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

Abstract

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 Epi-Skin™ 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.

Bioactivation of cinnamic alcohol in a reconstructed human epidermis model and evaluation of sensitizing potency of the identified metabolites

“…However, no correlation between in vitro skin sensitizing potential and degree of sulfate formation could be seen, indicating that the sulfate itself is not sensitizing. In the present study, the two in vitro systems tested that have the potential to form cinnamic sulfate—the human liver S9 fraction and RHE models—did not lead to the detection of this metabolite at any given time point, which is consistent with previous research findings ( Niklasson et al, 2014 ; Géniès et al, 2020 ). In this work, human S9 was used whereas Charpentier et al (2018) , in which cinnamic sulfate was detected, used rat S9.…”

“…There are two main studies available in which the bioactivation of cinnamic alcohol using RHE models has been investigated. The first study, conducted by Géniès et al (2020) , examined the formation of free metabolites and protein adducts after incubation of EpiSkin™ S9 subcellular fractions and in vitro human skin explants with 10 different compounds, including cinnamic alcohol. The authors only detected trace amounts of cinnamic acid in the RHE model, whereas multiple metabolites were detected in the skin explants, including hydroxycinnamic acid and cinnamic acid glucuronide.…”

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

“…Reliable and reproducible in vitro tools that adequately recapitulate the physiology and interactions of tissues are required to predict in vivo‐relevant systemic exposure, which is intrinsically accounted for in in vivo toxicity assays. To date, several in vitro bioavailability methods have been evaluated by the Cosmetics Europe ADME Task Force, including the measurement of penetration through human skin using a standardised protocol (Hewitt et al, 2020), metabolism in human skin explants (Géniès et al, 2019, 2020), as well as metabolism in S9 fractions from EpiSkin™ and human liver (Eilstein et al, 2020; Lester et al, 2020). Although each of the assays evaluated provides important information on the metabolism and bioavailability of topically applied chemicals, results from individual assays need to be combined in a modelling approach, for example, PBPK, to provide an estimation of systemic concentrations in humans.…”

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

“…Therefore, fit-for-purpose in vitro options are required to determine the metabolic fate and plasma/tissue distribution of an ingredient after application. The main route of exposure to cosmetics ingredients is via the skin; therefore, first-pass metabolism in the skin is relevant to ingredients as they penetrate the viable skin layers (Géniès et al, 2019(Géniès et al, , 2020, prior to liver metabolism after entering the systemic circulation. The metabolism and distribution kinetics of a compound in the body can be modelled using physiologically based kinetic (PBK) models (Moxon et al, 2020;Ouedraogo et al, 2022).…”