Integrating In Vitro Data and Physiologically Based Kinetic Modeling to Predict and Compare Acute Neurotoxic Doses of Saxitoxin in Rats, Mice, and Humans.

Current climate trends are likely to expand the geographic distribution of the toxigenic microalgae and concomitant phycotoxins, making intoxications by such toxins a global phenomenon. Among various phycotoxins, saxitoxin (STX) acts as a neurotoxin that might cause severe neurological symptoms in mammals following consumptions of contaminated seafood. To derive a point of departure (POD) for human health risk assessment upon acute neurotoxicity induced by oral STX exposure, a physiologically based kinetic (PBK) modeling-facilitated quantitative in vitro to in vivo extrapolation (QIVIVE) approach was employed. The PBK models for rats, mice, and humans were built using parameters from the literature, in vitro experiments, and in silico predictions. Available in vitro toxicity data for STX were converted to in vivo dose-response curves via the PBK models established for these three species, and POD values were derived from the predicted curves and compared to reported in vivo toxicity data. Interspecies differences in acute STX toxicity between rodents and humans were found, and they appeared to be mainly due to differences in toxicokinetics. The described approach resulted in adequate predictions for acute oral STX exposure, indicating that new approach methodologies, when appropriately integrated, can be used in a 3R-based chemical risk assessment paradigm.

Acetylcholinesterase Inhibition in Rats and Humans Following Acute Fenitrothion Exposure Predicted by Physiologically Based Kinetic Modeling-Facilitated Quantitative In Vitro to In Vivo Extrapolation.

Worldwide use of organophosphate pesticides as agricultural chemicals aims to maintain a stable food supply, while their toxicity remains a major public health concern. A common mechanism of acute neurotoxicity following organophosphate pesticide exposure is the inhibition of acetylcholinesterase (AChE). To support Next Generation Risk Assessment for public health upon acute neurotoxicity induced by organophosphate pesticides, physiologically based kinetic (PBK) modeling-facilitated quantitative in vitro to in vivo extrapolation (QIVIVE) approach was employed in this study, with fenitrothion (FNT) as an exemplary organophosphate pesticide. Rat and human PBK models were parametrized with data derived from in silico predictions and in vitro incubations. Then, PBK model-based QIVIVE was performed to convert species-specific concentration-dependent AChE inhibition obtained from in vitro blood assays to corresponding in vivo dose-response curves, from which points of departure (PODs) were derived. The obtained values for rats and humans were comparable with reported no-observed-adverse-effect levels (NOAELs). Humans were found to be more susceptible than rats toward erythrocyte AChE inhibition induced by acute FNT exposure due to interspecies differences in toxicokinetics and toxicodynamics. The described approach adequately predicts toxicokinetics and acute toxicity of FNT, providing a proof-of-principle for applying this approach in a 3R-based chemical risk assessment paradigm.

In vitro-in silico-based prediction of inter-individual and inter-ethnic variations in the dose-dependent cardiotoxicity of R- and S-methadone in humans.

New approach methodologies predicting human cardiotoxicity are of interest to support or even replace in vivo-based drug safety testing. The present study presents an in vitro-in silico approach to predict the effect of inter-individual and inter-ethnic kinetic variations in the cardiotoxicity of R- and S-methadone in the Caucasian and the Chinese population. In vitro cardiotoxicity data, and metabolic data obtained from two approaches, using either individual human liver microsomes or recombinant cytochrome P450 enzymes (rCYPs), were integrated with physiologically based kinetic (PBK) models and Monte Carlo simulations to predict inter-individual and inter-ethnic variations in methadone-induced cardiotoxicity. Chemical specific adjustment factors were defined and used to derive dose-response curves for the sensitive individuals. Our simulations indicated that Chinese are more sensitive towards methadone-induced cardiotoxicity with Margin of Safety values being generally two-fold lower than those for Caucasians for both methadone enantiomers. Individual PBK models using microsomes and PBK models using rCYPs combined with Monte Carlo simulations predicted similar inter-individual and inter-ethnic variations in methadone-induced cardiotoxicity. The present study illustrates how inter-individual and inter-ethnic variations in cardiotoxicity can be predicted by combining in vitro toxicity and metabolic data, PBK modelling and Monte Carlo simulations. The novel methodology can be used to enhance cardiac safety evaluations and risk assessment of chemicals.

Applicability of generic PBK modelling in chemical hazard assessment: A case study with IndusChemFate.

Toxicology is moving away from animal testing towards in vitro tools to assess chemical safety. This new testing framework requires a quantitative method, i.e. kinetic modelling, which extrapolates effective concentrations in vitro to a bioequivalent human dose in vivo and which can be applied on "high throughput screening" of a wide variety of chemicals. Generic physiologically based kinetic (PBK) models help account for the role of toxicokinetics in setting human toxic exposure levels. Furthermore these models may be parameterized only on in silico QSARs and in vitro metabolism assays, thereby circumventing the use of in vivo toxicokinetics for this purpose. Though several such models exist their applicability domains have yet to be comprehensively assessed. This study extends previous evaluations of the PBK model IndusChemFate and compares it with its more complex biological complement ("TNO Model"). Both models were evaluated with a broad span of chemicals, varying regarding physicochemical properties. The results reveal that the "simpler" performed best, illustrating that IndusChemFate can be a useful first-tier for simulating toxicokinetics based on QSARs and in vitro parameters. Finally, proper quantitative in vitro to in vivo extrapolation conditions were illustrated starting with acetaminophen induced in vitro cytotoxicity in human HepaRG cells.

Predicting the in vivo developmental toxicity of benzo[a]pyrene (BaP) in rats by an in vitro-in silico approach.

Developmental toxicity testing is an animal-intensive endpoints in toxicity testing and calls for animal-free alternatives. Previous studies showed the applicability of an in vitro-in silico approach for predicting developmental toxicity of a range of compounds, based on data from the mouse embryonic stem cell test (EST) combined with physiologically based kinetic (PBK) modelling facilitated reverse dosimetry. In the current study, the use of this approach for predicting developmental toxicity of polycyclic aromatic hydrocarbons (PAHs) was evaluated, using benzo[a]pyrene (BaP) as a model compound. A rat PBK model of BaP was developed to simulate the kinetics of its main metabolite 3-hydroxybenzo[a]pyrene (3-OHBaP), shown previously to be responsible for the developmental toxicity of BaP. Comparison to in vivo kinetic data showed that the model adequately predicted BaP and 3-OHBaP blood concentrations in the rat. Using this PBK model and reverse dosimetry, a concentration-response curve for 3-OHBaP obtained in the EST was translated into an in vivo dose-response curve for developmental toxicity of BaP in rats upon single or repeated dose exposure. The predicted half maximal effect doses (ED50) amounted to 67 and 45 mg/kg bw being comparable to the ED50 derived from the in vivo dose-response data reported for BaP in the literature, of 29 mg/kg bw. The present study provides a proof of principle of applying this in vitro-in silico approach for evaluating developmental toxicity of BaP and may provide a promising strategy for predicting the developmental toxicity of related PAHs, without the need for extensive animal testing.

Physiologically based kinetic modelling based prediction of in vivo rat and human acetylcholinesterase (AChE) inhibition upon exposure to diazinon.

The present study predicts in vivo human and rat red blood cell (RBC) acetylcholinesterase (AChE) inhibition upon diazinon (DZN) exposure using physiological based kinetic (PBK) modelling-facilitated reverse dosimetry. Due to the fact that both DZN and its oxon metabolite diazoxon (DZO) can inhibit AChE, a toxic equivalency factor (TEF) was included in the PBK model to combine the effect of DZN and DZO when predicting in vivo AChE inhibition. The PBK models were defined based on kinetic constants derived from in vitro incubations with liver fractions or plasma of rat and human, and were used to translate in vitro concentration-response curves for AChE inhibition obtained in the current study to predicted in vivo dose-response curves. The predicted dose-response curves for rat matched available in vivo data on AChE inhibition, and the benchmark dose lower confidence limits for 10% inhibition (BMDL10 values) were in line with the reported BMDL10 values. Humans were predicted to be 6-fold more sensitive than rats in terms of AChE inhibition, mainly because of inter-species differences in toxicokinetics. It is concluded that the TEF-coded DZN PBK model combined with quantitative in vitro to in vivo extrapolation (QIVIVE) provides an adequate approach to predict RBC AChE inhibition upon acute oral DZN exposure, and can provide an alternative testing strategy for derivation of a point of departure (POD) in risk assessment.

Integrating in vitro data and physiologically based kinetic modeling-facilitated reverse dosimetry to predict human cardiotoxicity of methadone.

Development of novel testing strategies to detect adverse human health effects is of interest to replace in vivo-based drug and chemical safety testing. The aim of the present study was to investigate whether physiologically based kinetic (PBK) modeling-facilitated conversion of in vitro toxicity data is an adequate approach to predict in vivo cardiotoxicity in humans. To enable evaluation of predictions made, methadone was selected as the model compound, being a compound for which data on both kinetics and cardiotoxicity in humans are available. A PBK model for methadone in humans was developed and evaluated against available kinetic data presenting an adequate match. Use of the developed PBK model to convert concentration-response curves for the effect of methadone on human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) in the so-called multi electrode array (MEA) assay resulted in predictions for in vivo dose-response curves for methadone-induced cardiotoxicity that matched the available in vivo data. The results also revealed differences in protein plasma binding of methadone to be a potential factor underlying variation between individuals with respect to sensitivity towards the cardiotoxic effects of methadone. The present study provides a proof-of-principle of using PBK modeling-based reverse dosimetry of in vitro data for the prediction of cardiotoxicity in humans, providing a novel testing strategy in cardiac safety studies.

Quantitative in vitro-to-in vivo extrapolation (QIVIVE) of estrogenic and anti-androgenic potencies of BPA and BADGE analogues.

The goal of the present study was to obtain an in vivo relevant prioritization method for the endocrine potencies of different polycarbonate monomers, by combining in vitro bioassay data with physiologically based kinetic (PBK) modelling. PBK models were developed for a selection of monomers, including bisphenol A (BPA), two bisphenol F (BPF) isomers and four different bisphenol A diglycidyl ethers (BADGEs), using in vitro input data. With these models, the plasma concentrations of the compounds were simulated, providing means to estimate the dose levels at which the in vitro endocrine effect concentrations are reached. The results revealed that, whereas the in vitro relative potencies of different BADGEs (predominantly anti-androgenic effects) can be up to fourfold higher than BPA, the estimated in vivo potencies based on the oral equivalent doses are one to two orders of magnitude lower than BPA because of fast detoxification of the BADGEs. In contrast, the relative potencies of 2,2-BPF and 4,4-BPF increase when accounting for the in vivo availability. 4,4-BPF is estimated to be fivefold more potent than BPA in humans in vivo in inducing estrogenic effects and both 2,2-BPF and 4,4-BPF are estimated to be, respectively, 7 and 11-fold more potent in inducing anti-androgenic effects. These relative potencies were considered to be first-tier estimates, particularly given that the potential influence of intestinal metabolism on the in vivo availability was not accounted for. Overall, it can be concluded that both 2,2-BPF and 4,4-BPF are priority compounds.

Quantification of freely dissolved effect concentrations in in vitro cell-based bioassays.

Improved understanding of chemical exposure in in vitro bioassays is required for quantitative in vitro-in vivo extrapolation (QIVIVE). In this study, we quantified freely dissolved concentrations in medium sampled from in vitro cell-based bioassays (Cfree,medium) for nine chemicals with different hydrophobicity and speciation at the time point of dosing and after an incubation period of 24 h using solid-phase microextraction. The chemicals were tested in two reporter gene assays, the AREc32 assay indicative of the oxidative stress response and the PPARγ-GeneBLAzer assay that responds to chemicals which bind to the peroxisome proliferator-activated receptor gamma. For seven of the nine chemicals, Cfree,medium did not change significantly over time in both assays and the experimentally determined Cfree,medium generally agreed well with predictions of a mass balance model that describes the partitioning between proteinaceous and lipidous medium constituents, cells and the aqueous phase. Two chemicals showed a decrease of Cfree,medium in the AREc32 assay over time that was probably caused by cellular metabolism. Furthermore, Cfree,medium of the acidic chemical diclofenac deviated from the model predictions by more than a factor of 10 at higher concentrations, which indicates nonlinear binding and saturation of the medium proteins. Bioassay results are typically reported as nominal effect concentrations (ECnom), although it is established that freely dissolved effect concentrations (ECfree) are a better measure for the bioavailable dose and the method developed here provides a simple experimental approach to measure and model ECfree in in vitro bioassay for improved QIVIVE models.

Expert opinions on the acceptance of alternative methods in food safety evaluations: Formulating recommendations to increase acceptance of non-animal methods for kinetics.

Inclusion of alternative methods that replace, reduce, or refine (3R) animal testing within regulatory safety evaluations of chemicals generally faces many hurdles. The goal of the current work is to i) collect responses from key stakeholders involved in food safety evaluations on what they consider the most relevant factors that influence the acceptance and use of 3R methods and to ii) use these responses to formulate activities needed to increase the acceptance and use of 3R methods, particularly for kinetics. The stakeholders were contacted by e-mail for their opinions, asking the respondents to write down three barriers and/or drivers and scoring these by distributing 5 points over the three factors. The main barriers that obtained the highest aggregated scores were i) uncertain predictability 3R methods/lack of validation, ii) insufficient guidance regulators/industry and iii) insufficient harmonization of legislation. The major driver identified was the possibility of 3R methods to provide more mechanistic information. Based on the results, recommendations are given to enhance the acceptance and application of 3R toxicokinetic methods in food safety evaluations. These include steering of regulatory data requirements as well as creating (funding) opportunities for development and validation of alternative methods for kinetics and development of guidances.

In vitro to in vivo extrapolation of effective dosimetry in developmental toxicity testing: Application of a generic PBK modelling approach.

Incorporation of kinetics to quantitative in vitro to in vivo extrapolations (QIVIVE) is a key step for the realization of a non-animal testing paradigm, in the sphere of regulatory toxicology. The use of Physiologically-Based Kinetic (PBK) modelling for determining systemic doses of chemicals at the target site is accepted to be an indispensable element for such purposes. Nonetheless, PBK models are usually designed for a single or a group of compounds and are considered demanding, with respect to experimental data needed for model parameterization. Alternatively, we evaluate here the use of a more generic approach, i.e. the so-called IndusChemFate model, which is based on incorporated QSAR model parametrization. The model was used to simulate the in vivo kinetics of three diverse classes of developmental toxicants: triazoles, glycol ethers' alkoxyacetic acid metabolites and phthalate primary metabolites. The model required specific input per each class of compounds. These compounds were previously tested in three alternative assays: the whole-embryo culture (WEC), the zebrafish embryo test (ZET), and the mouse embryonic stem cell test (EST). Thereafter, the PBK-simulated blood levels at toxic in vivo doses were compared to the respective in vitro effective concentrations. Comparisons pertaining to relative potency and potency ranking with integration of kinetics were similar to previously obtained comparisons. Additionally, all three in vitro systems produced quite comparable results, and hence, a combination of alternative tests is still preferable for predicting the endpoint of developmental toxicity in vivo. This approach is put forward as biologically more plausible since plasma concentrations, rather than external administered doses, constitute the most direct in vivo dose metric.

New probabilistic risk assessment of ethylhexyl methoxycinnamate: Comparing the genotoxic effects of trans- and cis-EHMC.

Ethylhexyl methoxycinnamate (EHMC) is a widely used UV filter present in a large number of personal care products (PCPs). Under normal conditions, EHMC occurs in a mixture of two isomers: trans-EHMC and cis-EHMC in a ratio of 99:1. When exposed to sunlight, the trans isomer is transformed to the less stable cis isomer and the efficiency of the UV filter is reduced. To date, the toxicological effects of the cis-EHMC isomer remain largely unknown. We developed a completely new method for preparing cis-EHMC. An EHMC technical mixture was irradiated using a UV lamp and 98% pure cis-EHMC was isolated from the irradiated solution using column chromatography. The genotoxic effects of the isolated cis-EHMC isomer and the nonirradiated trans-EHMC were subsequently measured using two bioassays (SOS chromotest and UmuC test). In the case of trans-EHMC, significant genotoxicity was observed using both bioassays at the highest concentrations (0.5 - 4 mg mL-1 ). In the case of cis-EHMC, significant genotoxicity was only detected using the UmuC test at concentrations of 0.25 - 1 mg mL-1 . Based on these results, the NOEC was calculated for both cis- and trans-EHMC, 0.038 and 0.064 mg mL-1 , respectively. Risk assessment of dermal, oral and inhalation exposure to PCPs containing EHMC was carried out for a female population using probabilistic simulation and by using Quantitative in vitro to in vivo extrapolation (QIVIVE). The risk of cis-EHMC was found to be ∼1.7 times greater than trans-EHMC. In the case of cis-EHMC, a hazard index of 1 was exceeded in the 92nd percentile. Based on the observed differences between the isomers, EHMC application in PCPs requires detailed reassessment. Further exploration of the toxicological effects and properties of cis-EHMC is needed in order to correctly predict risks posed to humans and the environment. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 569-580, 2017.

Protectiveness of NAM-based hazard assessment - which testing scope is required?

Hazard assessment (HA) requires toxicity tests to allow deriving protective points of departure (PoDs) for risk assessment irrespective of a compound's mode of action (MoA). The scope of in vitro test batteries (ivTB) thereby necessitated for systemic toxicity is still unclear. We explored the protectiveness regarding systemic toxicity of an ivTB with a scope, which was guided by previous findings from rodent studies, where examining six main targets, including liver and kidney, was sufficient to predict the guideline scope-based PoD with high probability. The ivTB comprises human in vitro models representing liver, kidney, lung and the neuronal system covering transcriptome, mitochondrial dysfunction and neuronal outgrowth. Additionally, 32 CALUX®- and 10 HepG2 BAC-GFP reporters cover a broad range of disturbance mechanisms. Eight compounds were chosen for causing adverse effects such as immunotoxicity or anemia in vivo, i.e., effects not directly covered by assays in the ivTB. PoDs derived from the ivTB and from oral repeated dose studies in rodents were extrapolated to maximum unbound plasma concentrations for comparison. The ivTB-based PoDs were one to five orders of magnitude lower than in vivo PoDs for six of eight compounds, implying that they were protective. The extent of in vitro response varied across test compounds. Especially for hematotoxic substances, the ivTB showed either no response or only cytotoxicity. Assays better capturing this type of hazard would be needed to complement the ivTB. This study highlights the potentially broad applicability of ivTBs for deriving protective PoDs of compounds with unknown MoA.

Animal tests are used to determine which amount of a chemical is toxic ('threshold of toxicity') and which organs are affected. In principle, the threshold can also be derived solely from tests with cultured cells. However, only a limited number of cell types can practically be tested, so one challenge is to determine how many and which types shall be tested. In animal studies, only few organs including liver and kidney are regularly among those most sensitively affected. We explored whether a cell-based test battery representing these sensitive organs and covering important mechanisms of toxicity can be used to derive protective human thresholds. To challenge this approach, eight chemicals were tested that primarily cause effects in organs not directly represented in our test battery. Results provided protective thresholds for most of the investigated compounds and gave indications how to further improve the approach towards a full-fledged replacement for animal tests.

Intestinal in vitro transport assay combined with physiologically based kinetic modeling as a tool to predict bile acid levels in vivo.

Bile acid homeostasis is vital for numerous metabolic and immune functions in humans. The enterohepatic circulation of bile acids is extremely efficient, with ~95% of intestinal bile acids being reabsorbed. Disturbing intestinal bile acid uptake is expected to substantially affect intestinal and systemic bile acid levels. Here, we aimed to predict the effects of apical sodium-dependent bile acid transporter (ASBT)-inhibition on systemic plasma levels. For this, we combined in vitro Caco-2 cell transport assays with physiologically based (PBK) modeling. We used the selective ASBT-inhibitor odevixibat (ODE) as a model compound. Caco-2 cells grown on culture inserts were used to obtain transport kinetic parameters of glycocholic acid (GCA). The apparent Michaelis-Menten constant (Km,app), apparent maximal intestinal transport rate (Vmax,app), and ODE's inhibitory constant (Ki) were determined for GCA. These kinetic parameters were incorporated into a PBK model and used to predict the ASBT inhibition effects on plasma bile acid levels. GCA is transported over Caco-2 cells in an active and sodium-dependent manner, indicating the presence of functional ASBT. ODE inhibited GCA transport dose-dependently. The PBK model predicted that oral doses of ODE reduced conjugated bile acid levels in plasma. Our simulations match in vivo data and provide a first proof-of-principle for the incorporation of active intestinal bile acid uptake in a bile acid PBK model. This approach could in future be of use to predict the effects of other ASBT-inhibitors on plasma and intestinal bile acid levels.

Bile acids regulate digestion and immune functions. Too little bile acid reuptake in the gut is related to several diseases, including inflammatory bowel disease. This study investigates how reducing bile acid absorption affects bile acid levels in humans using the drug odevixibat (ODE) as an example. ODE reduces bile acid absorption by blocking the intestinal bile acid transporter protein in gut cells. The transport of a bile acid through a gut cell line commonly used to model the intestinal barrier was measured with and without ODE, and mathematical modeling was used to translate the laboratory results to whole-body effects. This combined approach accurately predicted the known effects of ODE on intestinal and bloodstream bile acid levels in humans. This novel approach could be used to predict the effects of other chemicals on intestinal bile acid absorption and intestinal and bloodstream bile acid levels instead of animal testing.

Physiologically based Kinetic Modeling-Facilitated Quantitative In Vitro to In Vivo Extrapolation to Predict the Effects of Aloe-Emodin in Rats and Humans.

Aloe-emodin, a natural hydroxyanthraquinone, exerts both adverse and protective effects. This study aimed at investigating these potential effects of aloe-emodin in humans upon the use of food supplements and herbal medicines using a physiologically based kinetic (PBK) modeling-facilitated quantitative in vitro to in vivo extrapolation (QIVIVE) approach. For this, PBK models in rats and humans were established for aloe-emodin including its active metabolite rhein and used to convert in vitro data on hepatotoxicity, nephrotoxicity, reactive oxidative species (ROS) generation, and Nrf2 induction to corresponding in vivo dose-response curves, from which points of departure (PODs) were derived by BMD analysis. The derived PODs were subsequently compared to the estimated daily intakes (EDIs) resulting from the use of food supplements or herbal medicines. It is concluded that the dose levels of aloe-emodin from food supplements or herbal medicines are unlikely to induce toxicity, ROS generation, or Nrf2 activation in liver and kidney.

Physiologically-Based Pharmacokinetic Modeling of the Postbiotic Supplement Urolithin A Predicts its Bioavailability Is Orders of Magnitude Lower than Concentrations that Induce Toxicity, but also Neuroprotective Effects.

SCOPE: A range of health benefits are attributed to consuming urolithin A (UA), such as improved muscle health, anti-aging activity, and neuroprotection, whereas few studies raise possible adverse effects at high doses, including genotoxicity and estrogenic effects. Therefore, understanding UA bioactivity and safety depends on its pharmacokinetics. However, there is no physiologically-based pharmacokinetic (PBPK) model available for UA, thus limiting reliable assessment of effects observed from in vitro experimentation. METHODS AND RESULTS: We characterizes glucuronidation rates of UA by human S9 fractions. Partitioning and other physicochemical parameters are predicted using quantitative structure-activity relationship tools. Solubility and dissolution kinetics are determined experimentally. These parameters are used to construct a PBPK model, and results are compared with data from human intervention studies. We evaluates how different supplementation scenarios may influence UA plasma and tissue concentrations. Concentrations at which either toxic or beneficial effects are previously observed in vitro appear unlikely to be achieved in vivo. CONCLUSION: A first PBPK model for UA is established. It enables prediction of systemic UA concentrations and is critical for extrapolating in vitro results to in vivo uses. Results support the safety of UA, but also challenge the potential for readily achieving beneficial effects by postbiotic supplementation.

New approach methodologies: A quantitative in vitro to in vivo extrapolation case study with PFASs.

PER: and polyfluoroalkyl substances (PFASs) have been associated with increased blood lipids in humans. Perfluorooctanoic acid (PFOA) has been also linked with elevated alanine transferase (ALT) serum levels in humans, and in rodents the liver is a main target organ for many PFASs. With the focus on New Approach Methodologies, the chronic oral equivalent effect doses were calculated for PFOA, PFNA (perfluorononanoic acid), PFHxS (perfluorohexanesulfonic acid) and PFOS (perfluorooctane sulfonic acid) based on in vitro effects measured in the HepaRG cell line. Selected in vitro readouts were considered biomarkers for lipid disturbances and hepatotoxicity. Concentration-response data obtained from HepaRG cells on triglyceride (TG) accumulation and expression changes of 12 selected genes (some involved in cholesterol homeostasis) were converted into corresponding human dose-response data, using physiologically based kinetic (PBK) model-facilitated reverse dosimetry. Next to this, the biokinetics of the chemicals were studied in the cell system. The current European dietary PFASs exposure overlaps with the calculated oral equivalent effect doses, indicating that the latter may lead to interference with hepatic gene expression and lipid metabolism. These findings illustrate an in vitro-in silico methodology, which can be applied for more PFASs, to select those that should be prioritized for further hazard characterization.

Predicting acute paraquat toxicity using physiologically based kinetic modelling incorporating in vitro active renal excretion via the OCT2 transporter.

Including active renal excretion in physiologically based kinetic (PBK) models can improve their use in quantitative in vitro- in vivo extrapolation (QIVIVE) as a new approach methodology (NAM) for predicting the acute toxicity of organic cation transporter 2 (OCT2) substrates like paraquat (PQ). To realise this NAM, kinetic parameters Vmax and Km for in vitro OCT2 transport of PQ were obtained from the literature. Appropriate scaling factors were applied to translate the in vitro Vmax to an in vivo Vmax. in vitro cytotoxicity data were defined in the rat RLE-6TN and L2 cell lines and the human A549 cell line. The developed PQ PBK model was used to apply reverse dosimetry for QIVIVE translating the in vitro cytotoxicity concentration-response curves to predicted in vivo toxicity dose-response curves after which the lower and upper bound benchmark dose (BMD) for 50% lethality (BMDL50 and BMDU50) were derived by applying BMD analysis. Comparing the predictions to the in vivo reported LD50 values resulted in a conservative prediction for rat and a comparable prediction for human showing proof of principle on the inclusion of active renal excretion and prediction of PQ acute toxicity for the developed NAM.

Role of bioavailability and protein binding of four anionic perfluoroalkyl substances in cell-based bioassays for quantitative in vitro to in vivo extrapolations.

Perfluoroalkyl substances (PFAS) are persistent and pose a risk to human health. High throughput screening (HTS) cell-based bioassays may inform risk assessment of PFAS provided that quantitative in vitro to in vivo extrapolation (QIVIVE) can be developed. The QIVIVE ratio is the ratio of nominal (Cnom) or freely dissolved concentration (Cfree) in human blood to Cnom or Cfree in the bioassays. Considering that the concentrations of PFAS in human plasma and in vitro bioassays may vary by orders of magnitude, we tested the hypothesis that anionic PFAS bind to proteins concentration-dependently and therefore the binding differs substantially between human plasma and bioassays, which has an impact on QIVIVE. Solid phase microextraction (SPME) with C18-coated fibers served to quantify the Cfree of four anionic PFAS (perfluorobutanoate (PFBA), perfluorooctanoate (PFOA), perfluorohexane sulfonate (PFHxS) and perfluorooctane sulfonate (PFOS)) in the presence of proteins and lipid, medium components, cells and human plasma over five orders of magnitude in concentrations. The C18-SPME method was used to quantify the non-linear binding to proteins, human plasma and medium, and the partition constants to cells. These binding parameters were used to predict Cfree of PFAS in cell bioassays and human plasma by a concentration-dependent mass balance model (MBM). The approach was illustrated with a reporter gene assay indicating activation of the peroxisome proliferator-activated receptor gamma (PPARγ-GeneBLAzer). Blood plasma levels were collected from literature for occupational exposure and the general population. The QIVIVEnom ratios were higher than the QIVIVEfree ratios due to the strong affinity to proteins and large differences in protein contents between human blood and bioassays. For human health risk assessment, the QIVIVEfree ratios of many in vitro assays need to be combined to cover all health relevant endpoints. If Cfree cannot be measured, they can be estimated with the MBM and concentration-dependent distribution ratios.

Integrating in vitro chemical transplacental passage into a generic PBK model: A QIVIVE approach.

With the increasing application of cell culture models as primary tools for predicting chemical safety, the quantitative extrapolation of the effective dose from in vitro to in vivo (QIVIVE) is of increasing importance. For developmental toxicity this requires scaling the in vitro observed dose-response characteristics to in vivo fetal exposure, while integrating maternal in vivo kinetics during pregnancy, in particular transplacental transfer. Here the transfer of substances across the placental barrier, has been studied using the in vitro BeWo cell assay and six embryotoxic compounds of different kinetic complexity. The BeWo assay results were incorporated in an existing generic Physiologically Based Kinetic (PBK) model which for this purpose was extended with rat pregnancy. Finally, as a "proof of principle", the BeWo PBK model was used to perform a QIVIVE based on developmental toxicity as observed in various different in vitro toxicity assays. The BeWo results illustrated different transport profiles of the chemicals across the BeWo monolayer, allocating the substances into two distinct groups: the 'quickly-transported' and the 'slowly-transported'. BeWo PBK exposure simulations during gestation were compared to experimentally measured maternal blood and fetal concentrations and a reverse dosimetry approach was applied to translate in vitro observed embryotoxicity into equivalent in vivo dose-response curves. This approach allowed for a direct comparison of the in vitro dose-response characteristics as observed in the Whole Embryo Culture (WEC), and the Embryonic Stem Cell test (cardiac:ESTc and neural:ESTn) with in vivo rat developmental toxicity data. Overall, the in vitro to in vivo comparisons suggest a promising future for the application of such QIVIVE methodologies for screening and prioritization purposes of developmental toxicants. Nevertheless, the clear need for further improvements is acknowledged for a wider application of the approach in chemical safety assessment.