle-ci, il est même possible de cibler spécifiquement une strate cutanée.

Comparison of the metabolism of 10 cosmetics-relevant chemicals in EpiSkin™ S9 subcellular fractions and in vitro human skin explants.

An understanding of the bioavailability of topically applied cosmetics ingredients is key to predicting their local skin and systemic toxicity and making a safety assessment. We investigated whether short-term incubations with S9 from the reconstructed epidermal skin model, EpiSkin™, would give an indication of the rate of chemical metabolism and produce similar metabolites to those formed in incubations with human skin explants. Both have advantages: EpiSkin™ S9 is a higher-throughput assay, while the human skin explant model represents a longer incubation duration (24 hours) model integrating cutaneous distribution with metabolite formation. Here, we compared the metabolism of 10 chemicals (caffeine, vanillin, cinnamyl alcohol, propylparaben, 4-amino-3-nitrophenol, resorcinol, 4-chloroaniline, 2-amino-3-methyl-3H-imidazo[4,5-F]quinoline and 2-acetyl aminofluorene) in both models. Both models were shown to have functional Phase 1 and 2 enzymes, including cytochrome P450 activities. There was a good concordance between the models with respect to the level of metabolism (stable vs. slowly vs. extensively metabolized chemicals) and major early metabolites produced for eight chemicals. Discordant results for two chemicals were attributed to a lack of the appropriate cofactor (NADP+ ) in S9 incubations (cinnamyl alcohol) and protein binding influencing chemical uptake in skin explants (4-chloroaniline). These data support the use of EpiSkin™ S9 as a screening assay to provide an initial indication of the metabolic stability of a chemical applied topically. If required, chemicals that are not metabolized by EpiSkin™ S9 can be tested in longer-term incubations with in vitro human explant skin to determine whether it is slowly metabolized or not metabolized at all.

Use of human liver and EpiSkin™ S9 subcellular fractions as a screening assays to compare the in vitro hepatic and dermal metabolism of 47 cosmetics-relevant chemicals.

The abundance of xenobiotic metabolizing enzymes (XMEs) is different in the skin and liver; therefore, it is important to differentiate between liver and skin metabolism when applying the information to safety assessment of topically applied ingredients in cosmetics. Here, we have employed EpiSkin™ S9 and human liver S9 to investigate the organ-specific metabolic stability of 47 cosmetic-relevant chemicals. The rank order of the metabolic rate of six chemicals in primary human hepatocytes and liver S9 matched relatively well. XME pathways in liver S9 were also present in EpiSkin S9; however, the rate of metabolism tended to be lower in the latter. It was possible to rank chemicals into low-, medium- and high-clearance chemicals and compare rates of metabolism across chemicals with similar structures. The determination of the half-life for 21 chemicals was affected by one or more factors such as spontaneous reaction with cofactors or non-specific binding, but these technical issues could be accounted for in most cases. There were seven chemicals that were metabolized by liver S9 but not by EpiSkin S9: 4-amino-3-nitrophenol, resorcinol, cinnamyl alcohol and 2-acetylaminofluorene (slowly metabolized); and cyclophosphamide, benzophenone, and 6-methylcoumarin. These data support the use of human liver and EpiSkin S9 as screening assays to indicate the liver and skin metabolic stability of a chemical and to allow for comparisons across structurally similar chemicals. Moreover, these data can be used to estimate the systemic bioavailability and clearance of chemicals applied topically, which will ultimately help with the safety assessment of cosmetics ingredients.

Measurement of the penetration of 56 cosmetic relevant chemicals into and through human skin using a standardized protocol.

OECD test guideline 428 compliant protocol using human skin was used to test the penetration of 56 cosmetic-relevant chemicals. The penetration of finite doses (10 µL/cm2 ) of chemicals was measured over 24 hours. The dermal delivery (DD) (amount in the epidermis, dermis and receptor fluid [RF]) ranged between 0.03 ± 0.02 and 72.61 ± 8.89 µg/cm2 . The DD of seven chemicals was comparable with in vivo values. The DD was mainly accounted for by the amount in the RF, although there were some exceptions, particularly of low DD chemicals. While there was some variability due to cell outliers and donor variation, the overall reproducibility was very good. As six chemicals had to be applied in 100% ethanol due to low aqueous solubility, we compared the penetration of four chemicals with similar physicochemical properties applied in ethanol and phosphate-buffered saline. Of these, the DD of hydrocortisone was the same in both solvents, while the DD of propylparaben, geraniol and benzophenone was lower in ethanol. Some chemicals displayed an infinite dose kinetic profile; whereas, the cumulative absorption of others into the RF reflected the finite dosing profile, possibly due to chemical volatility, total absorption, chemical precipitation through vehicle evaporation or protein binding (or a combination of these). These investigations provide a substantial and consistent set of skin penetration data that can help improve the understanding of skin penetration, as well as improve the prediction capacity of in silico skin penetration models.

Dermal absorption study OECD TG 428 mass balance recommendations based on the EFSA database.

The European Food Safety Authority (EFSA) guidance (EFSA, 2017) for dermal absorption (DA) studies recommends stringent mass balance (MB) limits of 95-105%. EFSA suggested that test material can be lost after penetration and requires that for chemicals with <5% absorption the non-recovered material must be added to the absorbed dose if MB is <95%. This has huge consequences for low absorption pesticides. Indeed, one third of the MBs in the EFSA DA database are outside the refined criteria. This is also true for DA data generated by Cosmetics Europe (Gregoire et al., 2019), indicating that this criterion is often not achieved even when using highly standardized protocols. While EFSA hypothesizes that modern analytical and pipetting techniques would enable to achieve this criterion, no scientific basis was provided. We describe how protocol procedures impact MB and evaluate the EFSA DA database to demonstrate that MB is subject to random variation. Generic application of "the addition rule" skews the measured data and increases the DA estimate, which results in unnecessary risk assessment failure. In conclusion, "missing material" is just a random negative deviation to the nominal dose. We propose a data-driven MB criterion of 90-110%, fully in line with OECD recommendations.

Challenges in working towards an internal threshold of toxicological concern (iTTC) for use in the safety assessment of cosmetics: Discussions from the Cosmetics Europe iTTC Working Group workshop.

The Threshold of Toxicological Concern (TTC) is an important risk assessment tool which establishes acceptable low-level exposure values to be applied to chemicals with limited toxicological data. One of the logical next steps in the continued evolution of TTC is to develop this concept further so that it is representative of internal exposures (TTC based on plasma concentration). An internal TTC (iTTC) would provide threshold values that could be utilized in exposure-based safety assessments. As part of a Cosmetics Europe (CosEu) research program, CosEu has initiated a project that is working towards the development of iTTCs that can be used for the human safety assessment. Knowing that the development of an iTTC is an ambitious and broad-spanning topic, CosEu organized a Working Group comprised a balance of multiple stakeholders (cosmetics and chemical industries, the EPA and JRC and academia) with relevant experience and expertise and workshop to critically evaluate the requirements to establish an iTTC. Outcomes from the workshop included an evaluation on the current state of the science for iTTC, the overall iTTC strategy, selection of chemical databases, capture and curation of chemical information, ADME and repeat dose data, expected challenges, as well as next steps and ongoing work.

Use of a Simple in vitro Test to Assess Loss of Chemical due to Volatility during an in vitro Human Skin Absorption Study.

BACKGROUND: We tested the cutaneous distribution of 50 chemicals in frozen human skin. The mass balance (MB) values for 48% of the chemicals were < 90%, possibly due to evaporation. METHODS: We confirmed the reduction in MB was due to evaporation for two chemicals tested in skin penetration experiments using a carbon filter above the skin to trap airborne chemical. An in vitro assay was used to predict the reduction in MB due to evaporation by comparing the recovery of chemicals after 4 h of incubation at room temperature in open and closed vials. RESULTS: Evaporative losses in vitro correlated well with measured MBs (i.e., < 90%) in skin penetration experiments (R2 = 0.81). There was a correlation of the MB with the vapour pressure (VP) which could be used to group chemicals according to their likelihood to evaporate during the course of a skin penetration study. There was also a correlation of MB with Henry's law constants, melting and boiling points. CONCLUSION: Our data support the use of a quick and simple test for volatility to account for the loss of MB in skin penetration experiment due to volatility. The best parameter to indicate the potential of a chemical to evaporate is the VP.

Comparison of the Skin Penetration of 3 Metabolically Stable Chemicals Using Fresh and Frozen Human Skin.

BACKGROUND: The Cosmetics Europe ADME Task Force is developing in vitro and in silico tools for predicting skin and systemic concentrations after topical application of cosmetic ingredients. There are conflicting reports as to whether the freezing process affects the penetration of chemicals; therefore, we evaluated whether the storage of human skin used in our studies (8-12 weeks at -20°C) affected the penetration of model chemicals. METHODS: Finite doses of trans-cinnamic acid (TCA), benzoic acid (BA), and 6-methylcoumarin (6MC) (non-volatile, non-protein reactive and metabolically stable in skin) were applied to fresh and thawed frozen skin from the same donors. The amounts of chemicals in different skin compartments were analysed after 24 h. RESULTS: Although there were some statistical differences in some parameters for 1 or 2 donors, the penetration of TCA, BA, and 6MC was essentially the same in fresh and frozen skin, i.e., there were no biologically relevant differences in penetration values. Statistical differences that were evident indicated that penetration was marginally lower in frozen than in fresh skin, indicating that the barrier function of the skin was not lost. CONCLUSION: The penetration of the 3 chemicals was essentially unaffected by freezing the skin at -20°C for up to 12 weeks.

A Microfluidic Diffusion Cell for Fast and Easy Percutaneous Absorption Assays.

PURPOSE: Percutaneous absorption assays of molecules for pharmaceutical and cosmetology purposes are important to determine the bioavailability of new compounds, once topically applied. The current method of choice is to measure the rate of diffusion through excised human skin using a diffusion cell. This method however entails significant drawbacks such as scarce availability and poor reproducibility of the sample, low sampling rate, and tedious assay setup. METHODS: The objective of the present work is to propose an alternative method that overcomes these issues by integrating an experimental model of the skin (artificial stratum corneum) and online optical sensors into a microfluidic device. RESULTS: The measurement of the diffusion profile followed by the calculation of the permeability coefficients and time lag were performed on seven different molecules and obtained data positively fit with those available from literature on human skin penetration. The coating of the lipid mixture to generate the artificial stratum corneum also proved robust and reproducible. The results show that the proposed device is able to give fast, real-time, accurate, and reproducible data in a user-friendly manner, and can be produced at a large scale. CONCLUSION: These assets should help both the cosmetics and pharmaceutics fields where the skin is the target or a pathway of a formulated compound, by allowing more candidate molecules or formulations to be assessed during the various stages of their development.