Next Generation Risk Assessment of Human Exposure to Anti-Androgens Using Newly Defined Comparator Compound Values

Toxicology in Vitro 73 (2021) 105132

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Toxicology in Vitro

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Next generation risk assessment of human exposure to anti-androgens using newly defined comparator compound values

Tessa C.A. van Tongeren a,*, Thomas E. Moxon b, Matthew P. Dent b, Hequn Li b, Paul L. Carmichael b, Ivonne M.C.M. Rietjens a a Division of Toxicology, Wageningen University and Research, 6700, EA, Wageningen, the Netherlands b Unilever Safety and Environmental Assurance Centre, Colworth Science Park, Sharnbrook, Bedfordshire MK44 1LQ, UK

ARTICLE INFO ABSTRACT

Keywords: Next Generation Risk Assessment (NGRA) can use the so-called Dietary Comparator Ratio (DCR) to evaluate the Risk assessment safety of a definedexposure to a compound of interest. The DCR compares the Exposure Activity Ratio (EAR) for 3R compliant method the compound of interest, to the EAR of an established safe level of human exposure to a comparator compound Androgen receptor with the same putative mode of action. A DCR ≤ 1 indicates the exposure evaluated is safe. The present study Dietary comparator aimed at defining adequate and safe comparator compound exposures for evaluation of anti-androgenic effects, In vitro/in silico approaches using 3,3-diindolylmethane (DIM), from cruciferous vegetables, and the anti-androgenic drug bicalutamide (BIC). EAR values for these comparator compounds were definedusing the AR-CALUX assay. The adequacy of the new comparator EAR values was evaluated using PBK modelling and by comparing the generated DCRs of a series of test compound exposures to actual knowledge on their safety regarding in vivo anti-androgenicity. Results obtained supported the use of AR-CALUX-based comparator EARs for DCR-based NGRA for putative anti-androgenic compounds. This further validates the DCR approach as an animal free in silico/in vitro 3R compliant method in NGRA.

1. Introduction the same mode of action. The EAR compares an internal human expo sure concentration at a definedexternal dose level to a biological effect At the current state-of-the-art, toxicological risk and safety assess concentration such as the half maximal effective concentration (EC50) of ment is in many cases still based on the traditional use of animals for a chemical obtained from a relevant in vitro assay or in silico prediction, systemic toxicity testing in order to obtain points of departure (PoDs) to leading away from the use of in vivo data (Becker et al., 2015). When the define safe levels of human exposure. However, use of animal-based exposure scenario used to definethe EARcomparator is considered safe and testing strategies is under debate because of ethical, economic, and the DCR of the studied compound obtained is ≤1, it can be concluded legislative issues, while at the same time experimental animals may not that the exposure to the compound under study will also be safe. adequately represent the human situation. This initiates the need for Dent et al. (2019) described a proof-of-principle of the DCR approach alternative testing strategies to reduce, replace, and refine(3Rs) the use evaluating a series of human exposure scenarios to putative dietary anti- of animals for risk assessment purposes (Russell and Burch, 1959). Next androgenic compounds. In this study, the exposure to 3,3-diindolylme Generation Risk Assessment (NGRA) aims to not replace each existing thane (DIM, Fig. 1), resulting from the intake of a portion of 50 g animal test with an alternative but to assure human safety based on Brussels sprouts (Fujioka et al., 2016), was used as the comparator human data and in vitro and in silico approaches. One NGRA strategy is exposure because no anti-androgenic adverse effects are expected in based on the so-called Dietary Comparator Ratio (DCR) (Becker et al., humans after normal dietary consumption of this amount of the crucif 2015). erous vegetable (Dent et al., 2019). The DCR compares the Exposure Activity Ratio (EAR) for exposure to However, use of exposure to DIM from consumption of 50 g Brussels the compound of interest to the EAR of an established safe level of sprouts as the safe comparator exposure regimen, resulted in DCR values human exposure to a comparator compound (EARcomparator) exhibiting that exceeded the value of 1 for all test compounds evaluated. This

* Corresponding author at: Division of Toxicology, Wageningen University and Research, PO Box 8000, 6700, EA, Wageningen, the Netherlands. E-mail address: [email protected] (T.C.A. van Tongeren). https://doi.org/10.1016/j.tiv.2021.105132 Received 26 October 2020; Received in revised form 3 February 2021; Accepted 25 February 2021 Available online 2 March 2021 0887-2333/© 2021 The Author(s). Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license

(http://creativecommons.org/licenses/by-nc-nd/4.0/). T.C.A. van Tongeren et al. Toxicology in Vitro 73 (2021) 105132

◦ indicated that the exposure scenarios evaluated for the 9 putative anti- 37 C). The cells were routinely subcultured when reaching 85–95% androgenic compounds were all predicted to have higher chances to confluency (i.e. every 3 to 4 days), using trypsin-EDTA. results in in vivo anti-androgenic effects than resulting from the intake of 50 g Brussels sprouts. Whilst protective, this DCR approach was 2.1.3. AR-CALUX assay considered overly conservative, because for several of these exposure AR-CALUX assay data of DIM were taken from the literature (Dent scenarios data were available supporting the absence of anti-androgenic et al., 2019). The AR-CALUX assay of BIC was performed as described effects. previously (van der Burg et al., 2010; Sonneveld et al., 2005) by BDS. In Therefore, the aim of the present study was to explore the possibility short, the U2OS cells were plated in the inner wells of white, clear- to define less conservative comparator EARs based on alternative safe bottomed 96-well plates at a density of 1 × 105 cells/mL in a volume levels for human exposure to anti-androgenic active compounds and of 100 μL/well assay medium consisting of Phenol Red Free DMEM/F-12 newly defined EARcomparator values. To this end, an updated EAR supplemented with 5% DCC-FCS, 1% NEAAs, 10 units/mL penicillin, comparator was defined for DIM, as well as an EARcomparator for the anti- and 10 μg/mL streptomycin. 200 μL PBS was loaded in the outer wells to androgenic drug bicalutamide (BIC, Fig. 1). BIC is a nonsteroidal race prevent evaporation of the assay medium and the plate was placed for ◦ mate anti-androgen used in the treatment for prostate cancer. The 24 h in an incubator (5% CO2; 37 C). Afterwards, the assay medium was EARcomparator for BIC was defined for R-BIC, since R-BIC accounts for aspirated and 100 μL fresh assay medium was added to the cells and the ◦ 99% of the plasma concentration after dosing in humans and is a more plate was incubated for another 24 h in an incubator (5% CO2; 37 C). potent anti-androgen than S-BIC (Cockshott, 2004; AstraZeneca Phar The test compounds dissolved in DMSO (final concentration 0.1% maceuticals, 2005). In order to assure a 3R compliant method, in the DMSO) tested in triplicate (n = 3), the vehicle control (0.1% DMSO) (n present study the EARs were definedusing the AR-CALUX assay for anti- = 6), and the cytotoxicity control (0.01% Triton X-100) (n = 6) were androgenic activity. The newly defined EARcomparator values were used tested in the agonism assay and antagonism assay. After the incubation to obtain DCRs and an updated outcome of the NGRA for the series of time, the assay medium was removed and the cells were exposed by putative anti-androgen exposures. adding 100 μL fresh assay medium containing the appropriate concen tration of test compound to each well. In the agonism assay, the cells 2. Materials and methods were exposed to a concentration range of DHT (1 × 10 3 to 100 nM) or BIC (1 × 10 3 to 3 μM). In the antagonism assay, FLU as a quality control × 2 μ 2.1.1. Chemicals and BIC were tested in a concentration range of 1 10 to 30 M and 1 × 3 μ DHT (CAS no. 521–18-6), BIC (CAS no. 90357–06-5), FLU (CAS no. 10 to 3 M, respectively, in the presence of the EC50 (0.3 nM) of 13311–84-7), and Triton X-100 were purchased from Sigma–Aldrich DHT as the agonist. For the specificitycontrol, the cells were exposed to the same concentration range of FLU and BIC with 100 x EC50 (30 nM) of Chemie B.V. (Zwijndrecht, the Netherlands). Penicillin-streptomycin ◦ solution was purchased from Invitrogen (Breda, the Netherlands). DHT. After 24 h of exposure in an incubator (5% CO2; 37 C), the media μ Phosphate-buffered saline (PBS), trypsin EDTA (trypsin (0.025%)/EDTA were removed and the cells were washed with 100 L PBS in nanopore μ (0.01%)), Dulbecco’s modifiedEagle ’s Medium/Ham’s nutrient mixture water (1:1) and lysed with 30 L LSB. Luminescence was measured with ¨ F12 (DMEM/F12), Phenol Red Free DMEM/F-12, fetal calf serum (FCS), the Infinite200 PRO (Tecan, Mannedorf, Switzerland). Cytotoxicity was dextran-coated charcoal-treated (DCC) FCS, MEM (100×) non-essential measured as the lactate dehydrogenase (LDH) leakage into the culture amino acids (NEAAs), and geneticin (G-418) were purchased from Gibco medium quantified using the Cytotoxicity Detection Kit (Roche, = (Paisley, United Kingdom). Dimethyl sulfoxide (DMSO) was purchased 1,644,793). In short, cells were exposed as in the AR-CALUX assay (n 3) or to the positive control for cytotoxicity (0.01% Triton X-100) (n = 6) from Acros Organics (Geel, Belgium). Low salt buffer (LSB), consisted of ◦ 10 mM Tris (Invitrogen), 2 mM dithiothreitol (DTT) (Duchefa Biochemie for 24 h in an incubator (5% CO2; 37 C), after which the reaction bv, Haarlem, the Netherlands), and 2 mM 1, 2-diaminocyclohexanete mixture of the Cytotoxicity Detection Kit was added. After 5 to 30 min, triacetic acid monohydrate (CDTA) (Fluka, Munich, Germany). The the stop solution was added and absorbance was measured with a flashmix consisted of 20 mM tricine (Jansen chemica, Landsmeer, the microplate spectrophotometer system (Spectra max190-Molecular De vices, Wokingham, UK). Netherlands), 1.07 mM (MgCO3)4Mg(OH)2.5H2O (Sigma-Aldrich, 99% ¨ purity), 2.67 mM magnesium sulphate (MgSO4) (Ridel de Haen, Landsmeer, the Netherlands), 0.1 mM ethylenedinitrilotetraacetic acid 2.1.4. Data analysis disodium salt dihydrate (Titriplex III; Merck, Amsterdam, the Antagonism was defined as at least 20% decrease in relative induc Netherlands), 2 mM DTT (Duchefa Biochemie), 0.47 mM D-luciferin tion of the DHT induced response at a non-cytotoxic concentration of the ′ (Duchefa Biochemie), and 5 mM adenosine-5 -triphosphate (ATP, test compound and the response being confirmedas AR-specific.The test Boehringer, Alkmaar, the Netherlands). concentration was stated as cytotoxic when the percentage LDH leakage was higher than 15% comparted to the positive control of cytotoxicity 2.1.2. Cell culture set at 100%, and for these samples the observed reduction in lumines Cells from the stably transfected human osteosarcoma (U2OS) cell cence was considered not to be due to antagonism and excluded from the line expressing the human androgen receptor (AR) (BioDetection Sys analysis. The IC50 value of DIM was taken from Dent et al. (2019) and tems (BDS), Amsterdam, the Netherlands) were maintained in DMEM/F- the IC50 value of BIC was modelled with a nonlinear regression of log 12 supplemented with 7.5% FCS, 1% NEAAs, 10 units/mL penicillin, 10 (inhibitor) vs. response (four parameters) model using GraphPad Prism 5 (GraphPad, San Diego, USA). The BMCL05 value of DIM and BIC were μg/mL streptomycin, and 0.2 mg/mL G-418 in an incubator (5% CO2;

Fig. 1. Structure formulas of DIM, bicalutamide (BIC), and dihydrotestosterone (DHT).

2 T.C.A. van Tongeren et al. Toxicology in Vitro 73 (2021) 105132 derived from the AR-CALUX data using BMDS3.1.1 software (U.S. EPA) Table 1 using the benchmark response (BMR) of a 5% increase in the response Input parameters of the PBK model describing the kinetics of DIM and BIC in compared to the control. The benchmark concentration causing this humans. BMR (BMC05) and the upper (BMCU05) and lower (BMCL05) bound of Parameter Value DIM1 Value BIC its 95% confidenceinterval were determined. The acceptance criteria of MW 246.31a 430.37a > b a the BMCL05 values were a p-value 0.05, indicating support for a Log P 4.17 2.3 ◦ concentration-response. Furthermore, the model should describe the Solubility at 25 C (μg/mL) 12b 9.28a c data adequality in the BMR region reflectedby a BMC05: BMCL05 ratio pKa Acid 11.95 Base 4c lower than 3 (EFSA, 2017). 4 b 6 d Peff 2 × 10 cm/s 32 × 10 cm/s b b Fub in vivo 2.84% 10.52% b b 2.2. Evaluation of the BMCL05 for prediction of a safe comparator Rb2p 0.82 0.63 e exposure level CLsys (L/h) 0.32 f e,g CLren (L/h) 7.27 0.12 f,h e,g CLhep 0.931 L/h/kg 0.21 L/h The BMCL05 values of DIM and BIC for the induction of anti- androgenic effects were considered to reflect a safe internal exposure 1 Taken from Dent et al. (2019). a level without AR-mediated effects. This assumption is based on the fact Kim et al. (2016). b ADMET predictor™. that a BMCL05 value may be equivalent to what would be considered a c no observed adverse effect level (EFSA, 2017). To further evaluate this Wishart et al. (2006). d Bassetto et al. (2016). assumption, these in vitro safe levels were translated to their corre e AstraZeneca Pharmaceuticals (2005). sponding human urinary excretion and exposure levels using PBK f Dent et al. (2019). modelling-based quantitative in vitro to in vivo extrapolation (QIVIVE) g B.C. Cancer Agency (2001). and compared to the urine excretion level of DIM as a biomarker for the h Wu et al., 2015; Tang et al., 2007. intake of DIM from Brussels sprouts and the therapeutic dose of BIC, respectively. 2.2.2. PBK model validation For validation purposes, the PBK model predicting the DIM plasma 2.2.1. PBK model development concentration upon dosing of DIM was validated previously by Dent The PBK model of DIM was taken from Dent et al. (2019), linking et al. (2019) against corresponding clinical data from literature (Reed DIM plasma concentrations to urinary excretion levels of DIM (Fujioka et al., 2008). For validation of the PBK model of BIC, a simulation was et al., 2016). This PBK model and the PBK model of BIC were developed carried out using the GastroPlus™ Single Simulation Mode to obtain the ™ using the commercially available software GastroPlus version 9.6 time-dependent plasma concentration curves upon dosing a single im (Simulation Plus Inc., Lancaster, CA, USA). The built-in Population Es mediate release (IR) tablet of 50 mg BIC to a male with a bodyweight of ™ timates for Age-Related (PEAR) Physiology module was used to 85.53 kg with a simulation time of 25 days. The predicted time- parameterize the PBK model. For validation purposes, the model was dependent plasma concentration curve thus obtained was validated parameterized for a fasted 38-year-old female with a body weight of against pharmacokinetic data reported by McKillop et al. (1993) upon 71.2 kg and a fed 30-year-old male with a body weight of 85.53 kg of oral dosing of 50 mg in healthy male volunteers (n = 5). Similar, the DIM and BIC, respectively, to match the corresponding pharmacokinetic repeated IR tablet dosing of 10 mg/day BIC was simulated by the Gas data from literature (Reed et al., 2008; McKillop et al., 1993; Tyrrell troPlus™ Repeated Simulation Mode, with a simulation time of 12 et al., 1998). For QIVIVE, both PBK models were definedfor a 30-year- weeks and compared to corresponding pharmacokinetic data reported old male with a body weight of 70 kg. The physiochemical parameters by Tyrrell et al. (1998) in patients with advanced prostate cancer (n = were derived from PubChem databases (Kim et al., 2016), literature or 140) . predicted from their structure with the built-in ADMET Predictor™ version 9.6 (Simulation Plus Inc., Lancaster, CA) (Table 1). 2.2.3. Sensitivity analysis The effective permeability (peff) of BIC was simulated from the Caco- The PBK models were standardized by parameterizing for a 30-year- 2 value reported by Bassetto et al. (2016) using the built-in conversion old American male with a body weight of 70 kg to execute the sensitivity equation based on the Absorption Systems Caco-2 calibration (ABSCa). analysis with the built-in parameter sensitivity analysis (PSA) mode of The distribution of both compounds into tissues was assumed to be GastroPlus™. The sensitivity analysis was conducted with the previously perfusion limited and the tissue: plasma partition coefficients(Kps) were developed PBK model predicting the nominal plasma concentration of ™ calculated with the Lucakova method (GastroPlus ; Rodgers et al. 2005 DIM upon an oral dose of 50 mg DIM (Dent et al., 2019), and the PBK & 2006). The hepatic clearance (CLhep) of DIM was scaled from rat data model developed for BIC in the present study at an oral dose of 10 mg/ (Dent et al., 2019; Wu et al., 2015; Tang et al., 2007). The Extended day, both being dose levels used for the validation of the PBK models. Clearance Classification System (ECCS) method (Varma et al., 2015) The sensitivity coefficients (SCs) for the Cmax and AUC were calculated predicts renal clearance as the major route of clearance for BIC, though as the % change in model outcome divided by the % change in param evidence from studies also indicate metabolism can play a major role in eter value (Eq. (1)). the clearance (AstraZeneca Pharmaceuticals, 2005). Clinical studies %change in model outcome showed that severe hepatic impairment increases the accumulation of SC = (1) BIC in the plasma which is indicative of the physiological involvement of %change in parameter value hepatic clearance (CLhep) (Cockshott, 2004; AstraZeneca Pharmaceuti The % change in parameter value was set at 5% (Zhang et al., 2018; cals, 2005). No data on the in vitro hepatic clearance of BIC were found Moxon et al., 2020), leaving the rest of the parameters unchanged. Pa in literature, but from the study of AstraZeneca Pharmaceuticals (2005) rameters with a SC > 0.1 or < 0.1 were considered to be influentialon a nominal system clearance (CLsys) of 0.32 L/h was found. Since the the prediction of the Cmax and AUC (Zhang et al., 2018) . urinary excretion of BIC was observed to be 36% (B.C. Cancer Agency, × 2001), in this study the CLren was set to 0.12 L/h (0.36 0.32 L/h) and 2.2.4. PBK modelling-based QIVIVE the CLhep was calculated by subtracting CLren from the CLsys resulting in The nominal BMCL05 values of DIM and BIC derived from the in vitro a value of 0.21 L/h. AR-CALUX assay data were translated to the nominal maximum plasma concentration (Cmax) correcting for differences in protein binding in the

3 T.C.A. van Tongeren et al. Toxicology in Vitro 73 (2021) 105132 in vitro and in vivo situation following Eq. (2). This correction implies by Dent et al. (2019), resulting in distribution of the EARtest values from that the biological effect is related to the free concentrations and that the the corresponding percentiles of the respective test compound. When no free in vitro concentration in the assay medium is set equal to the free in biomarker distribution data were available, no distribution in the vivo concentration in the human plasma. nominal internal concentration was included and thus only one EARtest value was calculated. Furthermore in the present study, the EAR nominal BMCL05 (comparator)*fub in vitro (comparator) nominal Cmax = comparator was calculated following Eq. (5) using data obtained in the AR- fub in vivo (comparator) CALUX assay. (2)

nominal BMCL05 (comparator)*fub in vitro(comparator) EARcomparator (AR CALUX based) = (5) nominal IC50 (comparator)*fub in vitro (comparator)

The nominal BMCL05 values of DIM and BIC were derived from the AR-CALUX assay and the fub in vitro and the fub in vivo represent the fraction The nominal IC50 and nominal BMCL05 were both derived from the unbound in the medium used in the AR-CALUX assay and in human AR-CALUX concentration response curve of the respective comparator plasma, respectively, determined as described below. With the PBK compound and fub in vitro represents the fraction unbound of the models, QIVIVE was performed to predict the urinary excretion level or comparator compound in the medium of the assay, determined as the external dose level that would result in the nominal Cmax values of described hereafter. Since in this novel approach the EARcomparator is DIM and BIC, respectively, and the levels thus obtained were subse entirely based on the in vitro AR-CALUX assay, using the fub in vitro for quently compared to the urinary excretion from the intake of DIM via correction for protein binding does not affect the outcome of Eq. 5. The consumption of Brussels sprouts and the exposure to BIC at the thera EAR values obtained were used to calculate the DCR using Eq. (3). The peutic dose, respectively. DCR values thus calculated were compared to the DCR values reported previously by Dent et al. (2019) using intake of DIM via 50 g Brussels 2.3. Definition of the EAR and the DCR values sprouts as the comparator, with its mean, 5th, and 95th percentiles. The variability of the DCRs was calculated by comparing the EARtest from the In the DCR approach, the EAR of a compound under study (EARtest) is lowest percentile to the EARcomparator from the highest percentile and compared to the EARcomparator which is based on an established safe level vice versa for those test compounds with distribution data available. In of human exposure to a comparator compound (Eq. (3)). addition, since the DCR values previously reported were not corrected for protein binding, these DCR values were also considered taking the fub EARtest DCR = (3) in vitro and fub in vivo for definingthe EAR values into account (Eq. (4) and EARcomparator Eq. (6)).

nominal internal concentration at defined external dose level (comparator)*fub in vivo (comparator) EARcomparator (50 g Brussels sprouts)with correction for protein binding = nominal IC50 (comparator)*fub in vitro (comparator) (6)

In the present study, the EARtest was calculated using Eq. (4).

nominal internal concentration at defined external dose level (test)*fub in vivo (test) EARtest = (4) nominal IC50 (test)*fub in vitro (test)

2.4. Determination of fub in vivo In which the fub in vivo represents the fraction unbound of the test compound in human plasma, the fub in vitro the fraction unbound of the The fub in vivo values for the comparator and test compounds were test compound in the medium of the AR-CALUX assay. The fub values predicted by the commercially available software ADMET Predictor™ were determined as described below. The nominal IC50 was derived version 9.6 (Simulation Plus Inc.). This software predicts chemical from the AR-CALUX concentration response curve while the nominal dependent parameters based on SMILES using QSAR-methods. internal concentration at the defined external dose level of the test compound was calculated previously using a human PBK model for the 2.5. Determination of fub in vitro test compound or taken from reported pharmacokinetic data in litera ture (Dent et al., 2019). The variability of the biomarker and thus the The fub in vitro values were calculated assuming a linear relation be nominal internal concentration of the test compounds were included as tween the fraction unbound and the protein content in a biological the mean, the 5th or 25th, and the 95th or 75th percentiles, as reported matrix, with the fraction unbound being 1.0 in the absence of protein.

4 T.C.A. van Tongeren et al. Toxicology in Vitro 73 (2021) 105132

Table 2 Exposure scenarios of putative dietary anti-androgenic test compounds in humans evaluated by the DCR approach (adapted from Dent et al., 2019), including whether or not in vivo anti-androgenic effects at the respective expo sure scenarios are expected or where this is unknown.

Test compound Exposure In vivo anti- Source scenario androgenic effect expected

Resveratrol (RES) 25 mg orally; No Goldberg corresponding et al. (2003); to moderate Almeida et al. intake of red (2009) wine Fig. 2. The concentration dependent antagonistic activity of DIM (solid line Flutamide (FLU) & its metabolite Therapeutic oral Yes Radwanski and circles) and BIC (dashed line and squares), on the DHT-mediated luciferase hydroxyflutamide (HF) dose of 250 mg/ et al. (1989) induction in the U2OS AR-CALUX reporter gene assay. The data for DIM were day flutamide taken from Dent et al. (2019). The symbols present the mean ± SD values of 3 Bisphenol A (BPA), vinclozolin Oral RfD/TDI No Scaled by independent studies. (VIN), and methoxychlor (MX) & Dent et al. its metabolite HPTE (2019) from the Css values anti-androgenic response was confirmed to be AR-specific by the reported by observation of the right shift of the concentration response curve at a Wetmore higher agonist concentration (100 x EC of DHT) (data not shown). et al. (2012) 50 Dichlorodiphenyldichloroethylene Serum levels in Yes Aneck-Hahn From the concentration response data obtained the nominal BMCL05 and (3,3’-DDE) men from DDT- et al. (2006) IC50 values were derived. The results of the benchmark dose modelling sprayed area in to derive the BMCL05 values are presented in Supplementary material South Africa S1. The nominal BMCL05 and IC50 values thus obtained amounted to Dichlorodiphenyldichloroethylene serum levels in No Longnecker μ μ (3,3’-DDE) DDT-exposed et al. (2002); 5.13 nM and 1.27 M for DIM and to 0.10 nM and 0.07 M for BIC, women between Bhatia et al. respectively. 1959 and 1967 (2005); in the USA, n = respectively 3.2. Determination of the fub values 599 and n = 283 Bakuchiol (BAK) Exposure via Unknown Dent et al. shampoo & (2019) Table 3 compiles the ADMET predicted fub in vivo values. Using these body lotion once values at 8% protein, the protein content in human plasma, and per day as case- assuming the fub to equal 1.0 at 0% protein, the fub in vitro values repre study by Dent senting the fraction unbound in the assay medium of the in vitro AR- et al. (2019) CALUX assay containing 5% protein were calculated for the compar ator and test compounds of the present study. These values are also This assumption is supported by the linear relationship reported by presented in Table 3. Gülden et al. (2002) between the unbound fraction and albumin con Given the lower protein concentration in the in vitro AR-CALUX assay centrations added to the in vitro test system for some chlorophenols medium than in human plasma, the fub in vitro values were all higher than (Gülden et al., 2002). The protein content of human plasma has been the fub in vivo values. The method applied to obtain the fub in vitro data was reported to be 8% (Mescher, 2009; Mathew et al., 2020). The assay verifiedby comparison of the calculated fub in vitro of BPA of 0.43 to the medium used in the AR-CALUX assay contained 5% DCC-FCS (Sonne value obtained in the same in vitro medium using the Rapid Equilibrium veld et al., 2005; Dent et al., 2019) which was considered as a 5% Device (RED) assay amounting to 0.46 ± 0.04 (Zhang et al., 2018), protein content. Based on these assumptions, the fub in vitro values of the indicating a 1.1 fold difference. comparator and test compounds were calculated by linear extrapolation to the 5% protein content using the fub of 1.0 in the absence of protein 3.3. Evaluation of the BMCL05 for prediction of a safe comparator and the respective fub in vivo values generated with the ADMET Predic exposure level tor™ at 8% protein content in in vivo plasma. 3.3.1. PBK model development and validation 2.6. Definition of test compounds and their exposure scenarios In order to evaluate the use of the in vitro BMCL05 values to definea safe level of exposure to the comparator compounds, the values of DIM The exposure scenarios to the putative anti-androgenic test com pounds evaluated in the DCR-based NGRA were the same as the ones Table 3 previously selected by Dent et al. (2019) and are summarized in Table 2. The ADMET Predictor™ generated fub in vivo values and the calculated fub in vitro The table also presents whether at the exposure scenario evaluated anti- values of the comparator and test compounds of the present study. androgenic effects are expected to occur. This reveals that the data set Compound fub in vivo fub in vitro consists of both putative negative as well as positive test compound DIM 0.03 0.40 exposure scenarios for anti-androgenicity. BIC 0.11 0.44 BAK 0.05 0.41 3. Results RES 0.08 0.42 VIN 0.12 0.45 BPA 0.09 0.43 3.1. AR-CALUX assay MX 0.04 0.40 HPTE 0.05 0.40 Fig. 2 presents the concentration response curves for the anti- 3,3’-DDE 0.03 0.39 androgenic activity of DIM and BIC in the AR-CALUX assay. Both com FLU 0.20 0.50 HF 0.32 0.57 pounds did not show a response in the agonist AR-CALUX assay. Their

5 T.C.A. van Tongeren et al. Toxicology in Vitro 73 (2021) 105132

Fig. 3. PBK model predicted (lines) and reported (individual data points represented as the average values as reported in literature) for the time-dependent plasma concentration after A. a single oral dose of 50 mg BIC in humans (experimental data from Mckillop et al., 1993), and B. repeated oral doses of 10 mg/day BIC in humans (experimental data from Tyrrell et al., 1998).

Fig. 4. Sensitivity analysis of the parameters of the PBK model of A. DIM and B. BIC predicting the Cmax (open bars) and AUC (closed bars) following an oral dose of 50 mg DIM or an oral dose of 10 mg/day BIC, respectively. Parameters with a SC > 0.1 or < 0.1 are plotted. Permeability = intestinal permeability. Log D = distribution coefficient. Fub in vivo = fraction unbound in vivo. Rb2p = blood: plasma ratio. CLhep = hepatic clearance. Only parameters with a SC > 0.1 or < 0.1 are presented.

and BIC were translated to the in vivo urinary excretion and external values from the concentration response curves obtained in the AR- dose level, respectively, via PBK model facilitated reverse dosimetry CALUX assay were converted to the nominal Cmax values of DIM and enabling QIVIVE. The PBK model used for DIM was developed and BIC correcting for differences in protein binding in the in vitro and in vivo validated previously by Dent et al. (2019). The PBK model for BIC was situation using Eq. (2). Next, the PBK models defined were used to developed in the present study. To enable evaluation of this PBK model translate the nominal Cmax values of DIM and BIC to the corresponding for BIC, Fig. 3 reports the PBK-model predicted and literature reported urinary excretion and external dose level, respectively, that could be time-dependent plasma concentrations after a single oral dose of 50 mg compared to potential human exposure scenarios. The nominal Cmax BIC (Fig. 3A) and a repeated oral dose of 10 mg/day BIC (Fig. 3B) in values thus obtained amounted to 71 nM and 0.43 nM for DIM and BIC humans. From this comparison it appears that the model predicts the respectively, and the urinary excretion and external dose level resulting dose dependent plasma concentration of BIC well with a 1.3- to 1.6-fold in these Cmax values amounted to 0.64 mg DIM and 1.4 μg/day BIC, difference between the reported and predicted Cmax values of BIC. respectively. This urinary excretion level of DIM is 84- to 533-fold higher than the urinary excretion level resulting from consumption of 3.3.2. Sensitivity analysis 50 g Brussels sprouts which was estimated to amount to 1.2 × 10 3 to For further evaluation of the PBK models, a sensitivity analysis was 7.6 × 10 3 mg DIM (Dent et al., 2019). For BIC the dose level is performed at a dose of 50 mg for DIM and of 10 mg/day for BIC. Fig. 4 approximately 4 orders of magnitude lower than the therapeutic dose of presents the parameters with a SC > 0.1 or < 0.1 representing the PBK 25 mg/day used for treatment of hirsutism (Müderris et al., 2009) and model parameters that are most influentialon the PBK model output for 50 mg/day used in treatment of prostate cancer (B.C. Cancer Agency, Cmax and the AUC of DIM or BIC. The DIM PBK model prediction of the 2001). Cmax appears to be sensitive to the permeability, Log D, fub in vivo, and Rb2p, and the prediction of the AUC is sensitive to these latter three. The BIC PBK model prediction of Cmax is sensitive to Log D, CLhep, and fub in 3.4. Definition of the EAR values vivo whereas the prediction of the AUC is sensitive to only LogD and fub in vivo. The remaining parameters had an SC within 0.1 and 0.1. The EARcomparator of DIM and BIC calculated based on the AR-CALUX 3 3 were 4.04 × 10 and 1.51 × 10 , respectively. The EARtest values of 3.3.3. PBK modelling-based QIVIVE exposure scenarios to the putative anti-androgenic test compounds as To enable PBK modelling-based QIVIVE, first the nominal BMCL05 reported by Dent et al. (2019) were corrected for protein binding and are summarized in Supplementary material S2.

6 T.C.A. van Tongeren et al. Toxicology in Vitro 73 (2021) 105132

Fig. 5. The DCRs of the series of exposure scenarios to anti-androgenic test compounds (Table 2) based on different EARcomparator values including A. the EAR comparator for DIM intake from 50 g Brussels sprouts without correction for differences in protein binding as reported by Dent et al. (2019) calculated using an 4 4 4 EARcomparator of 3.51 × 10 at the mean and 1.16 × 10 and 7.43 × 10 at the 5th and 95th percentile, B. the EARcomparator for DIM intake from 50 g Brussels 5 6 5 sprouts with correction for differences in protein binding, calculated using an EARcomparator of 2.55 × 10 at the mean and 8.43 × 10 and 5.39 × 10 at the 5th 3 3 and 95th percentile C. the EARcomparator for DIM based on AR-CALUX data of 4.04 × 10 , and D. the EARcomparator for BIC based on AR-CALUX data of 1.51 × 10 . The comparator DCRs are represented as black symbols and by definition equal to 1 (log DCR = 0). The DCRs of test compounds where no in vivo anti-androgenic effects are expected at the corresponding exposure scenario (Table 2) are presented as green symbols and the DCRs of test compounds where in vivo anti-androgenic effects are expected or where this is unknown at the corresponding exposure scenario (Table 2) are presented as red and purple symbols, respectively. The dotted horizontal line marks the DCR of 1 (log DCR = 0). The vertical lines separate the exposure scenarios with DCR ≤ 1 from those with DCR > 1.

3.5. DCR values based on the different comparator exposure scenarios evaluated present a higher chance to result in in vivo anti-antagonistic effects than the intake of 50 g Brussels sprouts for which this effect is Using the EAR values obtained, the DCR values were calculated for absent. Comparison of the data in Fig. 5A and B reveals that the effect of the different putative anti-antagonist exposure scenarios using the the correction for the difference in protein binding in the in vitro and in newly defined EAR comparator values for DIM (Fig. 5C) and BIC vivo situation is limited, resulting in 0.9 to 7.6 fold higher DCR values (Fig. 5D). Fig. 5 also presents, for comparison, the DCR values as shown upon correction for differences in protein binding with the highest in previously using 50 g Brussels sprouts as the comparator without crease observed for compounds with relative high fub values. The DCRs correction for differences in protein binding (Fig. 5A) (Dent et al., 2019) calculated using the newly defined AR-CALUX based EARcomparator or with correction for differences in protein binding (Fig. 5B). Using the values for DIM (Fig. 5C) and BIC (Fig. 5D) were less conservative. To EAR based on the exposure to DIM via consumption of 50 g Brussels further evaluate the validity of these comparator values, the predictions sprouts as the comparator scenario, either without (Fig. 5A) or with made for the test compounds were compared to actual knowledge on the correction for differences in protein binding (Fig. 5B), resulted in DCRs anti-androgenicity of the respective exposure scenarios (Table 2). To this >1 for all test compounds, indicating that all the exposure scenarios end in Fig. 5, the DCRs of test compounds where no in vivo anti-

7 T.C.A. van Tongeren et al. Toxicology in Vitro 73 (2021) 105132 androgenic effects are expected at the corresponding exposure scenario of magnitude lower than the therapeutic dose of 25 mg/day in hirsutism (Table 2) are presented as green symbols. The DCRs of test compounds (Müderris et al., 2009) and 50 mg/day in prostate cancer (B.C. Cancer where in vivo anti-androgenic effects are expected or where this is un Agency, 2001) corroborating the adequacy of this exposure scenario and known at the corresponding exposure scenario (Table 2) are presented the AR-CALUX based method used to define this safe EARcomparator. as red and purple symbols, respectively. Given that in Fig. 5C and D all The EARcomparator and DCR values definedin the present study based exposure scenarios that have DCR values ≤1 relate to exposures that are on the in vitro AR-CALUX assay also included a correction for differences known to not result in anti-androgenic activity, supports the use of the in protein binding in the in vitro and in vivo situation. The capability of newly definedEAR comparator values as points of departure to definesafe chemicals to bind to proteins present in the surrounding medium in levels of exposure. There are even two of the test scenarios of DDE where fluencestheir availability for the biological target and the corresponding no in vivo anti-androgenic effects were observed with DCR values >1. potency (Gülden et al., 2002). Therefore, the free concentration of a chemical is considered to be a more appropriate dose metric than the 4. Discussion nominal concentration. This correction appeared to influence the EAR values to the largest extent for compounds with relatively high fub In the DCR approach, the EAR of an exposure scenario to a test values, for which the differences between the fub in vivo and fub in vitro were compound under study (EARtest) is compared to the EAR of a safe human most substantial. The comparison of the DCR values obtained using the exposure to a comparator compound (EARcomparator). A DCR cut-off newly defined EARcomparator values with actual knowledge on the anti- value of 1 is then used to evaluate the safety of the respective test androgenicity resulting from the exposure scenarios of the test com compound exposure. This DCR approach was introduced for the evalu pounds (Fig. 5C and D), provides further support for using the AR- ation of anti-androgenic effects by Dent et al. (2019) using the EAR for CALUX based BMCL05 and IC50 to define the EARcomparator value. All DIM resulting from consumption of 50 g Brussels sprouts as the together the results of the present study using newly defined EAR comparator. However, when using this EARcomparator the DCR of all test comparator values further validate the DCR approach as an animal free in compounds, even the ones for exposures known to be safe, were above 1, silico/in vitro 3R compliant method in NGRA. which indicates that use of this EARcomparator may be overprotective. Therefore in the present study, alternative EARcomparator values were Funding defined.The EARcomparator value of DIM was redefinedand an additional EARcomparator was definedusing the anti-androgenic drug BIC. Using BIC This work was funded via a grant from Unilever (United Kingdom) to as a comparator compound implies that formally the term dietary Wageningen University and Research (WUR, The Netherlands) for the comparator should be reconsidered but we preferred to maintain current PhD project of T.C.A. van Tongeren. terminology. To develop an NGRA compliant method to definethe new EARcomparator values, the values were determined using the BMCL05 and Declaration of Competing Interest IC50 for anti-androgenicity obtained in the in vitro AR-CALUX assay as the surrogate endpoints to define safe and effective in vivo plasma con The authors declare that they have no known competing financial centrations, respectively. Evaluation of the adequacy of the EAR values interests or personal relationships that could have appeared to influence thus obtained was based on PBK modelling-based translation of the the work reported in this paper. BMCL05 values to in vivo relevant biomarker levels and on the compar ison of the DCR values obtained for exposures to test compounds with Acknowledgment the actual knowledge on the anti-androgenicity of the respective expo sure scenarios. In contrast to the EARcomparator based on intake of 50 g The authors would like to express their graduate towards the people Brussels sprouts, with the newly defined EARcomparator values, test at BioDetection Systems (BDS, Amsterdam, the Netherlands) for their compound exposure scenarios with DCR values ≤1 were all negative for contribution to definingthe AR-CALUX concentration response data for in vivo AR antagonist mediated responses. All scenarios known to induce BIC and Beate Nicol (Unilever) for her helpful discussions valuable to in vivo anti-androgenic effects had DCR values >1, indicating the this work. method did not result in false negatives among the scenarios tested. There were even two of the test scenarios of DDE known to be without in Appendix A. Supplementary data vivo anti-androgenic activity with DCR values >1 which represent false positives. This result indicates that even the newly definedEAR comparator Supplementary data to this article can be found online at https://doi. values seem to be conservative. org/10.1016/j.tiv.2021.105132. Conversion of the AR-CALUX derived BMCL05 for the safe level of endogenous exposure to DIM and BIC to the corresponding in vivo levels References via PBK modelling-based QIVIVE resulted in a predicted urinary excre ˜ tion level of 0.64 mg DIM and a safe dose level of 1.4 μg/day BIC. This Almeida, L., Vaz-da-Silva, M., Falcao, A., Soares, E., Costa, R., Loureiro, A.I., Fernandes- Lopes, C., Rocha, J.-F., Nunes, T., Wright, L., Soares-da-Silva, P., 2009. urinary excretion level of DIM is 84- to 533-fold higher than the urinary Pharmacokinetic and safety profile of trans-resveratrol in a rising multiple-dose excretion level resulting from consumption of 50 g Brussels sprouts study in healthy volunteers. Mol. Nutr. Food Res. 53, S7–S15. https://doi.org/ which was estimated to amount to 1.2 × 10 3 to 7.6 × 10 3 mg DIM in 10.1002/mnfr.200800177. Aneck-Hahn, N.H., Schulenburg, G.W., Bornman, M.S., Farias, P., De Jager, C., 2006. urine (Dent et al., 2019). The reason to compare with urinary excretion Impaired semen quality associated with environmental DDT exposure in young men levels was because DIM is not consumed with Brussels sprouts as such living in a malaria area in the Limpopo Province, South Africa. J. Androl. 28, but has to be formed from indol-3-carbinol, with the corresponding 423–434. https://doi.org/10.2164/jandrol.106.001701. external dose which would result in this urinary excretion of DIM being AstraZeneca Pharmaceuticals, L.P., 2005. CASODEX (bicalutamide). In: Manufactured by Caroline, PR 00984: IPR Pharmaceuticals Inc., USA. For Wilmington, DE 19850: influenced by high variance in the processing and pharmacokinetics of AstraZeneca Pharmaceuticals LP. indol-3-carbinol following in the intake of Brussels sprouts (Fujioka B.C. Cancer Agency, 2001. Limited Revision (2008 & 2011). Bicalutamide monograph. et al., 2014; Barba et al., 2016; Thomson et al., 2016). However, com In: BCCA Cancer Drug Manual. Vancouver, British Columbia. Barba, F.J., Nikmaram, N., Roohinejad, S., Khelfa, A., Zhu, Z., Koubaa, M., 2016. parison of the urinary DIM levels corresponding to the established safe Bioavailability of glucosinolates and their breakdown products: impact of plasma levels to the urinary DIM levels resulting from intake of 50 g of processing. Front. Nutr. 3 (24) https://doi.org/10.3389/fnut.2016.00024. Brussels sprouts, indicates that the new comparator exposure scenario Bassetto, M., Ferla, S., Pertusati, F., Kandil, S., Westwell, W.D., Brancale, A., McGuigan, C., 2016. Design and synthesis of novel bicalutamide and enzalutamide for DIM is far less conservative than the one based on the intake of only derivatives as antiproliferative agents for the treatment of prostate cancer. Eur. J. 50 g Brussels sprouts. The modelled safe external dose of BIC is 4 orders Med. Chem. 118, 230–243. https://doi.org/10.1016/j.ejmech.2016.04.052.

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