Next generation risk assessment: an ab initio case study to assess the systemic safety of the cosmetic ingredient, benzyl salicylate, after dermal exposure.

We performed an ab initio next-generation risk assessment (NGRA) for a fragrance ingredient, benzyl salicylate (BSal), to demonstrate how cosmetic ingredients can be evaluated for systemic toxicity endpoints based on non-animal approaches. New approach methodologies (NAMs) used to predict the internal exposure included skin absorption assays, hepatocyte metabolism, and physiologically based pharmacokinetic (PBPK) modeling, and potential toxicodynamic effects were assessed using pharmacology profiling, ToxProfiler cell stress assay, transcriptomics in HepG2 and MCF-7 cells, ReproTracker developmental and reproductive toxicology (DART) assays, and cytotoxicity assays in human kidney cells. The outcome of the NGRA was compared to that of the traditional risk assessment approach based on animal data. The identification of the toxicologically critical entity was a critical step that directed the workflow and the selection of chemicals for PBPK modeling and testing in bioassays. The traditional risk assessment and NGRA identified salicylic acid (SA) as the "toxdriver." A deterministic PBPK model for a single-day application of 1.54 g face cream containing 0.5% BSal estimated the Cmax for BSal (1 nM) to be much lower than that of its major in vitro metabolite, SA (93.2 nM). Therefore, SA was tested using toxicodynamics bioassays. The lowest points of departure (PoDs) were obtained from the toxicogenomics assays. The interpretation of these results by two companies and methods were similar (SA only results in significant gene deregulation in HepG2 cells), but PoD differed (213 µM and 10.6 µM). A probabilistic PBPK model for repeated applications of the face cream estimated the highest Cmax of SA to be 630 nM. The resulting margins of internal exposure (MoIE) using the PoDs were 338 and 16, which were more conservative than those derived from external exposure and in vivo PoDs (margin of safety values were 9,705). In conclusion, both traditional and ab initio NGRA approaches concluded that the daily application of BSal in a cosmetic leave-on face cream at 0.5% is safe for humans. The processing and interpretation of toxicogenomics data can lead to different PoDs, which can subsequently affect the calculation of the MoIE. This case study supports the use of NAMs in a tiered NGRA ab initio approach.

Suitability of different reconstructed human skin models in the skin and liver Chip2 microphysiological model to investigate the kinetics and first-pass skin metabolism of the hair dye, 4-amino-2-hydroxytoluene.

The HUMIMIC skin-liver Chip2 microphysiological systems model using the epidermal model, EpiDerm™, was reported previously to mimic application route-dependent metabolism of the hair dye, 4-amino-2-hydroxytoluene (AHT). Therefore, we evaluated the use of alternative skin models-SkinEthic™, EpiDermFT™ and PhenionFT™-for the same purpose. In static incubations, AHT permeation was similar using SkinEthic™ and EpiDerm™ models. Older Day 21 (D21) SkinEthic™ models with a thicker stratum corneum did not exhibit a greater barrier to AHT (overall permeation was the same in D17 and D21 models). All epidermal models metabolised AHT, with the EpiDerm™ exhibiting higher N-acetylation than SkinEthic™ models. AHT metabolism by D21 SkinEthic™ models was lower than that by D17 SkinEthic™ and EpiDerm™ models, thus a thicker stratum corneum was associated with fewer viable cells and a lower metabolic activity. AHT permeation was much slower using PhenionFT™ compared to epidermal models and better reflected permeation of AHT through native human skin. This model also extensively metabolised AHT to N-acetyl-AHT. After a single topical or systemic application of AHT to Chip2 model with PhenionFT™, medium was analysed for parent and metabolites over 5 days. The first-pass metabolism of AHT was demonstrated, and the introduction of a wash step after 30 min decreased the exposure to AHT and its metabolites by 33% and 40%-43%, respectively. In conclusion, epidermal and FT skin models used in the Chip2 can mimic the first-pass skin metabolism of AHT. This highlights the flexibility of the Chip2 to incorporate different skin models according to the purpose.

Application of a skin and liver Chip2 microphysiological model to investigate the route-dependent toxicokinetics and toxicodynamics of consumer-relevant doses of genistein.

The HUMMIC skin-liver Chip2 microphysiological system using EpiDerm™ and HepaRG and stellate liver spheroids was used to evaluate the route-specific metabolism and toxicodynamic effects of genistein. Human-relevant exposure levels were compared: 60 nM representing the plasma concentration expected after topical application of a cosmetic product and 1 µM representing measured plasma concentrations after ingesting soya products. Genistein was applied as single and repeated topical and/or systemic doses. The kinetics of genistein and its metabolites were measured over 5 days. Toxicodynamic effects were measured using transcriptional analyses of skin and liver organoids harvested on Days 2 and 5. Route-specific differences in genistein's bioavailability were observed, with first-pass metabolism (sulfation) occurring in the skin after topical application. Only repeated application of 1 µM, resembling daily oral intake of soya products, induced statistically significant changes in gene expression in liver organoids only. This was concomitant with a much higher systemic concentration of genistein which was not reached in any other dosing scenario. This suggests that single or low doses of genistein are rapidly metabolised which limits its toxicodynamic effects on the liver and skin. Therefore, by facilitating longer and/or repeated applications, the Chip2 can support safety assessments by linking relevant gene modulation with systemically available parent or metabolite(s). The rate of metabolism was in accordance with the short half-life observed in in vivo in humans, thus supporting the relevance of the findings. In conclusion, the skin-liver Chip2 provides route-specific information on metabolic fate and toxicodynamics that may be relevant to safety assessment.

Development of a microphysiological skin-liver-thyroid Chip3 model and its application to evaluate the effects on thyroid hormones of topically applied cosmetic ingredients under consumer-relevant conditions.

All cosmetic ingredients registered in Europe must be evaluated for their safety using non-animal methods. Microphysiological systems (MPS) offer a more complex higher tier model to evaluate chemicals. Having established a skin and liver HUMIMIC Chip2 model demonstrating how dosing scenarios impact the kinetics of chemicals, we investigated whether thyroid follicles could be incorporated to evaluate the potential of topically applied chemicals to cause endocrine disruption. This combination of models in the HUMIMIC Chip3 is new; therefore, we describe here how it was optimized using two chemicals known to inhibit thyroid production, daidzein and genistein. The MPS was comprised of Phenion® Full Thickness skin, liver spheroids and thyroid follicles co-cultured in the TissUse HUMIMIC Chip3. Endocrine disruption effects were determined according to changes in thyroid hormones, thyroxine (T4) and 3,3',5-triiodothyronine (T3). A main part of the Chip3 model optimization was the replacement of freshly isolated thyroid follicles with thyrocyte-derived follicles. These were used in static incubations to demonstrate the inhibition of T4 and T3 production by genistein and daidzein over 4 days. Daidzein exhibited a lower inhibitory activity than genistein and both inhibitory activities were decreased after a 24 h preincubation with liver spheroids, indicating metabolism was via detoxification pathways. The skin-liver-thyroid Chip3 model was used to determine a consumer-relevant exposure to daidzein present in a body lotion based on thyroid effects. A "safe dose" of 0.235 µg/cm2 i.e., 0.047% applied in 0.5 mg/cm2 of body lotion was the highest concentration of daidzein which does not result in changes in T3 and T4 levels. This concentration correlated well with the value considered safe by regulators. In conclusion, the Chip3 model enabled the incorporation of the relevant exposure route (dermal), metabolism in the skin and liver, and the bioactivity endpoint (assessment of hormonal balance i.e., thyroid effects) into a single model. These conditions are closer to those in vivo than 2D cell/tissue assays lacking metabolic function. Importantly, it also allowed the assessment of repeated doses of chemical and a direct comparison of systemic and tissue concentrations with toxicodynamic effects over time, which is more realistic and relevant for safety assessment.

Identification of dihydromyricetin as a natural DNA methylation inhibitor with rejuvenating activity in human skin.

Changes in DNA methylation patterning have been reported to be a key hallmark of aged human skin. The altered DNA methylation patterns are correlated with deregulated gene expression and impaired tissue functionality, leading to the well-known skin aging phenotype. Searching for small molecules, which correct the aged methylation pattern therefore represents a novel and attractive strategy for the identification of anti-aging compounds. DNMT1 maintains epigenetic information by copying methylation patterns from the parental (methylated) strand to the newly synthesized strand after DNA replication. We hypothesized that a modest inhibition of this process promotes the restoration of the ground-state epigenetic pattern, thereby inducing rejuvenating effects. In this study, we screened a library of 1800 natural substances and 640 FDA-approved drugs and identified the well-known antioxidant and anti-inflammatory molecule dihydromyricetin (DHM) as an inhibitor of the DNA methyltransferase DNMT1. DHM is the active ingredient of several plants with medicinal use and showed robust inhibition of DNMT1 in biochemical assays. We also analyzed the effect of DHM in cultivated keratinocytes by array-based methylation profiling and observed a moderate, but significant global hypomethylation effect upon treatment. To further characterize DHM-induced methylation changes, we used published DNA methylation clocks and newly established age predictors to demonstrate that the DHM-induced methylation change is associated with a reduction in the biological age of the cells. Further studies also revealed re-activation of age-dependently hypermethylated and silenced genes in vivo and a reduction in age-dependent epidermal thinning in a 3-dimensional skin model. Our findings thus establish DHM as an epigenetic inhibitor with rejuvenating effects for aged human skin.

Read-across and new approach methodologies applied in a 10-step framework for cosmetics safety assessment - A case study with parabens.

Parabens are esters of para-hydroxybenzoic acid that have been used as preservatives in many types of products for decades including agrochemicals, pharmaceuticals, food and cosmetics. This illustrative case study with propylparaben (PP) demonstrates a 10-step read-across (RAX) framework in practice. It aims at establishing a proof-of-concept for the value added by new approach methodologies (NAMs) in read-across (RAX) for use in a next-generation risk assessment (NGRA) in order to assess consumer safety after exposure to PP-containing cosmetics. In addition to structural and physico-chemical properties, in silico information, toxicogenomics, in vitro toxicodynamic, toxicokinetic data from PBK models, and bioactivity data are used to provide evidence of the chemical and biological similarity of PP and analogues and to establish potency trends for observed effects in vitro. The chemical category under consideration is short (C1-C4) linear chain n-alkyl parabens: methylparaben, ethylparaben, propylparaben and butylparaben. The goal of this case study is to illustrate how a practical framework for RAX can be used to fill a hypothetical data gap for reproductive toxicity of the target chemical PP.

Use of in vitro metabolism and biokinetics assays to refine predicted in vivo and in vitro internal exposure to the cosmetic ingredient, phenoxyethanol, for use in risk assessment.

A novel approach was developed to help characterize the biokinetics of the cosmetic ingredient, phenoxyethanol, to help assess the safety of the parent and its major stable metabolite. In the first step of this non-animal tiered approach, primary human hepatocytes were used to confirm or refute in silico predicted metabolites, and elucidate the intrinsic clearance of phenoxyethanol. A key result was the identification of the major metabolite, phenoxyacetic acid (PAA), the exposure to which in the kidney was subsequently predicted to far exceed that of phenoxyethanol in blood or other tissues. Therefore, a novel aspect of this approach was to measure in the subsequent step the formation of PAA in the cells dosed with phenoxyethanol that were used to provide points of departure (PoDs) and express the intracellular exposure as the Cmax and AUC24. This enabled the calculation of the intracellular concentrations of parent and metabolite at the PoD in the cells used to derive this value. These concentrations can be compared with in vivo tissue levels to conclude on the safety margin. The lessons from this case study will help to inform the design of other non-animal safety assessments.

A 10-step framework for use of read-across (RAX) in next generation risk assessment (NGRA) for cosmetics safety assessment.

This paper presents a 10-step read-across (RAX) framework for use in cases where a threshold of toxicological concern (TTC) approach to cosmetics safety assessment is not possible. RAX builds on established approaches that have existed for more than two decades using chemical properties and in silico toxicology predictions, by further substantiating hypotheses on toxicological similarity of substances, and integrating new approach methodologies (NAM) in the biological and kinetic domains. NAM include new types of data on biological observations from, for example, in vitro assays, toxicogenomics, metabolomics, receptor binding screens and uses physiologically-based kinetic (PBK) modelling to inform about systemic exposure. NAM data can help to substantiate a mode/mechanism of action (MoA), and if similar chemicals can be shown to work by a similar MoA, a next generation risk assessment (NGRA) may be performed with acceptable confidence for a data-poor target substance with no or inadequate safety data, based on RAX approaches using data-rich analogue(s), and taking account of potency or kinetic/dynamic differences.

New framework for a non-animal approach adequately assures the safety of cosmetic ingredients - A case study on caffeine.

This case study on the model substance caffeine demonstrates the viability of a 10-step read-across (RAX) framework in practice. New approach methodologies (NAM), including RAX and physiologically-based kinetic (PBK) modelling were used to assess the consumer safety of caffeine. Appropriate animal systemic toxicity data were used from the most relevant RAX analogue while assuming that no suitable animal toxicity data were available for caffeine. Based on structural similarities, three primary metabolites of the target chemical caffeine (theophylline, theobromine and paraxanthine) were selected as its most relevant analogues, to estimate a point of departure in order to support a next generation risk assessment (NGRA). On the basis of the pivotal mode of action (MOA) of caffeine and other methylxanthines, theophylline appeared to be the most potent and suitable analogue. A worst-case aggregate exposure assessment determined consumer exposure to caffeine from different sources, such as cosmetics and food/drinks. Using a PBK model to estimate human blood concentrations following exposure to caffeine, an acceptable Margin of Internal Exposure (MOIE) of 27-fold was derived on the basis of a RAX using theophylline animal data, which suggests that the NGRA approach for caffeine is sufficiently conservative to protect human health.

Demonstration of the first-pass metabolism in the skin of the hair dye, 4-amino-2-hydroxytoluene, using the Chip2 skin-liver microphysiological model.

We used TissUse's HUMIMIC Chip2 microfluidic model, incorporating reconstructed skin models and liver spheroids, to investigate the impact of consumer-relevant application scenarios on the metabolic fate of the hair dye, 4-amino-2-hydroxytoluene (AHT). After a single topical or systemic application of AHT to Chip2 models, medium was analysed for parent and metabolites over 5 days. The metabolic profile of a high dose (resulting in a circuit concentration of 100 µM based on 100% bioavailability) of AHT was the same after systemic and topical application to 96-well EpiDerm™ models. Additional experiments indicated that metabolic capacity of EpiDerm™ models were saturated at this dose. At 2.5 µM, concentrations of AHT and several of its metabolites differed between application routes. Topical application resulted in a higher Cmax and a 327% higher area under the curve (AUC) of N-acetyl-AHT, indicating a first-pass effect in the EpiDerm™ models. In accordance with in vivo observations, there was a concomitant decrease in the Cmax and AUC of AHT-O-sulphate after topical, compared with systemic application. A similar alteration in metabolite ratios was observed using a 24-well full-thickness skin model, EpiDermFT™, indicating that a first-pass effect was also possible to detect in a more complex model. In addition, washing the EpiDermFT™ after 30 min, thus reflecting consumer use, decreased the systemic exposure to AHT and its metabolites. In conclusion, the skin-liver Chip2 model can be used to (a) recapitulate the first-pass effect of the skin and alterations in the metabolite profile of AHT observed in vivo and (b) provide consumer-relevant data regarding leave-on/rinse-off products.

Use of Physiologically-Based Kinetics Modelling to Reliably Predict Internal Concentrations of the UV Filter, Homosalate, After Repeated Oral and Topical Application.

Ethical and legal considerations have led to increased use of non-animal methods to evaluate the safety of chemicals for human use. We describe the development and qualification of a physiologically-based kinetics (PBK) model for the cosmetic UV filter ingredient, homosalate, to support its safety without the need of generating further animal data. The intravenous (IV) rat PBK model, using PK-Sim®, was developed and validated using legacy in vivo data generated prior to the 2013 EU animal-testing ban. Input data included literature or predicted physicochemical and pharmacokinetic properties. The refined IV rat PBK model was subject to sensitivity analysis to identify homosalate-specific sensitive parameters impacting the prediction of Cmax (more sensitive than AUC(0-∞)). These were then considered, together with population modeling, to calculate the confidence interval (CI) 95% Cmax and AUC(0-∞). Final model parameters were established by visual inspection of the simulations and biological plausibility. The IV rat model was extrapolated to oral administration, and used to estimate internal exposures to doses tested in an oral repeated dose toxicity study. Next, a human PBK dermal model was developed using measured human in vitro ADME data and a module to represent the dermal route. Model performance was confirmed by comparing predicted and measured values from a US-FDA clinical trial (Identifier: NCT03582215, https://clinicaltrials.gov/). Final exposure estimations were obtained in a virtual population and considering the in vitro and input parameter uncertainty. This model was then used to estimate the Cmax and AUC(0-24 h) of homosalate according to consumer use in a sunscreen. The developed rat and human PBK models had a good biological basis and reproduced in vivo legacy rat and human clinical kinetics data. They also complied with the most recent WHO and OECD recommendations for assessing the confidence level. In conclusion, we have developed a PBK model which predicted reasonably well the internal exposure of homosalate according to different exposure scenarios with a medium to high level of confidence. In the absence of in vivo data, such human PBK models will be the heart of future completely non-animal risk assessments; therefore, valid approaches will be key in gaining their regulatory acceptance. Clinical Trial Registration: https://clinicaltrials.gov/, identifier, NCT03582215.

Metabolism and plasma protein binding of 16 straight- and branched-chain parabens in in vitro liver and skin models.

Parabens are alkyl esters of 4-hydroxybenzoic acid (4-HBA), with short-chain parabens used as antimicrobials in cosmetics. We investigated the impact of chain structure on skin and liver metabolism. Incubations with primary human hepatocytes and human liver S9 indicated that methyl-, ethyl-, propyl- and butylparaben were rapidly metabolized to similar metabolites, including 4-HBA plus the corresponding alcohols. Liver and EpiSkin™ S9 were used to investigate the metabolism of 16 short and long straight- and branched-chain parabens. The rate of hydrolysis generally decreased with increasing chain length in liver S9, whereas the reverse was true for EpiSkin™ S9. Chain length also correlated with the number of metabolites, with more oxidized metabolites detected from longer chain parabens. The identity of the alcohol group impacted metabolism the most, in terms of the rate of metabolism and the contribution of cofactors. The majority of parabens (13/16) exhibited high plasma protein binding (PPB) (>90%); whereas, 4-HBA PPB was 38%. PPB was related to the LogP of the parabens. In conclusion, the major and common paraben metabolite in PHH, liver S9 and EpiSkin™ S9 was 4-HBA. The rate of metabolism, type of metabolite and contribution of hydrolysis was tissue-specific (liver, skin) and was influenced by the chain length (and hence LogP), structural isomeric form (straight vs branched), and/or the identity of the alkyl group. SHORT ABSTRACT: We investigated how the chain structure of parabens affects their metabolism by liver and EpiSkin™ S9. The major and common metabolite in primary human hepatocytes, liver S9 and EpiSkin™ S9 was 4-HBA plus the corresponding alcohols. The rate of metabolism, type of metabolite and contribution of hydrolysis was tissue-specific and influenced by the chain length, structural isomeric form (straight vs branched), and/or the identity of the alkyl group. Most parabens exhibited high PPB (>90%), whereas the PPB of 4-HBA was 38%.

Characterization of application scenario-dependent pharmacokinetics and pharmacodynamic properties of permethrin and hyperforin in a dynamic skin and liver multi-organ-chip model.

Microphysiological systems (MPS) aim to mimic the dynamic microenvironment and the interaction between tissues. While MPS exist for investigating pharmaceuticals, the applicability of MPS for cosmetics ingredients is yet to be evaluated. The HUMIMIC Chip2 ("Chip2″), is the first multi-organ chip technology to incorporate skin models, allowing for the topical route to be tested. Therefore, we have used this model to analyze the impact of different exposure scenarios on the pharmacokinetics and pharmacodynamics of two topically exposed chemicals, hyperforin and permethrin. The Chip2 incorporated reconstructed human epidermis models (EpiDerm™) and HepaRG-stellate spheroids. Initial experiments using static incubations of single organoids helped determine the optimal dose. In the Chip2 studies, parent and metabolites were analyzed in the circuit over 5 days after application of single and repeated topical or systemic doses. The gene expression of relevant xenobiotic metabolizing enzymes in liver spheroids was measured to reflect toxicodynamics effects of the compounds in liver. The results show that 1) metabolic capacities of EpiDerm™ and liver spheroids were maintained over five days; 2) EpiDerm™ model barrier function remained intact; 3) repeated application of compounds resulted in higher concentrations of parent chemicals and most metabolites compared to single application; 4) compound-specific gene induction e.g. induction of CYP3A4 by hyperforin depended on the application route and frequency; 5) different routes of application influenced the systemic concentrations of both parents and metabolites in the chip over the course of the experiment; 6) there was excellent intra- and inter-lab reproducibility. For permethrin, a process similar to the excretion in a human in vivo study could be simulated which was remarkably comparable to the in vivo situation. These results support the use of the Chip2 model to provide information on parent and metabolite disposition that may be relevant to risk assessment of topically applied cosmetics ingredients.

Partition coefficient and diffusion coefficient determinations of 50 compounds in human intact skin, isolated skin layers and isolated stratum corneum lipids.

A standard protocol was used to determine partition (K) and diffusion (D) coefficients in dermatomed human skin and isolated human skin layers for 50 compounds relevant to cosmetics ingredients. K values were measured in dermatomed skin, isolated dermis, whole epidermis, intact stratum corneum (SC), delipidized SC and SC lipids by direct measurements of the radioactivity in the tissue layers/lipid component vs. buffer samples. D determinations were made in dermatomed skin, isolated dermis, whole epidermis and intact SC using a non-linear regression of the cumulative receptor fluid content of radiolabeled compound, fit to the solution of Fick's 2nd Law. Correlation analysis was completed between K, D, and physicochemical properties. The amount of interindividual (donor) and intraindividual (replicate) variability in the K and D data was characterized for each skin layer and chemical. These data can be further used to help inform the factors that influence skin bioavailability and to help improve in silico models of dermal penetration.

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.

Internal Threshold of Toxicological Concern (iTTC): Where We Are Today and What Is Possible in the Near Future.

The Threshold of Toxicological Concern (TTC) is a risk assessment tool for evaluating low-level exposure to chemicals with limited toxicological data. A next step in the ongoing development of TTC is to extend this concept further so that it can be applied to internal exposures. This refinement of TTC based on plasma concentrations, referred to as internal TTC (iTTC), attempts to convert the chemical-specific external NOAELs (in mg/kg/day) in the TTC database to an estimated internal exposure. A multi-stakeholder collaboration formed, with the aim of establishing an iTTC suitable for human safety risk assessment. Here, we discuss the advances and future directions for the iTTC project, including: (1) results from the systematic literature search for metabolism and pharmacokinetic data for the 1,251 chemicals in the iTTC database; (2) selection of ~350 chemicals that will be included in the final iTTC; (3) an overview of the in vitro caco-2 and in vitro hepatic metabolism studies currently being generated for the iTTC chemicals; (4) demonstrate how PBPK modeling is being utilized to convert a chemical-specific external NOAEL to an internal exposure; (5) perspective on the next steps in the iTTC project.

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.

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.