# Induction of human cytochrome P450 enzyme activities by metabolism disrupting chemicals in the hepatic cell line HepaRG

Elodie Person, Nicolas J Cabaton, Laurie Urien, Marlène Z Lacroix, Jorke H

Kamstra, Louisa Mediouni, Barbara Kubickova, Agnès Jamin, Sylvie

Chevolleau, Sebastian Hoffmann, et al.

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## To cite this version:

Elodie Person, Nicolas J Cabaton, Laurie Urien, Marlène Z Lacroix, Jorke H Kamstra, et al. Induction of human cytochrome P450 enzyme activities by metabolism disrupting chemicals in the hepatic cell line HepaRG. Toxicology, 2026, Volume 519, pp.154300. 10.1016/j.tox.2025.154300. hal-05315562v2

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# Induction of human cytochrome P450 enzyme activities by metabolism disrupting chemicals in the hepatic cell line HepaRG

Elodie Person<sup>a, b</sup>, Nicolas J. Cabaton<sup>a, b</sup>, Laurie Urien<sup>a, b</sup>, Marlène Z. Lacroix<sup>b</sup>, Jorke H. Kamstra<sup>c, d</sup>, Louisa Mediouni<sup>a</sup>, Barbara Kubickova<sup>d, e</sup>, Agnès Jamin<sup>e, d</sup>, Sylvie Chevolleau<sup>a, f</sup>, Sebastian Hoffmann<sup>g, d</sup>, Juliette Legler<sup>c, d</sup>, Miriam N. Jacobs<sup>d, g</sup>, Daniel Zalko<sup>a, *, d</sup>

## ARTICLE INFO

Handling Editor: Mathieu Vinken

**Keywords:**

Cytochrome P450

Metabolism

Metabolism disrupting chemicals

Hepatic cells

NAMs

## ABSTRACT

Metabolism disrupting chemicals (MDCs) are a class of endocrine disrupting substances that promote metabolic changes leading to metabolic disorders in humans. Central to assessing their adverse effects is the need to better understand their modes of action (MoA). Cytochrome P450 (CYP) enzymes play a major role in xenobiotic metabolism, but also catalyse many endogenous metabolic reactions. Therefore, modulation of CYP functionality may impact homeostasis, contributing to adverse outcomes. At the functional level, alteration of the activity of human CYPs by MDCs largely remains unexplored. In this study we investigated the capability of six candidate MDCs, bisphenol A (BPA), perfluorooctanoic acid (PFOA), tributyltin (TBT), dichlorodiphenyldichloroethylene (p,p'-DDE), triclosan (TCS) and triphenylphosphate (TPP) to induce CYP1A2, CYP2B6 and CYP3A4 activities in the human hepatic HepaRG cell line. The CYP induction test method previously validated for pharmaceuticals was optimised and selected MDCs were tested in the context of the European Horizon 2020 GOLIATH project. Induction was revealed using a cocktail of CYP-selective probe substrates, followed by probe metabolite quantification by mass spectrometry. All MDCs except TCS induced CYP activities. PFOA, TBT, p,p'-DDE and TPP induced CYP1A2, TPP being the most potent inducer. BPA, PFOA, TBT and TPP induced CYP2B6, PFOA being the most potent inducer. BPA, PFOA, TBT, p,p'-DDE and TPP all induced CYP3A4, p,p'-DDE and BPA being the most potent inducers. These results highlight the capability of candidate MDCs to induce key CYP activities in a human relevant hepatic model, paving the way for a better understanding of MDCs mechanisms of action.

## 1. Introduction

Metabolism disrupting chemicals (MDCs) are substances, mostly of anthropogenic origin, that have the ability to promote metabolic changes that may contribute to the etiology of metabolic disorders in humans (Heindel et al., 2015), including obesity, type 2 diabetes, insulin resistance and metabolic dysfunction-associated steatotic liver disease.

The concept of metabolic disruption gradually emerged from studies carried out on endocrine disrupting chemicals (EDCs), which led researchers involved in this field to formalise the MDC hypothesis (Heindel et al., 2015; Lind et al., 2016). It is now acknowledged that the worldwide pandemic of obesity and metabolic disorders is not solely linked with genetic factors, sedentary lifestyle and nutritional factors (Heindel et al., 2023, 2017). Many chemicals have been demonstrated to

<footnote><sup>a</sup> Toxalim, Université de Toulouse, INRAE, ENVT, El-Purpan, Toulouse, France</footnote>

<footnote><sup>b</sup> Intheres, Université de Toulouse, INRAE, ENVT, Toulouse, France</footnote>

<footnote><sup>c</sup> Division of Toxicology, Department of Population Health Sciences, Faculty of Veterinary Medicine, Institute for Risk Assessment Sciences, Utrecht University, Utrecht, the Netherlands</footnote>

<footnote><sup>d</sup> Radiation, Chemical, Climate and Environmental Hazards (RCCE), Department of Toxicology, UK Health Security Agency (UKHSA), Harwell Science and Innovation Campus, Chilton OX110RQ, United Kingdom</footnote>

<footnote><sup>e</sup> Biopredic International, Parc d'activité de la Bretèche Bâtiment A4, Saint Grégoire 35760, France</footnote>

<footnote><sup>f</sup> MetaboHUB-MetaToul, National Infrastructure of Metabolomics and Fluxomics, Metatoul-AXIOM, Toulouse 31077, France</footnote>

<footnote><sup>g</sup> seh consulting + services, Paderborn 33106, Germany</footnote>

<footnote>* Correspondence to: Toxalim, 180 chemin de Tournefeuille, BP93173, Cedex 3, Toulouse 31027, France.
E-mail address: daniel.zalko@inrae.fr (D. Zalko).</footnote>
act as obesogens in animal studies, including diethylstilbestrol (DES), bisphenol A (BPA), dichlorodiphenyltrichloroethane (DDT), organotins, per- and polyfluoroalkyl substances (PFAS) as well as phthalates (reviewed by Heindel et al., 2015). Direct evidence for "obesogenic" effects in human are more difficult to assess, but several studies support this hypothesis (Heindel et al., 2022; Tang-Péronard et al., 2011; Valvi et al., 2014). Central to assessing the adverse effects of candidate MDCs is the need to better understand their mechanisms and modes of action (MoA). Moreover, new approach methodologies (NAMs) are required to characterise MDCs' MoA in human relevant *in vitro* models. In this context, major efforts have been undertaken at the international level to address the challenges linked with metabolic disruption, including within the EURION cluster of projects (European cluster to improve identification of endocrine disruptors, Horizon 2020, 2019–2024, https://eurion-cluster.eu/).

One key mechanism of metabolic disruption, that is difficult to assess and quantify *in vitro* and remains largely unexplored for candidate MDCs is their potential to modulate the functional activity of metabolizing enzymes.

Xenobiotic metabolizing enzymes (XMEs) catalyse key metabolic reactions, which mainly occur in the liver. Several activities are also expressed in different extra-hepatic tissues. XMEs constitute a pivotal system largely involved in the protection against exogenous compounds, through metabolic processes that generally promote xenobiotic detoxification and elimination. However, XMEs also catalyse numerous anabolic and catabolic physiological reactions essential to endogenous metabolism and signalling pathways, including energy metabolism and hormone metabolism (Nebert et al., 2013). In particular, phase I reactions encompass a broad panel of enzyme families catalysing reduction, hydrolysis, hydration, and oxidation reactions (Pearson and Wienkers, 2008). Although these reactions generally lead to xenobiotic detoxification, the formation of reactive metabolites may also occur, potentially leading to adverse effects. The human cytochrome P450 (CYP) enzyme superfamily includes 57 functional enzymes, with high catalytic versatility (Guengerich, 2006). These heme-containing enzymes are able to metabolise many chemical groups. In human liver, CYP-dependent xenobiotic biotransformation are primarily catalysed by enzymes from the CYP3A, CYP2C, CYP2D, CYP1A, CYP2E and CYP2B subfamilies (Guengerich, 2006). Many factors can influence CYP activities, including genetic factors, gender and age. In addition, some isoforms are constitutive, while others are inducible and therefore prone to a functional modulation of their activity by environmental factors. In this study, we focused on three inducible CYP isoforms: CYP1A2, CYP2B6 and CYP3A4.

CYP1A2 plays an important role in the biotransformation of xenobiotics, including drugs (analgesics, antipyretics, antipsychotics, anti-depressants, anti-inflammatory and cardiovascular drugs) and other substances of concern such as carcinogenic/mutagenic compounds (polycyclic aromatic hydrocarbons, aflatoxin B1) and various aromatic amines/amides. In parallel, CYP1A2 plays a major role in endogenous metabolism, with substrates including arachidonic acid, prostaglandins, oestrogens, melatonin and retinoic acid (Nebert and Dalton, 2006; Zanger and Schwab, 2013). CYP2B6 is expressed in human liver to a lesser extent, but is well known to contribute to the metabolism of various drugs (such as antidepressants, antiretrovirals, anaesthetics) as well as pesticides (chlorpyrifos, methoxychlor, diethyltoluamide) and other chemicals (Hodgson and Rose, 2007; Wang and Tompkins, 2008). In parallel, CYP2B6 plays a significant role in the endogenous metabolism of arachidonic acid, lauric acid, 17-β-estradiol, estrone, ethinyl-estradiol, and testosterone (16α- and 16β-hydroxylation) (Zanger and Klein, 2013). CYP3A4 is a key CYP isoform abundantly expressed in the liver. It metabolises a larger array of xenobiotics than any other CYP. CYP3A4 is involved in the biotransformation of over half of all drugs and xenobiotics (Guengerich, 1999; Pelkonen et al., 2008), but also takes part in the endogenous synthesis and metabolism of bile acids, steroid hormones, and vitamin D, amongst others.

CYP enzyme induction is a complex process that can involve different mechanisms (Hakkola et al., 2020; Preiss et al., 2021). Induction can result from *de novo* protein synthesis following receptor-mediated transcriptional activation, from an increased translation of mRNA, or from a better stabilisation of enzymes (Klaassen, 2013). In particular, CYP genes may be activated by xenobiotics, via interactions and complex cross-talk with specific ligand-activated nuclear receptors (NR) and/or transcriptional factors acting as "xenosensors", leading to the increase of CYP activity. The aryl hydrocarbon receptor (AhR) and two NRs, namely the constitutive androstane receptor (CAR) and the pregnant X receptor (PXR), are the major regulators of CYP1A2, CYP2B6 and CYP3A4, respectively. Determining the potential of xenobiotics to induce these key CYPs is therefore pivotal both from a pharmacological and toxicological point of view. Moreover, there is a growing awareness of the intersectional positioning of CYP metabolic activities, i.e. CYP being simultaneously involved in exogenous and endogenous metabolic reactions (Guengerich, 2017; Kretschmer and Baldwin, 2005). Thus, any modulation of CYP activities may impact (1) the metabolic fate and kinetics of exogenous substances metabolised by these CYPs, (2) the detoxification/bioactivation balance of these substances, and, (3) the efficiency of endogenous metabolic routes in which these CYP enzymes are involved. The latter is of utmost concern in the case of MDCs, since these metabolic pathways encompass numerous anabolic and catabolic reactions involved in energy and hormone metabolism. Any significant modulation of XMEs activity may have a notable impact on homeostasis, and should be carefully considered as a possible MoA of MDCs. Therefore, it is essential to accurately assess the impact of xenobiotics on the activity level of major CYP isoforms, using human relevant and validated *in vitro* approaches.

Methods allowing to assess CYP activity using specific probe substrates exist, but some of these methods are now standardised (Bernasconi et al., 2019). The most relevant and reliable test method proposed following a detailed review paper by the OECD (Jacobs et al., 2008; OECD, 2014) and successfully validated so far is the *in vitro* CYP enzyme induction test method. It was first validated with pharmaceutical chemicals to induce CYP1A2, CYP2B6 and CYP3A4 activities. This development was led by the European Union Reference Laboratory for Alternatives to Animal Testing (EURL ECVAM, 2014), followed by the publication of Bernasconi et al. (2019). The method is based on the use of the human hepatoma cell line HepaRG™. Among *in vitro* hepatic models, HepaRG cells are increasingly used in the field of toxicology as an alternative model to primary human hepatocytes (PHH) (Kanebratt and Andersson, 2008; Stanley and Wolf, 2022). HepaRG cells, derived from a female patient, are able to proliferate and differentiate into biliary-like and hepatocyte-like cells (Gripon et al., 2002). These metabolically competent cells have been shown to express a variety of nuclear receptors including AhR, PXR, CAR, PPARα (Peroxisome Proliferator-Activated Receptor alpha), as well as most of the major xenobiotic metabolizing enzymes (e.g. CYP, UGT, transporters). This *in vitro* system allows to investigate liver-related functions like metabolism, biotransformation pathways as well as the effects of xenobiotics on human liver (Aninat et al., 2006; Anthérieu et al., 2012; Gripon et al., 2002). Previous studies have demonstrated the relevance of using this cell model for CYP induction studies (Gerets et al., 2012) and the capability of the HepaRG cell line to predict the induction of CYP enzymes *in vivo* in humans (Kanebratt and Andersson, 2008). Following a comparison of HepaRG cells with PHH based on the assessment of ten pharmaceutical chemicals, HepaRG cells displayed a better predictivity than PHH as regards CYP induction results (EURL ECVAM, 2014).

In this study, we first tested the same set of ten pharmaceutical proficiency chemicals to assess the implementation of the CYP induction test method and demonstrate proficiency for testing, and compared our results with previous reports (Bernasconi et al., 2019; EURL ECVAM, 2014). Then, we tested the six model MDCs, with the aim to investigate their potential to induce major human CYP activities. This set of chemicals was selected by the GOLIATH consortium, a project which is
part of the European EURION cluster and focuses on metabolic disruption (Legler et al., 2020). The six model candidate MDCs assessed in this study are bisphenol A (BPA), perfluorooctanoic acid (PFOA), tributyltin chloride (TBT), dichlorodiphenyldichloroethylene (p,p'-DDE), triclosan (TCS) and triphenylphosphate (TPP). These chemicals are all candidate MDCs to which humans are exposed to through diet, air or skin, and that have raised questions with respect to their putative adverse effects in humans, related to metabolic disruption.

## 2. Materials and methods

### 2.1. Chemicals and reagents

Dimethyl sulfoxide (DMSO, reference D2650, ≥ 99.7 % purity), bisphenol A (239658, ≥ 99 %), tributyltin chloride (T50202, 96 %), perfluorooctanoic acid ammonium salt (77262, ≥ 98.0 %), triphenylphosphate (241288, ≥ 99 %), dichlorodiphenyldichloroethylene (123897, 99 %), triclosan (PHR1338, 99.9 %), omeprazole (19329, ≥ 98.0 %), carbamazepine (94496, ≥ 99.0 %), phenytoin sodium (PHR1492, ≥ 98.6 %), penicillin G sodium (P3032, ≥ 1477 units per milligram), (±)-sulfinpyrazone (S9509, ≥ 99 %), bosentan hydrate (SML1265, ≥ 98 %), artemisinin (361593, 98 %), metoprolol (COM448643953, 96 %), (±)-sotalol hydrochloride (39863, ≥ 98.0 %), rifampicin (R3501 ≥ 97 %), β-naphthoflavone (N3633, ≥ 98 %), phenacetin (77440, ≥ 98.0 %), bupropion hydrochloride (B102, ≥ 98 %), acetaminophen (A5000, 98.0-102.0 %), doxorubicin hydrochloride (D1515, 98.0-102.0 %), LiChropur for LC-MS formic acid, internal standards (±)-hydroxybupropion-D6 (H-062) and acetaminophen-D4 (P-909), phenobarbital sodium salt (P5178) and midazolam hydrochloride (1443602) with authorisation for purchase, detention and use of psychotropic substances under international control (N°A-2019-6-638-SP from Agence Nationale de Sécurité du Médicament et des produits de santé, ANSM, France) were purchased from Sigma-Aldrich Merck (Saint Quentin Fallavier, France). Hydroxybupropion (9000522, ≥ 98 %) and 1'-hydroxymidazolam (10385, ≥ 95 %) were from Bertin Bioreagent (Montigny-le-Bretonneux, France). Sodium hydroxide was purchased from VWR (Fontenay-sous-Bois, France), Ca²⁺ and Mg²⁺-free Dulbecco's phosphate-buffered saline

(DPBS) from PAN-Biotech (Aidenbach, Germany). Optima LC/MS-grade acetonitrile and LC-MS-grade methanol were from Thermo Fisher Scientific (Illkirch, France). Ultrapure water, produced using a Millipore Milli-Q system (Saint-Quentin-en-Yvelines, France), was used for the preparation of HPLC mobile phases.

The ten pharmaceutical proficiency chemicals tested were omeprazole, carbamazepine, phenytoin sodium, penicillin G sodium, sulfinpyrazone, bosentan hydrate, artemisinin, rifampicin, metoprolol and sotalol hydrochloride. This list includes chemicals recognised either as CYP inducers or as non-inducers in human (Bernasconi et al., 2019). The selected set of six MDCs is detailed in Table 1.

### 2.2. Cells and media

Cryopreserved HPR116 cells (HepaRG cells at their differentiated state at passage 16, HPR116-TA08 batches #284, #295, #305, for biological replicates) were provided by Biopredic International (Saint-Grégoire, France). Following provider's recommendations, media used were "HepaRG thaw, plate and general purpose medium with antibiotics" (basal medium MIL600C + supplement ADD670C), "HepaRG serum-free induction medium with antibiotics" (basal medium MIL600C + supplement ADD650C) from Biopredic International and incubation medium, consisting of Williams' E without phenol red supplemented with HEPES 1 M (25 mM) and L-glutamine 200 mM (2 mM) from Thermo Fisher Scientific.

### 2.3. Cell culture protocol

The Standard Operating Procedure (SOP) that was followed for this study was based on EURL ECVAM test method TM2009-14 (EURL ECVAM, 2009) and Bernasconi et al. (2019), with slight modifications and extension of its chemical applicability domain (Jacobs et al. in prep). Briefly, HPR116 cells were thawed and seeded in monolayer at 7.2 × 10⁴ viable cells per well in a type I collagen coated 96-well plate and maintained in a humidified incubator at 37 °C with 5 % CO₂. As recommended by the manufacturer, a cell viability after thawing above 80 % was required and confirmed by trypan blue exclusion test. Six hours after seeding, cell morphology was observed and "HepaRG thaw,

<table><caption>**Table 1**

Overview of the set of six Metabolism Disrupting Chemicals (MDCs).</caption><thead><tr><th>MDC</th><th>CAS</th><th>MW (g/mol)</th><th>Structure</th><th>Properties</th></tr></thead><tbody><tr><td>Bisphenol A (BPA)</td><td>80-05-7</td><td>228.29</td><td></td><td>Plasticizer, color developer</td></tr><tr><td>Perfluorooctanoic acid ammonium salt (PFOA)</td><td>3825-26-1</td><td>431.10</td><td></td><td>Non-stick agent, water and oil repellent</td></tr><tr><td>Tributyltin chloride (TBT)</td><td>1461-22-9</td><td>325.51</td><td></td><td>Biocide agent</td></tr><tr><td>Dichlorodiphenyldichloroethylene (p,p'-DDE)</td><td>72-55-9</td><td>318.03</td><td></td><td>Insecticide metabolite</td></tr><tr><td>Triclosan (TCS)</td><td>3380-34-5</td><td>289.54</td><td></td><td>Anti-bacterial agent</td></tr><tr><td>Triphenylphosphate (TPP)</td><td>115-86-6</td><td>326.28</td><td></td><td>Plasticizer, flame retardant</td></tr></tbody></table>
plate and general purpose medium" was renewed for further sixty-five hours at least, before starting the assay. The time spent in the laminar airflow hood was minimised, and 96-well plates were kept on a preheated surface during this period, to reduce temperature fluctuations.

## 2.4. Solubility and cytotoxicity assays

For the set of ten pharmaceutical proficiency chemicals, solubility and cytotoxicity assays were not performed, as concentration ranges were already provided (Bernasconi et al., 2019; EURL ECVAM, 2014). The highest concentrations assayed in serum-free induction medium were 40 µg/mL dissolved in DMSO (0.1 % v/v), except for phenytoin sodium (30 µg/mL dissolved in DMSO/H₂O (0.1 % v:1 v/v)). For the six GOLIATH chemicals, the solubility was determined as prescribed in the SOP by visual inspection in DMSO and in serum-free induction medium after centrifugation. The highest concentration was set at 40 mg/mL in DMSO and at 80 µg/mL in the medium. The highest soluble concentration was used as the starting concentration for cytotoxicity assessment. Cytotoxicity assays were carried out at eight concentrations using the CellTiter-Blue® Cell Viability Assay (Promega, WI, USA) according to the manufacturer's protocol and using three different cell batches, as prescribed in the SOP. This fluorescent method measures the ability of living hepatic cells to convert resazurin into resorufin. Doxorubicin at 8 µM was used as positive control. Following cytotoxicity experiments, the highest concentration producing a cellular viability higher than 90 % after 48 h of incubation was used as the highest concentration for cytochrome P450 enzyme induction assessment.

## 2.5. Cytochrome P450 induction test method

According to the SOP, two test chemicals were assessed per plate, at six concentrations with a dilution factor of 1:2, starting from the highest soluble and non-cytotoxic concentration. The marker reactions were phenacetin O-deethylation catalysed by CYP1A2, bupropion hydroxyl-ation catalysed by CYP2B6 and midazolam 1'-hydroxylation catalysed by CYP3A4. Reference positive inducers β-naphthoflavone (CYP1A2, 25 µM), phenobarbital (CYP2B6, 500 µM) and rifampicin (CYP3A, 10 µM), as well as solvent controls (100 µL/well) were assessed on every plate. The final solvent concentration was 0.1 % (v/v) in "HepaRG serum-free induction medium". The plate design included three technical replicates per condition. Outer wells were filled with DPBS. 72 h after thawing, the assay was initiated by the addition of chemicals in serum-free induction medium. Cells were cultured in serum-free conditions during the 48-hour treatment, reducing potential interferences and providing greater consistency. After 24 h of treatment, the treatment solutions were renewed and replaced by freshly prepared solutions for 24 h. At 48 h, exposure medium was removed, and all wells were washed twice with 100 µL incubation medium. A cocktail of three CYP specific probe substrates (phenacetin, bupropion and midazolam) was prepared in methanol in a tube, which was wrapped with aluminum foil since bupropion is light-sensitive and evaporated under a stream of nitrogen at room temperature. The dried residue was reconstituted in 6 mL of incubation medium and vortexed to obtain the following final concentrations: phenacetin 26 µM, bupropion 100 µM and midazolam 3 µM. Each plate column was treated with 50 µL of the substrate cocktail with the same time-interval between columns. Substrate incubation was carried out during 60 ± 3 min at 37 °C in a humidified incubator with 5 % CO₂. At the end of the incubation time, the reaction was quenched by stop solution containing (±)-hydroxybupropion-D6 (15 pg/µL) and acetaminophen-D4 (90 pg/µL) in acetonitrile. 40 µL of cell medium were transferred to and mixed with 40 µL of ice-cold stop solution in a stop solution 96-well plate placed on ice.

## 2.6. Sample preparation

Calibration standards and quality controls were independently

prepared by fortifying the incubation medium with intermediate solutions of probe metabolites in acetonitrile, as described in Supplementary Data Tables 1, 2 and 3. 40 µL of calibration standards, quality controls in matrix and blank and internal standard (ISTD) samples (incubation medium) were added into the outer wells of each stop solution plate and mixed with 40 µL stop solution at the same time as unknown samples, except for blank samples mixed with acetonitrile. This plate was centrifuged 10 min at 2200 x g, 4 °C. 30 µL of the particle-free supernatant was transferred into the corresponding wells of an analysis plate, thermo-sealed and stored at -20 °C for further LC-MS/MS analysis, which took place within a week. An example of an analysis plate is presented in Supplementary Data Fig. 1. After thawing for analysis, 70 µL H₂O was added and mixed directly in the plate (final acetonitrile content 15 % v/v in a total volume of 100 µL). Analysis plate was thermo-sealed and agitated for 20 s before introduction in a thermostated (nominal 10 °C) autosampler for further LC-MS/MS analysis.

## 2.7. LC-MS/MS analysis

Probe metabolites (i.e. acetaminophen, hydroxybupropion and 1'-hydroxymidazolam) were assayed using an U-HPLC system (Nexera LC40) coupled to a triple quadrupole (8045) mass spectrometer (Shimadzu, Kyoto, Japan). Metabolites were separated using a Phenomenex Synergi 2.5 µm Polar-RP 100 A 100 mm Ø 2.0 mm thermostated at 40 °C. The autosampler was maintained at 10 °C and the injection volume was 10 µL, from a 96-well plate covered with peelable heat sealing foil. Optimal separation was achieved with a binary mobile phase (solvent A: 0.1 % formic acid in water and solvent B: 0.1 % formic acid in methanol) at a flow rate of 0.35 mL/min. The gradient elution program was: 0-0.5 min, 30 % B; 0.5-2 min, 30-95 % B; 2-3.5 min, 95 % B; 3.5-3.6 min, 95-30 % B; 3.6-6 min, 30 % B. Metabolites were ionised with an ElectroSpray Ionisation source (ESI) in positive mode (ESI+). The source parameters were set at 3, 10 and 10 L/min for nebulising, heating and drying gases, respectively. Temperatures were set at 300 °C for the interface, 250 °C for the desolvation line (DL), and 400 °C for the heat block. Metabolites were detected by multiple reaction monitoring (MRM) using two transitions. Collision energies were optimised for each transition (quantification and confirmation) using the LabSolutions Connect software, and chromatographic data were monitored using the LabSolutions Insight software (software obtained from Shimadzu, Kyoto, Japan). Quality controls at three different concentration levels (low, medium and high, Supplementary Data Table 3), were included at the beginning, the middle and the end of each analytical run to ensure the integrity and quality of the data. Quantification was obtained using the peak-area ratio of the analyte to the respective deuterated internal standard: (±)-hydroxybupropion-D6 for hydroxybupropion and 1'-hydroxymidazolam, and acetaminophen-D4 for acetaminophen.

## 2.8. Protein content quantification

Residual incubation medium was aspirated off the cell plate and cells were lysed by the addition of 50 µL of 1 M sodium hydroxide and by mixing each column. The plate was covered and gently agitated for two hours at room temperature. Aliquots of the lysates were diluted with H₂O (1:20) in duplicate, vortexed and stored at -20 °C until further analysis for protein content using the micro-BCA protein assay kit (#23235, Thermo Fisher Scientific). Experiments were conducted as per the manufacturer's instructions, consisting of a 2h-incubation at 37 °C followed by plate reading at a wavelength of 562 nm.

## 2.9. Result processing

CYP activities were evaluated after protein content normalisation, and expressed in pmol of metabolite/min/mg of total protein. Each CYP enzyme activity fold-change was determined with respect to solvent controls. To validate the proper performance of the test system, positive
controls (reference chemicals) were run on each cell plate, concurrently with test chemicals. Chemicals demonstrating a ≥ 2-fold CYP enzyme activity induction in at least at two consecutive concentrations in a minimum of one out of three cell batches were considered to be inducers of the CYP isoform, here called 'positive'. Otherwise, chemicals were considered 'negative' (Bernasconi et al., 2019).

## 3. Results

We first describe the development of the LC-MS/MS method within the INRAE laboratory, to quantify probe metabolite formation, since this step was not part of the original SOP (EURL ECVAM, 2009). Next, we provide the results obtained for the ten pharmaceutical proficiency chemicals and the six MDCs assayed.

### 3.1. LC-MS/MS method

The development of a robust and reproducible validated LC-MS/MS method to quantify metabolite formation was a major prerequisite (Bernasconi et al., 2019), essential to conduct the CYP induction test method. We developed and optimised a LC-MS/MS method, for which mass spectrometry parameters and results are detailed in Table 2.

Samples were quantified using a linear model weighted by 1/X², with concentrations ranging from the lower limit of quantification (LLOQ) to 933 µg/L, 1167 µg/L and 1633 µg/L for hydroxybupropion, 1'-hydroxymidazolam and acetaminophen, respectively (Supplementary Data Table 3). The LLOQ were validated at 0.89, 1.11 and 1.56 µg/L in the cell incubation medium, for hydroxybupropion, 1'-hydroxymidazolam and acetaminophen, respectively. The accuracy of the method ranged from 93 % to 127 % for all probe metabolites; Whatever the concentration, the intra- and inter-day precision values were < 23 % and < 28 %, respectively. The method, including the sample extraction step, enabled to assay 200 samples per day. As we encountered stability issues while measuring acetaminophen formation in cell medium, we selected deuterated acetaminophen as an internal standard and proceeded to LC-MS/MS analysis within a week following the experiments.

### 3.2. CYP induction of positive controls

Positive controls (reference chemicals) induced the specific CYP isoform for which they were used as control substances in all the plates assessed. They demonstrated a > 2-fold CYP enzyme activity induction, with respect to solvent controls, validating the proper performance of the test system (Supplementary Data Fig. 2).

### 3.3. CYP enzyme induction of the ten pharmaceutical proficiency chemicals

A non-cytotoxic concentration range was tested for each chemical on three different cell batches. CYP activity fold-inductions were determined after protein content normalisation of enzyme activities. Results for the pharmaceutical proficiency chemicals are shown in Table 3 and further detailed in Supplementary Data Fig. 3.

Of note, the induction of CYP2B6 by omeprazole was previously excluded from proficiency testing due to the earlier report of a discrepancy between its predicted *in vitro* activity and observed clinical

in vivo evidence (EURL ECVAM, 2014). The overall between-batch reproducibility, i.e. concordance of batch results for each pharmaceutical proficiency chemical and CYP isoform, was 86 % (25/29). Greater cell batch reproducibility was observed for CYP1A2 (100 % between-batch reproducibility) and CYP3A4 (90 % between-batch reproducibility), compared to CYP2B6 induction (67 % between-batch reproducibility). Results were compared to the reference *in vitro*, according to Bernasconi et al. (2019) and validation results (EURL ECVAM, 2014). Classification was 100 % (10/10) concordant for CYP1A2 and CYP3A4 and 89 % (8/9) concordant for CYP2B6. Concordance with the validation ring trial results between 80 % and 100 % was considered acceptable for the demonstration of proficiency and was achieved for all three CYP isoforms. CYP2B6 induction was expected for bosentan hydrate (Bernasconi et al., 2019; EURL ECVAM, 2014), but was consistently negative in our study. Whilst some induction was observed at the lowest concentrations, the values did not reach the 2-fold induction threshold. We applied a 1:2 dilution factor, compliant with EURL ECVAM test method TM2009-14. In the case of CYP2B6's induction by bosentan hydrate, a dilution factor of 1:3 (as in Bernasconi et al., 2019) would likely have led, similarly, to a positive induction response. Altogether our results were found to be consistent with previous publications (Bernasconi et al., 2019, EURL ECVAM, 2014), demonstrating successful implementation of the test method.

### 3.4. Solubility and cytotoxicity assays

For the six MDCs, a full development was carried out, starting with the solubility assays. At the highest concentrations tested, solubility was assessed at 40 mg/mL in DMSO and 80 µg/mL in serum-free induction medium. After cytotoxicity experiments, concentration ranges were established for each chemical. Results are presented in Table 4. Of note, cytotoxic effects of TBT were observed at a lower concentration compared to other MDCs, which explains its lower test concentration range.

### 3.5. CYP enzyme induction of the six MDCs

A non-cytotoxic concentration range was tested for each MDC using the same three different cell batches used for proficiency testing. CYP activity fold-inductions were determined after protein content normalisation of enzyme activities. Concentration-response curves for the six MDCs are shown in Fig. 1, the classification results are provided in Table 5, and corresponding values are presented in Supplementary Data Table 4.

Out of the six MDCs tested, five were determined to induce several specific CYP isoforms. Only TCS was found to have no effect on any of the measured CYP isoforms (Fig. 1-e). In all cell batches CYP1A2 induction was < 1.6-fold, CYP2B6 induction was < 1.6-fold and CYP3A4 induction was < 1.8-fold. BPA induced CYP2B6 and CYP3A4 activities (Fig. 1-a). At the highest concentration tested, the induction was about 3-fold, 5-fold and 9-fold for CYP1A2, 2B6 and 3A4 activities, respectively. An increase of CYP1A2 activity was only observed at the highest concentration tested (13.3 µg/mL; 58.4 µM). However, the criteria for an overall positive test were not met in this case (only a single concentration delivered a positive response in the three cell batches), leading to conclude that BPA is not an inducer of the CYP1A2 activity *in vitro*. PFOA, TBT and TPP induced the three CYP activities according to

<table><caption>**Table 2**
Mass spectrometry parameters for the detection of probe substance metabolites.</caption><thead><tr><th rowspan="2">Metabolite</th><th rowspan="2">Precursor ion (m/z)</th><th colspan="2">Product ions (m/z)</th><th rowspan="2">Dwell (ms)</th><th colspan="2">Collision energy (V)</th><th rowspan="2">Retention time (min)</th></tr><tr><th>Quantification</th><th>Confirmation</th><th>Quantification</th><th>Confirmation</th></tr></thead><tbody><tr><td>Acetaminophen</td><td>152.2</td><td>110</td><td>65.05 / 93.05</td><td>22</td><td>-18</td><td>-31/-23</td><td>1.47</td></tr><tr><td>Hydroxybupropion</td><td>256.2</td><td>139.05</td><td>238.1 / 131.1</td><td>22</td><td>-26</td><td>-13/-27</td><td>2.45</td></tr><tr><td>1'-Hydroxymidazolam</td><td>342.2</td><td>324.1</td><td>203.1 / 168.15</td><td>22</td><td>-20</td><td>-27/-28</td><td>2.91</td></tr></tbody></table>
<caption>**Table 3**</caption>

CYP induction results for the ten pharmaceutical proficiency chemicals, three cell batches. P = Positive; N = Negative. *: excluded from proficiency testing. For the three independent cell batches assayed, “Positive” (P) is mentioned if the chemical was found to be an inducer and “Negative” (N) if the chemical was not found to be an inducer of this isoform. The summary result across the three batches was ‘Positive’ (P) when at least one batch was ‘Positive’.

<table><caption>**Table 4**</caption><thead><tr><th rowspan="2">Chemical</th><th colspan="4">CYP1A2 induction</th><th colspan="4">CYP2B6 induction</th><th colspan="4">CYP3A4 induction</th></tr><tr><th>Batch 1</th><th>Batch 2</th><th>Batch 3</th><th>Result</th><th>Batch 1</th><th>Batch 2</th><th>Batch 3</th><th>Result</th><th>Batch 1</th><th>Batch 2</th><th>Batch 3</th><th>Result</th></tr></thead><tbody><tr><td>Omeprazole</td><td>P</td><td>P</td><td>P</td><td>P</td><td>P</td><td>P</td><td>P</td><td>P*</td><td>N</td><td>N</td><td>N</td><td>N</td></tr><tr><td>Carbamazepine</td><td>P</td><td>P</td><td>P</td><td>P</td><td>P</td><td>P</td><td>P</td><td>P</td><td>N</td><td>P</td><td>P</td><td>P</td></tr><tr><td>Phenytoin sodium</td><td>P</td><td>P</td><td>P</td><td>P</td><td>P</td><td>P</td><td>P</td><td>P</td><td>P</td><td>P</td><td>P</td><td>P</td></tr><tr><td>Penicillin G sodium</td><td>N</td><td>N</td><td>N</td><td>N</td><td>N</td><td>N</td><td>N</td><td>N</td><td>N</td><td>N</td><td>N</td><td>N</td></tr><tr><td>Sulfapyrazone</td><td>P</td><td>P</td><td>P</td><td>P</td><td>P</td><td>P</td><td>N</td><td>P</td><td>P</td><td>P</td><td>P</td><td>P</td></tr><tr><td>Bosentan hydrate</td><td>P</td><td>P</td><td>P</td><td>P</td><td>N</td><td>N</td><td>N</td><td>N</td><td>P</td><td>P</td><td>P</td><td>P</td></tr><tr><td>Artemisinin</td><td>N</td><td>N</td><td>N</td><td>N</td><td>P</td><td>N</td><td>P</td><td>P</td><td>N</td><td>N</td><td>N</td><td>N</td></tr><tr><td>Rifampicin</td><td>P</td><td>P</td><td>P</td><td>P</td><td>N</td><td>N</td><td>P</td><td>P</td><td>P</td><td>P</td><td>P</td><td>P</td></tr><tr><td>Metoprolol</td><td>N</td><td>N</td><td>N</td><td>N</td><td>N</td><td>N</td><td>N</td><td>N</td><td>N</td><td>N</td><td>N</td><td>N</td></tr><tr><td>Sotalol hydrochloride</td><td>N</td><td>N</td><td>N</td><td>N</td><td>N</td><td>N</td><td>N</td><td>N</td><td>N</td><td>N</td><td>N</td><td>N</td></tr></tbody></table>

Solubility results and non-cytotoxic concentration ranges for the six MDCs.

<table><thead><tr><th rowspan="2">MDC</th><th colspan="2">Solubility in DMSO</th><th colspan="2">Solubility in medium</th><th colspan="2">Test concentration range (in medium)</th><th rowspan="2">Final solvent concentration</th></tr><tr><th>mg/mL</th><th>mM</th><th>µg/mL</th><th>µM</th><th>µg/mL</th><th>µM</th></tr></thead><tbody><tr><td>BPA</td><td>40</td><td>175.2</td><td>80</td><td>350.4</td><td>0.42–13.3</td><td>1.8–58.4</td><td>0.1 % DMSO</td></tr><tr><td>PFOA</td><td>40</td><td>92.8</td><td>80</td><td>185.6</td><td>1.25–40</td><td>2.9–92.8</td><td>0.1 % DMSO</td></tr><tr><td>TBT</td><td>40</td><td>122.9</td><td>80</td><td>245.8</td><td>0.023–0.74</td><td>0.071–2.3</td><td>0.1 % DMSO</td></tr><tr><td>p,p'-DDE</td><td>40</td><td>125.8</td><td>80</td><td>251.5</td><td>1.25–40</td><td>3.9–126</td><td>0.1 % DMSO</td></tr><tr><td>TCS</td><td>40</td><td>138.2</td><td>80</td><td>276.3</td><td>0.42–13.3</td><td>1.4–45.9</td><td>0.1 % DMSO</td></tr><tr><td>TPP</td><td>40</td><td>122.6</td><td>80</td><td>245.2</td><td>1.25–40</td><td>3.8–123</td><td>0.1 % DMSO</td></tr></tbody></table>

<figure><img src="image_3.png" /><figcaption>**Fig. 1.** CYP induction graphs for the six MDCs (n = 3 cell batches). (a) BPA, (b) PFOA, (c) TBT, (d) p,p'-DDE, (e) TCS, (f) TPP. Concentration-response curves (mean ± SD), GraphPad Prism software V10.</figcaption></figure>

the criteria of the test, i.e. demonstrating a ≥ 2-fold CYP enzyme activity induction at least at two consecutive concentrations in a minimum of one out of three cell batches. PFOA demonstrated an increasing concentration-dependent induction for all tested CYPs (Fig. 1-b). At the

highest concentrations tested, the induction was about 2-fold, 5-fold and 3-fold for CYP1A2, 2B6 and 3A4 activities, respectively. TBT also induced all CYP activities (Fig. 1-c). The highest induction values reached a level of 2.2-fold, 3.8-fold and 3.6-fold for CYP1A2, 2B6 and
<caption>**Table 5**</caption>

CYP induction results for the six MDCs, three cell batches. P = Positive; N = Negative. The overall result was positive when at least one positive was obtained.

<table><thead><tr><th rowspan="2">MDC</th><th colspan="4">CYP1A2 induction</th><th colspan="4">CYP2B6 induction</th><th colspan="4">CYP3A4 induction</th></tr><tr><th>Batch 1</th><th>Batch 2</th><th>Batch 3</th><th>Result</th><th>Batch 1</th><th>Batch 2</th><th>Batch 3</th><th>Result</th><th>Batch 1</th><th>Batch 2</th><th>Batch 3</th><th>Result</th></tr></thead><tbody><tr><td>BPA</td><td>N</td><td>N</td><td>N</td><td>N</td><td>P</td><td>P</td><td>P</td><td>P</td><td>P</td><td>P</td><td>P</td><td>P</td></tr><tr><td>PFOA</td><td>N</td><td>N</td><td>P</td><td>P</td><td>P</td><td>P</td><td>P</td><td>P</td><td>P</td><td>P</td><td>P</td><td>P</td></tr><tr><td>TBT</td><td>P</td><td>P</td><td>N</td><td>P</td><td>N</td><td>P</td><td>P</td><td>P</td><td>N</td><td>P</td><td>P</td><td>P</td></tr><tr><td>p,p'-DDE</td><td>P</td><td>P</td><td>P</td><td>P</td><td>N</td><td>N</td><td>N</td><td>N</td><td>P</td><td>P</td><td>P</td><td>P</td></tr><tr><td>TCS</td><td>N</td><td>N</td><td>N</td><td>N</td><td>N</td><td>N</td><td>N</td><td>N</td><td>N</td><td>N</td><td>N</td><td>N</td></tr><tr><td>TPP</td><td>N</td><td>P</td><td>P</td><td>P</td><td>P</td><td>N</td><td>P</td><td>P</td><td>P</td><td>N</td><td>P</td><td>P</td></tr></tbody></table>

3A4 activities at 1.14 µM, respectively. For all activities a drop was observed at the highest concentration (2.7 µM). p,p'-DDE induced CYP1A2 and CYP3A4 activities, with a marked concentration-dependent increase (Fig. 1-d). At the two highest concentrations tested, the induction was about 3-fold, 0.6-fold and 7-fold for CYP1A2, 2B6 and 3A4 activities, respectively. For TPP, the observed induction profiles were clearly different as regards CYP1A2 and CYP3A4 activities on the one hand, and the CYP2B6 activity on the other hand (Fig. 1-f). The latter was found to reach a peak around 15 µM, but decreased at higher concentrations. The highest induction values reached a level of 4.1-fold, 2.6-fold and 3.0-fold for CYP1A2, 2B6 and 3A4 activities at 123 µM, 15.3 µM and 30.6 µM, respectively.

We further provide F2 values for each of these chemicals, i.e., the concentrations resulting in a 2-fold induction of the considered CYP enzyme activity in at least one cell batch (Kanebratt and Andersson, 2008). These are reported for the six MDCs in Table 6.

In addition to the 2-fold induction criteria for the readout of the test, we further categorised the induction potential of the tested MDCs on a preliminary basis, pending the additional chemical augmentation data generated in further multilaboratory studies (Jacobs et al. in prep; Quartermain et al. in prep). CYP induction data were categorised according to the fold-induction values obtained in HPR116 cells, using the following potency categories: "not an inducer", "uncertain", "weak", "moderate" or "strong" inducer, by selecting induction thresholds (two additional thresholds set at 3- and 4.5-fold change, to be consistent with those proposed by Jacobs et al. 2022), as detailed in Table 7. For the CYP1A2 activity, TPP was found to be a "strong" inducer and p,p'-DDE a "moderate" inducer. PFOA and TBT, both positive, were "weak" inducers, BPA was classified as an "uncertain" inducer and TCS had no effect. Regarding the CYP2B6 activity, PFOA was found to be a "strong" inducer, TPP and BPA were classified as "moderate" inducers, and TBT as a "weak" inducer. This activity was not modulated by p,p'-DDE nor by TCS. For the CYP3A4 activity, p,p'-DDE and TPP were found to be "strong" inducers. BPA, PFOA and TBT were classified as "moderate" inducers of the CYP3A4 activity and TCS had no effect.

## 4. Discussion

The liver plays a pivotal role in the biotransformation and elimination of xenobiotics, including natural compounds (e.g. toxins, phytohormones, combustion by-products, etc.), drugs and other substances of anthropogenic origin (e.g. plasticizers, biocides, surfactants, flame retardants, etc.).

<caption>**Table 6**</caption>

F2 values: concentrations resulting in a 2-fold CYP enzyme activity induction, in at least one HPR116 cell batch. N.A.: not applicable (no induction).

<table><caption>**Table 7**</caption><thead><tr><th rowspan="2">MDC</th><th colspan="3">F2 values (µM)</th></tr><tr><th>CYP1A2</th><th>CYP2B6</th><th>CYP3A4</th></tr></thead><tbody><tr><td>BPA</td><td>58.4</td><td>29.2-58.4</td><td>29.2-58.4</td></tr><tr><td>PFOA</td><td>23.2-92.8</td><td>2.90-92.8</td><td>23.2-92.8</td></tr><tr><td>TBT</td><td>0.568-2.27</td><td>0.284-1.14</td><td>0.568-1.14</td></tr><tr><td>p,p'-DDE</td><td>15.7-126</td><td>N.A.</td><td>15.7-126</td></tr><tr><td>TCS</td><td>N.A.</td><td>N.A.</td><td>N.A.</td></tr><tr><td>TPP</td><td>7.66-123</td><td>3.83-15.3</td><td>7.66-123</td></tr></tbody></table>

CYP induction results for the six MDCs. N: not an inducer; ~: uncertain inducer: one concentration generating a response ≥ 2-fold induction in at least one cell batch; +: weak inducer (2 consecutive concentrations generating a response ≥ 2 to ≤ 3-fold induction in at least one cell batch); ++: moderate inducer (2 consecutive concentrations generating a response > 3 to ≤ 4.5-fold induction in at least one cell batch); +++: strong inducer (2 consecutive concentrations generating a response > 4.5-fold induction in at least one cell batch).

<table><thead><tr><th rowspan="2">MDC</th><th colspan="3">Enzyme induction</th></tr><tr><th>CYP1A2</th><th>CYP2B6</th><th>CYP3A4</th></tr></thead><tbody><tr><td>BPA</td><td>~</td><td>++</td><td>++</td></tr><tr><td>PFOA</td><td>+</td><td>+++</td><td>++</td></tr><tr><td>TBT</td><td>+</td><td>+</td><td>++</td></tr><tr><td>p,p'-DDE</td><td>++</td><td>N</td><td>+++</td></tr><tr><td>TCS</td><td>N</td><td>N</td><td>N</td></tr><tr><td>TPP</td><td>+++</td><td>++</td><td>+++</td></tr></tbody></table>

The data generated from the human HepaRG *in vitro* CYP enzyme induction test method applied here, contributes to a better understanding of the molecular mechanisms and mode(s) of action underlying EDC effects on metabolic activities. The chemicals we tested are also candidate MDCs (BPA, PFOA, TBT, p,p'-DDE, TPP and TCS), and were demonstrated to have the capability to induce key CYP enzyme activities (CYP1A2, CYP2B6 and CYP3A4), in the way that the OECD Validation Management Group-Non Animal were calling for over 12 years ago (Jacobs et al., 2013).

The CYP induction *in vitro* test method was originally developed and standardised with the intention of assessing the induction of major human CYP activities, to support regulatory purposes and human relevance needs (EURL ECVAM, 2014). Among other advantages, it opens the possibility to run high throughput screening studies, and will support the reduction of animal testing, which also suffers translational interpretation due to inter-species variation in CYP expression and regulation (Lehmann et al., 1998). Indeed, its development may contribute to support the phasing out of animal testing for chemical safety assessments (e.g. in the European Union, Walder et al., 2025). The test methodology is based upon the monitoring of the metabolites of probe substrates phenacetin (for CYP1A2), bupropion (for CYP2B6) and midazolam (for CYP3A4), all of which were recommended for their high selectivity in the recent ICH guideline on drug interaction studies M12 (ICH Harmonised guideline, 2024). It enables the simultaneous measurement of CYP activities using a single analytical LC-MS/MS run. This test method relies on the use of HPR116 cells (differentiated HepaRG cells), a metabolically competent human model representing an alternative to primary human hepatocytes, that overcomes the problems retardants, etc.). Liver cells express a large panel of XMEs, many of which are inducible by xenobiotics (Hakkola et al., 2020). The intrinsic adaptive nature of the XME system aims at enabling an effective response to xenobiotic insult, by adapting the capability of an organism to metabolise potentially harmful substances. However, a lasting modulation of the functionality of XMEs, and notably of key CYP enzymes, may also impact general metabolism, since many of these enzymes catalyse biochemical reactions that contribute to endogenous metabolic pathways and homeostasis (Nebert et al., 2013).

The CYP induction *in vitro* test method was originally developed and standardised with the intention of assessing the induction of major human CYP activities, to support regulatory purposes and human relevance needs (EURL ECVAM, 2014). Among other advantages, it opens the possibility to run high throughput screening studies, and will support the reduction of animal testing, which also suffers translational interpretation due to inter-species variation in CYP expression and regulation (Lehmann et al., 1998). Indeed, its development may contribute to support the phasing out of animal testing for chemical safety assessments (e.g. in the European Union, Walder et al., 2025). The test methodology is based upon the monitoring of the metabolites of probe substrates phenacetin (for CYP1A2), bupropion (for CYP2B6) and midazolam (for CYP3A4), all of which were recommended for their high selectivity in the recent ICH guideline on drug interaction studies M12 (ICH Harmonised guideline, 2024). It enables the simultaneous measurement of CYP activities using a single analytical LC-MS/MS run. This test method relies on the use of HPR116 cells (differentiated HepaRG cells), a metabolically competent human model representing an alternative to primary human hepatocytes, that overcomes the problems retardants, etc.). Liver cells express a large panel of XMEs, many of which are inducible by xenobiotics (Hakkola et al., 2020). The intrinsic adaptive nature of the XME system aims at enabling an effective response to xenobiotic insult, by adapting the capability of an organism to metabolise potentially harmful substances. However, a lasting modulation of the functionality of XMEs, and notably of key CYP enzymes, may also impact general metabolism, since many of these enzymes catalyse biochemical reactions that contribute to endogenous metabolic pathways and homeostasis (Nebert et al., 2013).
associated with the availability and variability of human hepatic tissue. As a cell model was used, it should be noted that tissue architecture and homeostatic regulations are not similar to intact whole organism *in vivo* conditions, and that intraspecies variability is not taken into account.

The CYP induction test method assesses specific Phase I enzyme activities at the functional level. Alternatively, such exploration can be carried out *in vitro* based on the quantification of mRNAs (transcriptional level) or on peptide quantification or immunoblotting (protein level). Some studies have demonstrated discrepancies between these different approaches (Abass et al., 2012; Choi et al., 2013; Fujita et al., 2024; Sumida et al., 1999; Takahashi et al., 2021). Despite this, at the functional level, it cannot be ruled out, that a concomitant inhibition may occur and partly mask enzyme induction (ICH Harmonised guideline, 2024), only functional approaches can take into account factors such as post-translational regulatory mechanisms.

The CYP induction test method was previously demonstrated to provide a robust *in vitro* tool to characterise CYP activities induction using a selection of 10 pharmaceutical proficiency chemicals (Bernasconi et al., 2019). In the current study, we first assessed the same list of pharmaceutical proficiency chemicals, with largely concordant results. On this basis, the test method was extended, within the EU Horizon 2020 GOLIATH project, to six candidate MDCs, whose selection was guided by the availability of human biomonitoring data as well as positive evidence from epidemiological studies documenting an association between exposure and the occurrence of metabolic disorders (reviewed by Legler et al., 2020). All MDCs except TCS were demonstrated to induce the activity of at least one CYP isoform. Following 48 h exposure, PFOA, TBT and TPP significantly induced all three tested CYP activities in HPR116 cells. Both BPA and p,p'-DDE induced the CYP3A4 activity, in addition to the CYP2B6 activity (BPA) or the CYP1A2 activity (p,p'-DDE). The most potent CYP1A2 inducer was TPP, with a n-fold induction above 4, at 123 µM. The most potent CYP2B6 inducer was PFOA, with a n-fold induction above 5, at 93 µM and 46 µM, and the most potent CYP3A4 inducers were p,p'-DDE (n-fold induction above 7, at 63 µM), TPP (n-fold induction above 4, at 31 µM and 61 µM in one cell batch) and BPA (n-fold induction above 9, at 58 µM).

The appropriateness of using a 2-fold threshold (relative to solvent control) to assess the induction of CYP activities in HepaRG cells was first demonstrated by Kanebratt and Andersson (2008). Within the frame of the CYP induction method, this threshold should be reached for at least two consecutive concentrations, ensuring that a concentration-dependent CYP induction occurs, to reduce the risk of false positives. This data interpretation rule results in a positive/-negative readout (Bernasconi et al., 2019; EURL ECVAM, 2009). We suggest an additional classification of CYP induction results according to the fold induction response, based on the use of two additional thresholds set at 3- and 4.5-fold change, as proposed by Jacobs et al. (2022), resulting in the classification of positive chemicals as "weak", "moderate" and "strong" inducers. For further comparison purposes, quantitative information about the concentrations necessary to produce a 2-fold CYP induction (F2 values) were also provided.

Studies documenting the functional modulation of CYP activities in human *in vivo* are scarce, especially as regards environmental pollutants (compared to drugs). For candidate MDCs, few *in vitro* human data are available and models as well as approaches do vary, illustrating the need to develop and standardise robust methods. BPA is a model endocrine disruptor (Beausoleil et al., 2018). Its capability to modulate XMEs activities in human and animal models was reviewed by Quesnot et al. (2014). Notably, BPA was reported to induce CYP3A4 activity via PXR in human hepatoma DPX2 cells exposed to 5–50 µM during 24 h (Kuzbari et al., 2013). Despite the differences between models (DPX2 are HepG2-derived cells), our results using HepaRG cells are fully consistent with this study, with induction observed around 30 µM but already commencing at lower concentrations. PFOA is a widespread perfluoroalkyl substance (PFAS) with endocrine disrupting properties (Mokra, 2021). Several studies have reported significant gene

expression upregulation for *Cyp3a4*, *Cyp4a11*, *Cyp2b6* and *Cyp4x1* in HepaRG cells consecutive to PFOA treatment (Behr et al., 2020; Louisse et al., 2020; Murase et al., 2023). Our results support these findings at the functional level, with PFOA assessed as a "moderate" inducer of the CYP3A4 activity, in addition to being a "strong" inducer of the CYP2B6 activity. p,p'-DDE is the main metabolite of DDT. It is an extremely persistent chemical characterised by marked endocrine disrupting properties (Burgos-Aceves et al., 2021) and was hypothesised to be obesogenic in humans (Cano-Sancho et al., 2017). Using the CYP induction method, p,p'-DDE was identified in this study as a "strong" and "moderate" inducer of the CYP3A4 and CYP1A2 activities, respectively. The mechanisms underlying the disruption of steroid hormone biotransformation through the induction of hepatic XMEs by xenobiotics, including p,p'-DDE, have previously been reviewed by You (2004). Although most existing evidence documenting CYP activities induction by p,p'-DDE have been obtained in animals (You, 2004), it should be stressed that a positive association between p,p'-DDE exposure and the induction of CYP3A4 activity was reported in healthy Faroese men (Petersen et al., 2007). TCS is used in personal care products and its endocrine disrupting properties have been documented (Milanović et al., 2023). However, this chemical did not induce any of the tested CYP activities in our study. The hypothesis that TCS would be quickly metabolised by HepaRG cells into metabolites lacking any modulatory effect on CYPs functionality cannot be ruled out, as this method does not provide information on the number or nature of possible (reactive/non-reactive) metabolites. Still, it should be mentioned that in the only clinical study examining the question in human *in vivo* (following exposure to TCS via toothpaste) no effect of this chemical on CYP3A4 activity was observed (Allmyr et al., 2009), consistent with the results returned by the CYP induction test method.

To our knowledge, no data was published so far as regards the effects of TPP on CYP activities, although this chemical, widely used as a plasticizer and flame retardant, is classified as a suspected MDC *via* PPARγ (Peroxisome Proliferator-Activated Receptor gamma) signaling (Beausoleil et al., 2024). TPP was found to be one of the most potent CYP inducers assayed in this study. It was classified as a strong inducer of both the CYP1A2 and the CYP3A4 activities, and as a moderate inducer of the CYP2B6 activity, for which induction was observed at the lowest concentrations tested (3.8–15.3 µM). The apparent lack of induction of CYP2B6 activity at higher TPP concentrations might rely on concomitant inhibitory mechanisms. Such question may be solved by measuring CYP2B6 mRNA levels in exposed cells. TBT is a highly toxic model MDC and one of the earliest substances demonstrated to be an environmental obesogen (Grün and Blumberg, 2006). With the exception of one study suggesting TBT's concentration-dependent induction of CYP3A4 in Atlantic salmon (Mortensen and Arukwe, 2007), no data has been published so far as regards its modulatory effects on XMEs. TBT was found to induce the three CYP activities assessed, and a drop of all activities was observed at the highest non-cytotoxic concentration tested (2.3 µM). Nebbia et al. (1999) reported that organotin compounds, especially triphenyltin, can generate a concentration-dependent *in vitro* inactivation of CYPs functionality via interaction with critical sulfhydryl groups of the hemoprotein (Nebbia et al., 1999). Mechanisms of action leading to these enzymatic down regulations at the highest TBT concentration remain to be further elucidated.

In the context of Adverse Outcome Pathways (AOPs), the results reported herein support mechanistic data generation at the functional Phase I metabolic level, i.e., integrating molecular-level activity beyond nuclear receptor activation (e.g., AhR, PXR, or CAR, which are major upstream regulators for the herein studied inducible CYP isoforms) (Hakkola et al., 2018). Hence, we propose CYP enzyme activity induction as an early molecular key event with relevance in AOPs for at least endocrine and metabolic disruption, due to their potential impact on hormone balance and (energy) homeostasis via modulating endogenous metabolism. Indeed, CYP activities, and notably the key enzymes targeted by the CYP induction test method, are involved in numerous
exogenous and endogenous pathways. The latter is of utmost relevance in the case of MDCs, with CYPs playing a major role in the metabolism of steroid hormones, fat-soluble vitamins, and free fatty acids, among others (Amacher, 2010; Hakkola et al., 2018; Nebert et al., 2013). Accordingly, broad functions such as hepatic energy metabolism, alongside thyroid function and steroid-regulated pathways may be affected by CYP induction, with far-reaching consequences on homeostasis, but also on development and reproduction. Exposure to CYP inducers is known to increase the rate of steroid hormone oxidation by CYP enzymes (reviewed by Jacobs et al., 2013; Klaassen, 2013). An impaired redox balance resulting in oxidative stress is one of the major mechanistic hypothesis proposed to explain MDCs adverse effects (He et al., 2024; Heindel et al., 2024), and the increase of CYP activities has previously been linked with increasing reactive oxygen species (ROS) production through the uncoupling of the CYP enzymatic cycle (De Almeida et al., 2022; Veith and Moorthy, 2018). CYP-driven ω-oxidation of fatty acids decouples catalytic cycling, releasing ROS and lipid peroxides (Berlanga et al., 2014; Hardwick et al., 2009), and it was hypothesised that the activation of these oxidative pathways may lead to endoplasmic reticulum stress and hepatic lipid accumulation (Angrish et al., 2016). It should also be mentioned that the upregulation of CYP3A4 was previously proposed as a key event in the AOP for cholestasis (Vinken, 2013). Such examples illustrate why any sustained and significant modulation of CYP activities may have an important and potentially adverse health impact on organisms.

## 5. Conclusions

The CYP induction test method provides an optimised and standadised in vitro test method able to reliably identify chemicals inducing CYP enzyme activity (CYP1A2, CYP2B6 and CYP3A4) in the human liver model HepaRG. In this study, the test method was extended for the first time beyond pharmaceutical chemicals to six candidate MDCs with documented human exposure. All MDCs, with the exception of TCS were clearly demonstrated to induce part (BPA, p,p'-DDE) or all (PFOA, TBT, TPP) the CYP activities assessed. The results of this study, which provide novel information about the capability of candidate MDCs to induce major CYP activities, should facilitate substantial progress in our understanding of the possible modes of action of these chemicals, and the method is intended to support relevance of in vitro testing strategies for chemical safety assessment.

## Funding

This project has received funding from the European Union's Horizon 2020 research and innovation program under grant agreement GOLIATH No. 825489.

## CRediT authorship contribution statement

**Laurie Urien:** Writing – review & editing, Validation, Methodology, Investigation.
**Daniel Zalko:** Writing – review & editing, Writing – original draft, Validation, Supervision, Project administration, Methodology, Investigation, Funding acquisition, Formal analysis, Conceptualization.
**Nicolas J Cabaton:** Writing – review & editing, Visualization, Validation, Methodology, Investigation, Formal analysis.
**Miriam N Jacobs:** Writing – review & editing, Methodology, Funding acquisition, Conceptualization.
**Elodie Person:** Writing – review & editing, Writing – original draft, Visualization, Validation, Methodology, Investigation, Formal analysis, Conceptualization.
**Juliette Legler:** Writing – review & editing, Project administration, Funding acquisition, Conceptualization.
**Sebastian Hoffmann:** Writing – review & editing, Formal analysis.
**Sylvie Chevolleau:** Resources, Investigation.
**Agnès Jamin:** Resources.
**Barbara Kubickova:** Writing – review & editing.
**Louisa Mediouni:** Validation, Methodology, Investigation.
**Jorke H Kamstra:** Writing – review & editing, Project administration,

**Methodology, Conceptualization.** Marlène Z Lacroix: Writing – review & editing, Validation, Resources, Methodology, Investigation, Formal analysis.

## Declaration of Competing Interest

The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Elodie PERSON reports financial support was provided by European Commission. Nicolas J. CABATON reports financial support was provided by European Commission. Laurie URIEN reports financial support was provided by European Commission. Marlene Z. LACROIX declares she has no competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Jorke H. KAMSTRA reports financial support was provided by European Commission. Louisa MEDIOUNI reports financial support was provided by European Commission. Barbara KUBICKOVA reports financial support was provided by European Commission. Further, Barbara KUBICKOVA also reports a relationship with Syngenta Jealott's Hill International Research Centre that includes: employment since November 2023. However, during the whole period related to her work on the test method submitted for this manuscript, Dr KUBICKOVA was not yet working for this company, and was a full-time employee of the UK Health Security Agency, which was one of the partners of the EU Goliath project consortium. Thus, for Dr KUBICKOVA, this work is solely affiliated with the UK Health Security Agency. Agnes JAMIN reports financial support was provided by European Commission. Sylvie CHEVOLLEAU reports financial support was provided by European Commission. Sebastian HOFFMANN reports financial support was provided by European Commission. Juliette LEGLER reports financial support was provided by European Commission. Miriam N. JACOBS reports financial support was provided by European Commission. Further, Miriam N. JACOBS reports that she has been also funded by the UK Government Department of Environment, Food and Rural Affairs, to conduct additional experimental work at UKHSA on this test method. This work will be published separately and does not overlap with this work. Daniel ZALKO reports financial support was provided by European Commission.

## Appendix A. Supporting information

Supplementary data associated with this article can be found in the online version at doi:10.1016/j.tox.2025.154300.

## Data availability

Data will be made available on request.

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