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.

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.

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.

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 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.

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.

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.

Skin Doctor: Machine Learning Models for Skin Sensitization Prediction that Provide Estimates and Indicators of Prediction Reliability.

The ability to predict the skin sensitization potential of small organic molecules is of high importance to the development and safe application of cosmetics, drugs and pesticides. One of the most widely accepted methods for predicting this hazard is the local lymph node assay (LLNA). The goal of this work was to develop in silico models for the prediction of the skin sensitization potential of small molecules that go beyond the state of the art, with larger LLNA data sets and, most importantly, a robust and intuitive definition of the applicability domain, paired with additional indicators of the reliability of predictions. We explored a large variety of molecular descriptors and fingerprints in combination with random forest and support vector machine classifiers. The most suitable models were tested on holdout data, on which they yielded competitive performance (Matthews correlation coefficients up to 0.52; accuracies up to 0.76; areas under the receiver operating characteristic curves up to 0.83). The most favorable models are available via a public web service that, in addition to predictions, provides assessments of the applicability domain and indicators of the reliability of the individual predictions.

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.

Gluteal and abdominal subcutaneous adipose tissue depots as stroma cell source: gluteal cells display increased adipogenic and osteogenic differentiation potentials.

Human adipose-derived stroma cells (ADSCs) have successfully been employed in explorative therapeutic studies. Current evidence suggests that ADSCs are unevenly distributed in subcutaneous adipose tissue; therefore, the anatomical origin of ADSCs may influence clinical outcomes. This study was designed to investigate proliferation and differentiation capacities of ADSCs from the gluteal and abdominal depot of 8 females. All had normal BMI (22.01 ± 0.39 kg/m(2) ) and waist circumference (81.13 ± 2.33 cm). Examination by physicians and analysis of 31 laboratory parameters did not reveal possibly confounding medical disorders. Gluteal and abdominal adipose tissue was sampled by en bloc resection on day 7 (±1) after the last menses. Histological examination did not reveal significant depot-specific differences. As assessed by BrdU assay, proliferation of cells from both depots was similar after 24 h and analysis of 15 cell surface markers by flow cytometry identified the isolated cells as ADSCs, again without depot-specific differences. ADSCs from both depots differentiated poorly to chondroblasts. Gluteal ADSCs displayed significantly higher adipogenic differentiation potential than abdominal cells. Osteogenic differentiation was most pronounced in gluteal cells, whereas differentiation of abdominal ADSCs was severely impaired. Our data demonstrate a depot-specific difference in ADSC differentiation potential with abdominal cells failing to meet the criteria of multipotent ADSCs. This finding should be taken into account in future explorations of ADSC-derived therapeutic strategies.

Next-generation risk assessment read-across case study: application of a 10-step framework to derive a safe concentration of daidzein in a body lotion.

Introduction: We performed an exposure-based Next Generation Risk Assessment case read-across study using New Approach Methodologies (NAMs) to determine the highest safe concentration of daidzein in a body lotion, based on its similarities with its structural analogue, genistein. Two assumptions were: (1) daidzein is a new chemical and its dietary intake omitted; (2) only in vitro data were used for daidzein, while in vitro and legacy in vivo data for genistein were considered. Methods: The 10-step tiered approach evaluating systemic toxicity included toxicokinetics NAMs: PBPK models and in vitro biokinetics measurements in cells used for toxicogenomics and toxicodynamic NAMs: pharmacology profiling (i.e., interaction with molecular targets), toxicogenomics and EATS assays (endocrine disruption endpoints). Whole body rat and human PBPK models were used to convert external doses of genistein to plasma concentrations and in vitro Points of Departure (PoD) to external doses. The PBPK human dermal module was refined using in vitro human skin metabolism and penetration data. Results: The most relevant endpoint for daidzein was from the ERα assay (Lowest Observed Effective Concentration was 100 ± 0.0 nM), which was converted to an in vitro PoD of 33 nM. After application of a safety factor of 3.3 for intra-individual variability, the safe concentration of daidzein was estimated to be 10 nM. This was extrapolated to an external dose of 0.5 µg/cm2 for a body lotion and face cream, equating to a concentration of 0.1%. Discussion: When in vitro PoD of 33 nM for daidzein was converted to an external oral dose in rats, the value correlated with the in vivo NOAEL. This increased confidence that the rat oral PBPK model provided accurate estimates of internal and external exposure and that the in vitro PoD was relevant in the safety assessment of both chemicals. When plasma concentrations estimated from applications of 0.1% and 0.02% daidzein were used to calculate bioactivity exposure ratios, values were >1, indicating a good margin between exposure and concentrations causing adverse effects. In conclusion, this case study highlights the use of NAMs in a 10-step tiered workflow to conclude that the highest safe concentration of daidzein in a body lotion is 0.1%.

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.

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.