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

Abstract

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.

Combining a microphysiological system of three organ equivalents and transcriptomics to assess toxicological endpoints for cosmetic ingredients

“…The implications of our MPS model extend not only to the cosmetic industry, but also to food, pharmaceutical, and agrochemical industries, where it can play a vital role in the development of new ingredients to evaluate their toxicological potential, particularly when dealing with unpublished molecules lacking available data. 12,93 As future perspectives, additional tests using different reference substances that act through diverse biological mechanisms should be conducted to comprehensively evaluate the model's robustness. Furthermore, in vitro to in vivo extrapolation should be applied to determine human-relevant doses, aiming to establish a reliable point of departure that, through a weight of evidence approach, can be integrated into the risk assessment process to ensure consumer safety.…”

“…This shift reflects a broader trend away from traditional one-to-one animal test replacements towards more comprehensive, multifaceted testing approaches. 11,12 In this context, here we developed a three-organ MPS model comprising equivalents of the liver, intestinal barrier, and human reconstructed skin. Through the integration of gene expression analysis, we demonstrated the potential of this model for safety assessment of chemicals, suggesting a new assay which may be applied in industry for this purpose.…”

“…This shift reflects a broader trend away from traditional one-to-one animal test replacements towards more comprehensive, multifaceted testing approaches. 11,12…”

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

“…The Chip2 integrates liver organoids to mimic liver clearance, while daily medium exchanges can be considered (to some extent) as a surrogate for 'excretion'. More recent work has incorporated an in vitro model for a third organ, namely, the thyroid, which was used to evaluate the highest concentration of daidzein that could be topically applied in a body lotion without affecting the production of thyroid hormones (Tao et al, 2023). All experiments so far conducted have provided information on route-specific effects after single and repeated applications of test chemicals.…”

“…The integration of biological barriers in MPS also facilitates options for realistic exposure, that is, topical application of cosmetic ingredients or formulations on skin models. More realistic exposure partially integrates important parameters for cosmetic ingredients such as formulationdependent release of substances, evaporation, skin penetration and first-pass metabolism (Tao et al, 2021(Tao et al, , 2023.…”