Report of the First ONTOX Stakeholder Network Meeting: Digging Under the Surface of ONTOX Together With the Stakeholders.

The first Stakeholder Network Meeting of the EU Horizon 2020-funded ONTOX project was held on 13-14 March 2023, in Brussels, Belgium. The discussion centred around identifying specific challenges, barriers and drivers in relation to the implementation of non-animal new approach methodologies (NAMs) and probabilistic risk assessment (PRA), in order to help address the issues and rank them according to their associated level of difficulty. ONTOX aims to advance the assessment of chemical risk to humans, without the use of animal testing, by developing non-animal NAMs and PRA in line with 21st century toxicity testing principles. Stakeholder groups (regulatory authorities, companies, academia, non-governmental organisations) were identified and invited to participate in a meeting and a survey, by which their current position in relation to the implementation of NAMs and PRA was ascertained, as well as specific challenges and drivers highlighted. The survey analysis revealed areas of agreement and disagreement among stakeholders on topics such as capacity building, sustainability, regulatory acceptance, validation of adverse outcome pathways, acceptance of artificial intelligence (AI) in risk assessment, and guaranteeing consumer safety. The stakeholder network meeting resulted in the identification of barriers, drivers and specific challenges that need to be addressed. Breakout groups discussed topics such as hazard versus risk assessment, future reliance on AI and machine learning, regulatory requirements for industry and sustainability of the ONTOX Hub platform. The outputs from these discussions provided insights for overcoming barriers and leveraging drivers for implementing NAMs and PRA. It was concluded that there is a continued need for stakeholder engagement, including the organisation of a 'hackathon' to tackle challenges, to ensure the successful implementation of NAMs and PRA in chemical risk assessment.

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.

The role of receptor-mediated activities of 4- and 5-ring unsubstituted and methylated polycyclic aromatic hydrocarbons (PAHs) in developmental toxicity.

The present study evaluated the aryl hydrocarbon receptor (AhR), estrogen receptor-α (ER-α), and retinoic acid receptor (RAR) mediated activities of nine 4- and 5-ring unsubstituted and monomethylated polycyclic aromatic hydrocarbons (PAHs) using a series of Chemical-Activated LUciferase gene eXpression (CALUX) assays. The potential role of these aforementioned receptors in relation to the developmental toxicity of these PAHs was further assessed in the zebrafish embryotoxicity test (ZET). The results show that all nine tested PAHs were AhR agonists, benz[a]anthracene (BaA) and 8-methyl-benz[a]anthracene (8-MeBaA) were ER-α agonists, and none of the tested PAHs induced ER-α antagonistic or RAR (ant)agonistic activities. In the AhR CALUX assay, all the methylated PAHs showed higher potency (lower EC50) in activating the AhR than their respective unsubstituted PAHs, implying that the addition of a methyl substituent on the aromatic ring of PAHs could enhance their AhR-mediated activities. Co-exposure of zebrafish embryos with each individual PAH and an AhR antagonist (CH223191) counteracted the observed developmental retardations and embryo lethality to a certain extent, except for 8-methyl-benzo[a]pyrene (8-MeBaP). Co-exposure of zebrafish embryos with either of the two estrogenic PAHs (i.e., BaA and 8-MeBaA) and an ER-α antagonist (fulvestrant) neutralized embryo lethality induced by 50 µM BaA and the developmental retardations induced by 15 µM 8-MeBaA. Altogether, our findings suggest that the observed developmental retardations in zebrafish embryos by the PAH tested may partially be AhR- and/or ER-α-mediated, whereas the RAR seems not to be relevant for the PAH-induced developmental toxicity in the ZET.

Drug Metabolism of Hepatocyte-like Organoids and Their Applicability in In Vitro Toxicity Testing.

Emerging advances in the field of in vitro toxicity testing attempt to meet the need for reliable human-based safety assessment in drug development. Intrahepatic cholangiocyte organoids (ICOs) are described as a donor-derived in vitro model for disease modelling and regenerative medicine. Here, we explored the potential of hepatocyte-like ICOs (HL-ICOs) in in vitro toxicity testing by exploring the expression and activity of genes involved in drug metabolism, a key determinant in drug-induced toxicity, and the exposure of HL-ICOs to well-known hepatotoxicants. The current state of drug metabolism in HL-ICOs showed levels comparable to those of PHHs and HepaRGs for CYP3A4; however, other enzymes, such as CYP2B6 and CYP2D6, were expressed at lower levels. Additionally, EC50 values were determined in HL-ICOs for acetaminophen (24.0−26.8 mM), diclofenac (475.5−>500 µM), perhexiline (9.7−>31.5 µM), troglitazone (23.1−90.8 µM), and valproic acid (>10 mM). Exposure to the hepatotoxicants showed EC50s in HL-ICOs comparable to those in PHHs and HepaRGs; however, for acetaminophen exposure, HL-ICOs were less sensitive. Further elucidation of enzyme and transporter activity in drug metabolism in HL-ICOs and exposure to a more extensive compound set are needed to accurately define the potential of HL-ICOs in in vitro toxicity testing.

Towards best use and regulatory acceptance of generic physiologically based kinetic (PBK) models for in vitro-to-in vivo extrapolation (IVIVE) in chemical risk assessment.

With an increasing need to incorporate new approach methodologies (NAMs) in chemical risk assessment and the concomitant need to phase out animal testing, the interpretation of in vitro assay readouts for quantitative hazard characterisation becomes more important. Physiologically based kinetic (PBK) models, which simulate the fate of chemicals in tissues of the body, play an essential role in extrapolating in vitro effect concentrations to in vivo bioequivalent exposures. As PBK-based testing approaches evolve, it will become essential to standardise PBK modelling approaches towards a consensus approach that can be used in quantitative in vitro-to-in vivo extrapolation (QIVIVE) studies for regulatory chemical risk assessment based on in vitro assays. Based on results of an ECETOC expert workshop, steps are recommended that can improve regulatory adoption: (1) define context and implementation, taking into consideration model complexity for building fit-for-purpose PBK models, (2) harmonise physiological input parameters and their distribution and define criteria for quality chemical-specific parameters, especially in the absence of in vivo data, (3) apply Good Modelling Practices (GMP) to achieve transparency and design a stepwise approach for PBK model development for risk assessors, (4) evaluate model predictions using alternatives to in vivo PK data including read-across approaches, (5) use case studies to facilitate discussions between modellers and regulators of chemical risk assessment. Proof-of-concepts of generic PBK modelling approaches are published in the scientific literature at an increasing rate. Working on the previously proposed steps is, therefore, needed to gain confidence in PBK modelling approaches for regulatory use.

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.

Acetylcholinesterase inhibition in electric eel and human donor blood: an in vitro approach to investigate interspecies differences and human variability in toxicodynamics.

In chemical risk assessment, default uncertainty factors are used to account for interspecies and interindividual differences, and differences in toxicokinetics and toxicodynamics herein. However, these default factors come with little scientific support. Therefore, our aim was to develop an in vitro method, using acetylcholinesterase (AChE) inhibition as a proof of principle, to assess both interspecies and interindividual differences in toxicodynamics. Electric eel enzyme and human blood of 20 different donors (12 men/8 women) were exposed to eight different compounds (chlorpyrifos, chlorpyrifos-oxon, phosmet, phosmet-oxon, diazinon, diazinon-oxon, pirimicarb, rivastigmine) and inhibition of AChE was measured using the Ellman method. The organophosphate parent compounds, chlorpyrifos, phosmet and diazinon, did not show inhibition of AChE. All other compounds showed concentration-dependent inhibition of AChE, with IC50s in human blood ranging from 0.2-29 µM and IC20s ranging from 0.1-18 µM, indicating that AChE is inhibited at concentrations relevant to the in vivo human situation. The oxon analogues were more potent inhibitors of electric eel AChE compared to human AChE. The opposite was true for carbamates, pointing towards interspecies differences for AChE inhibition. Human interindividual variability was low and ranged from 5-25%, depending on the concentration. This study provides a reliable in vitro method for assessing human variability in AChE toxicodynamics. The data suggest that the default uncertainty factor of ~ 3.16 may overestimate human variability for this toxicity endpoint, implying that specific toxicodynamic-related adjustment factors can support quantitative in vitro to in vivo extrapolations that link kinetic and dynamic data to improve chemical risk assessment.

Time-Resolved Freely Dissolved Concentrations of Semivolatile and Hydrophobic Test Chemicals in In Vitro Assays-Measuring High Losses and Crossover by Headspace Solid-Phase Microextraction.

In vitro assays are normally conducted in plastic multiwell plates open to exchange with the ambient air. The concentration of test substances freely available to cells is often not known, can change over time, and is difficult to measure in the small volumes in microplates. However, even a well-characterized toxicological response is of limited value if it cannot be linked to a well-defined exposure level. The aim of this study was to develop and apply an approach for determining time-resolved freely dissolved concentrations of semivolatile and hydrophobic organic chemicals (SVHOCs) in in vitro assays: (1) free fractions were measured by a new medium dilution method and (2) time-resolved loss curves were obtained by measurements of total concentrations in 96-well plates during incubations at 37 °C. Headspace solid-phase microextraction was used as an analytical technique for 24 model chemicals spanning 6 chemical groups and 4-5 orders of magnitude in Kow and Kaw. Free fractions were >30% for chemicals with log Kow < 3.5 and then decreased with increasing log Kow. Medium concentrations declined significantly (>50%) within 24 h of incubation for all 20 chemicals having log Kow > 4 or log Kaw > -3.5 in serum-free medium. Losses of chemicals were lower for medium containing 10% fetal bovine serum, most significantly for chemicals with log Kow > 4. High crossover to neighboring wells also was observed below log Kow of 4 and log Kaw of -3.5. Sealing the well plates had limited effect on the losses but clearly reduced crossover. The high losses and crossover of most tested chemicals question the suitability of multiwell plates for in vitro testing of SVHOCs and call for (1) test systems that minimize losses, (2) methods to control in vitro exposure, (3) analytical confirmation of exposure, and (4) exposure control and confirmation being included in good in vitro reporting standards.

Influence of in Vitro Assay Setup on the Apparent Cytotoxic Potency of Benzalkonium Chlorides.

The nominal concentration is generally used to express concentration-effect relationships in in vitro toxicity assays. However, the nominal concentration does not necessarily represent the exposure concentration responsible for the observed effect. Surfactants accumulate at interphases and likely sorb to in vitro system components such as serum protein and well plate plastic. The extent of sorption and the consequences of this sorption on in vitro readouts is largely unknown for these chemicals. The aim of this study was to demonstrate the effect of sorption to in vitro components on the observed cytotoxic potency of benzalkonium chlorides (BAC) varying in alkyl chain length (6-18 carbon atoms, C6-18) in a basal cytotoxicity assay with the rainbow trout gill cell line (RTgill-W1). Cells were exposed for 48 h in 96-well plates to increasing concentration of BACs in exposure medium containing 0, 60 µM bovine serum albumin (BSA) or 10% fetal bovine serum (FBS). Before and after exposure, BAC concentrations in exposure medium were analytically determined. Based on freely dissolved concentrations at the end of the exposure, median effect concentrations (EC50) decreased with increasing alkyl chain length up to 14 carbons. For BAC with alkyl chains of 12 or more carbons, EC50's based on measured concentrations after exposure in supplement-free medium were up to 25-times lower than EC50's calculated using nominal concentrations. When BSA or FBS was added to the medium, a decrease in cytotoxic potency of up to 22 times was observed for BAC with alkyl chains of eight or more carbons. The results of this study emphasize the importance of expressing the in vitro readouts as a function of a dose metric that is least influenced by assay setup to compare assay sensitivities and chemical potencies.

Characterizing the coverage of critical effects relevant in the safety evaluation of food additives by AOPs.

There is considerable interest in adverse outcome pathways (AOPs) as a means of organizing biological and toxicological information to assist in data interpretation and method development. While several chemical sectors have shown considerable progress in applying this approach, this has not been the case in the food sector. In the present study, safety evaluation reports of food additives listed in Annex II of Regulation (EC) No 1333/2008 of the European Union were screened to qualitatively and quantitatively characterize toxicity induced in laboratory animals. The resulting database was used to identify the critical adverse effects used for risk assessment and to investigate whether food additives share common AOPs. Analysis of the database revealed that often such scrutiny of AOPs was not possible or necessary. For 69% of the food additives, the report did not document any adverse effects in studies based on which the safety evaluation was performed. For the remaining 31% of the 326 investigated food additives, critical adverse effects and related points of departure for establishing health-based guidance values could be identified. These mainly involved effects on the liver, kidney, cardiovascular system, lymphatic system, central nervous system and reproductive system. AOPs are available for many of these apical endpoints, albeit to different degrees of maturity. For other adverse outcomes pertinent to food additives, including gastrointestinal irritation and corrosion, AOPs are lacking. Efforts should focus on developing AOPs for these particular endpoints.

A novel hypothesis for an alkaline phosphatase 'rescue' mechanism in the hepatic acute phase immune response.

The liver isoform of the enzyme alkaline phosphatase (AP) has been used classically as a serum biomarker for hepatic disease states such as hepatitis, steatosis, cirrhosis, drug-induced liver injury, and hepatocellular carcinoma. Recent studies have demonstrated a more general anti-inflammatory role for AP, as it is capable of dephosphorylating potentially deleterious molecules such as nucleotide phosphates, the pathogenic endotoxin lipopolysaccharide (LPS), and the contact clotting pathway activator polyphosphate (polyP), thereby reducing inflammation and coagulopathy systemically. Yet the mechanism underlying the observed increase in liver AP levels in circulation during inflammatory insults is largely unknown. This paper hypothesizes an immunological role for AP in the liver and the potential of this system for damping generalized inflammation along with a wide range of ancillary pathologies. Based on the provided framework, a mechanism is proposed in which AP undergoes transcytosis in hepatocytes from the canalicular membrane to the sinusoidal membrane during inflammation and the enzyme's expression is upregulated as a result. Through a tightly controlled, nucleotide-stimulated negative feedback process, AP is transported in this model as an immune complex with immunoglobulin G by the asialoglycoprotein receptor through the cell and secreted into the serum, likely using the receptor's State 1 pathway. The subsequent dephosphorylation of inflammatory stimuli by AP and uptake of the circulating immune complex by endothelial cells and macrophages may lead to decreased inflammation and coagulopathy while providing an early upstream signal for the induction of a number of anti-inflammatory gene products, including AP itself.

Use of Physiologically Based Kinetic Modeling to Predict Deoxynivalenol Metabolism and Its Role in Intestinal Inflammation and Bile Acid Kinetics in Humans.

Current points of departure used to derive health-based guidance values for deoxynivalenol (DON) are based on studies in laboratory animals. Here, an animal-free testing approach was adopted in which a reverse dosimetry physiologically based kinetic (PBK) modeling is used to predict in vivo dose response curves for DON's effects on intestinal pro-inflammatory cytokine secretion and intestinal bile acid reabsorption in humans from concentration-effect relationships for DON in vitro. The calculated doses for inducing a 5% added effect above the background level (ED5) of DON for increasing IL-1ß secretion in intestinal tissue and for increasing the amounts in the colon lumen of glycochenodeoxycholic acid (GCDCA) were 246 and 36 µg/kg bw/day, respectively. These in vitro-in silico-derived ED5 values were compared to human dietary DON exposure levels, indicating that the risk of DON's effects on these end points occurring in various human populations cannot be excluded. This in vitro-in silico approach provides a novel testing strategy for hazard and risk assessment without using laboratory animals.

The effects of hexabromocyclododecane on the transcriptome and hepatic enzyme activity in three human HepaRG-based models.

The disruption of thyroid hormone homeostasis by hexabromocyclododecane (HBCD) in rodents is hypothesized to be due to HBCD increasing the hepatic clearance of thyroxine (T4). The extent to which these effects are relevant to humans is unclear. To evaluate HBCD effects on humans, the activation of key hepatic nuclear receptors and the consequent disruption of thyroid hormone homeostasis were studied in different human hepatic cell models. The hepatoma cell line, HepaRG, cultured as two-dimensional (2D), sandwich (SW) and spheroid (3D) cultures, and primary human hepatocytes (PHH) cultured as sandwich were exposed to 1 and 10 µM HBCD and characterized for their transcriptome changes. Pathway enrichment analysis showed that 3D models, followed by SW, had a stronger transcriptome response to HBCD, which is explained by the higher expression of hepatic nuclear receptors but also greater accumulation of HBCD measured inside cells in these models. The Pregnane X receptor pathway is one of the pathways most upregulated across the three hepatic models, followed by the constitutive androstane receptor and general hepatic nuclear receptors pathways. Lipid metabolism pathways had a downregulation tendency in all exposures and in both PHH and the three cultivation modes of HepaRG. The activity of enzymes related to PXR/CAR induction and T4 metabolism were evaluated in the three different types of HepaRG cultures exposed to HBCD for 48 h. Reference inducers, rifampicin and PCB-153 did affect 2D and SW HepaRG cultures' enzymatic activity but not 3D. HBCD did not induce the activity of any of the studied enzymes in any of the cell models and culture methods. This study illustrates that for nuclear receptor-mediated T4 disruption, transcriptome changes might not be indicative of an actual adverse effect. Clarification of the reasons for the lack of translation is essential to evaluate new chemicals' potential to be thyroid hormone disruptors by altering thyroid hormone metabolism.

Integrating distribution kinetics and toxicodynamics to assess repeat dose neurotoxicity in vitro using human BrainSpheres: a case study on amiodarone.

For ethical, economical, and scientific reasons, animal experimentation, used to evaluate the potential neurotoxicity of chemicals before their release in the market, needs to be replaced by new approach methodologies. To illustrate the use of new approach methodologies, the human induced pluripotent stem cell-derived 3D model BrainSpheres was acutely (48 h) or repeatedly (7 days) exposed to amiodarone (0.625-15 µM), a lipophilic antiarrhythmic drug reported to have deleterious effects on the nervous system. Neurotoxicity was assessed using transcriptomics, the immunohistochemistry of cell type-specific markers, and real-time reverse transcription-polymerase chain reaction for various genes involved in the lipid metabolism. By integrating distribution kinetics modeling with neurotoxicity readouts, we show that the observed time- and concentration-dependent increase in the neurotoxic effects of amiodarone is driven by the cellular accumulation of amiodarone after repeated dosing. The development of a compartmental in vitro distribution kinetics model allowed us to predict the change in cell-associated concentrations in BrainSpheres with time and for different exposure scenarios. The results suggest that human cells are intrinsically more sensitive to amiodarone than rodent cells. Amiodarone-induced regulation of lipid metabolism genes was observed in brain cells for the first time. Astrocytes appeared to be the most sensitive human brain cell type in vitro. In conclusion, assessing readouts at different molecular levels after the repeat dosing of human induced pluripotent stem cell-derived BrainSpheres in combination with the compartmental modeling of in vitro kinetics provides a mechanistic means to assess neurotoxicity pathways and refine chemical safety assessment for humans.

Quantifying processes determining the free concentration of phenanthrene in Basal cytotoxicity assays.

Difficulties may arise when extrapolating in vitro derived toxicity data to in vivo toxicity data because of the high variability and occasional low sensitivity of in vitro results. Differences in the free concentration of a test compound between in vitro and in vivo systems and between different in vitro systems may in part explain this variability and sensitivity difference. The aim of this study was to determine what assay components influence the free concentration of phenanthrene in a Balb/c 3T3 and RTgill-W1 MTT assay. Partition coefficients of phenanthrene to serum, well plate plastic, cells, and headspace were measured and subsequently used to model the free concentration of the compound in vitro. The estimated free concentration was compared to the free concentration measured in the assays using solid phase microextraction (SPME). Results indicate that the free concentration of phenanthrene, a relatively volatile and hydrophobic compound, is significantly reduced in a typical in vitro setup as it binds to matrices such as serum protein and well plate plastic. A reduction in free concentration due to increasing serum protein levels is accompanied by an increase in the median effect concentration (EC(50)) and can be modeled, with the exception of evaporation, using the partition coefficients of the compound to assay components.

Predicting adult fish acute lethality with the zebrafish embryo: relevance of test duration, endpoints, compound properties, and exposure concentration analysis.

The zebrafish embryo toxicity test has been proposed as an alternative for the acute fish toxicity test, which is required by various regulations for environmental risk assessment of chemicals. We investigated the reliability of the embryo test by probing organic industrial chemicals with a wide range of physicochemical properties, toxicities, and modes of toxic action. Moreover, the relevance of using measured versus nominal (intended) exposure concentrations, inclusion of sublethal endpoints, and different exposure durations for the comparability with reported fish acute toxicity was explored. Our results confirm a very strong correlation of zebrafish embryo to fish acute toxicity. When toxicity values were calculated based on measured exposure concentrations, the slope of the type II regression line was 1 and nearly passed through the origin (1 to 1 correlation). Measured concentrations also explained several apparent outliers. Neither prolonged exposure (up to 120 h) nor consideration of sublethal effects led to a reduced number of outliers. Yet, two types of compounds were less lethal to embryos than to adult fish: a neurotoxic compound acting via sodium channels (permethrin) and a compound requiring metabolic activation (allyl alcohol).

Dynamic Mass Balance Modeling for Chemical Distribution Over Time in In Vitro Systems With Repeated Dosing.

As toxicologists and risk assessors move away from animal testing and more toward using in vitro models and biological modeling, it is necessary to produce tools to quantify the chemical distribution within the in vitro environment prior to extrapolating in vitro concentrations to human equivalent doses. Although models predicting chemical distribution in vitro have been developed, very little has been done for repeated dosing scenarios, which are common in prolonged experiments where the medium needs to be refreshed. Failure to account for repeated dosing may lead to inaccurate estimations of exposure and introduce bias into subsequent in vitro to in vivo extrapolations. Our objectives were to develop a dynamic mass balance model for repeated dosing in in vitro systems; to evaluate model accuracy against experimental data; and to perform illustrative simulations to assess the impact of repeated doses on predicted cellular concentrations. A novel dynamic in vitro partitioning mass balance model (IV-MBM DP v1.0) was created based on the well-established fugacity approach. We parameterized and applied the dynamic mass balance model to single dose and repeat dosing scenarios, and evaluated the predicted medium and cellular concentrations against available empirical data. We also simulated repeated dosing scenarios for organic chemicals with a range of partitioning properties and compared the in vitro distributions over time. In single dose scenarios, for which only medium concentrations were available, simulated concentrations predicted measured concentrations with coefficients of determination (R 2) of 0.85-0.89, mean absolute error within a factor of two and model bias of nearly one. Repeat dose scenario simulations displayed model bias <2 within the cell lysate, and ∼1.5-3 in the medium. The concordance between simulated and available experimental data supports the predictive capacity of the IV-MBM DP v1.0 tool, but further evaluation as empirical data becomes available is warranted, especially for cellular concentrations.

Application of the Adverse Outcome Pathway Concept to In Vitro Nephrotoxicity Assessment: Kidney Injury due to Receptor-Mediated Endocytosis and Lysosomal Overload as a Case Study.

Application of adverse outcome pathways (AOP) and integration of quantitative in vitro to in vivo extrapolation (QIVIVE) may support the paradigm shift in toxicity testing to move from apical endpoints in test animals to more mechanism-based in vitro assays. Here, we developed an AOP of proximal tubule injury linking a molecular initiating event (MIE) to a cascade of key events (KEs) leading to lysosomal overload and ultimately to cell death. This AOP was used as a case study to adopt the AOP concept for systemic toxicity testing and risk assessment based on in vitro data. In this AOP, nephrotoxicity is thought to result from receptor-mediated endocytosis (MIE) of the chemical stressor, disturbance of lysosomal function (KE1), and lysosomal disruption (KE2) associated with release of reactive oxygen species and cytotoxic lysosomal enzymes that induce cell death (KE3). Based on this mechanistic framework, in vitro readouts reflecting each KE were identified. Utilizing polymyxin antibiotics as chemical stressors for this AOP, the dose-response for each in vitro endpoint was recorded in proximal tubule cells from rat (NRK-52E) and human (RPTEC/TERT1) in order to (1) experimentally support the sequence of key events (KEs), to (2) establish quantitative relationships between KEs as a basis for prediction of downstream KEs based on in vitro data reflecting early KEs and to (3) derive suitable in vitro points of departure for human risk assessment. Time-resolved analysis was used to support the temporal sequence of events within this AOP. Quantitative response-response relationships between KEs established from in vitro data on polymyxin B were successfully used to predict in vitro toxicity of other polymyxin derivatives. Finally, a physiologically based kinetic (PBK) model was utilized to transform in vitro effect concentrations to a human equivalent dose for polymyxin B. The predicted in vivo effective doses were in the range of therapeutic doses known to be associated with a risk for nephrotoxicity. Taken together, these data provide proof-of-concept for the feasibility of in vitro based risk assessment through integration of mechanistic endpoints and reverse toxicokinetic modelling.

A regression-based QSAR-model to predict acute toxicity of aromatic chemicals in tadpoles of the Japanese brown frog (Rana japonica): Calibration, validation, and future developments to support risk assessment of chemicals in amphibians.

Amphibian populations are undergoing a global decline worldwide. Such decline has been attributed to their unique physiology, ecology, and exposure to multiple stressors including chemicals, temperature, and biological hazards such as fungi of the Batrachochytrium genus, viruses such as Ranavirus, and habitat reduction. There are limited toxicity data for chemicals available for amphibians and few quantitative structure-activity relationship (QSAR) models have been developed and are publicly available. Such QSARs provide important tools to assess the toxicity of chemicals particularly in a data poor context. QSARs provide important tools to assess the toxicity of chemicals particularly when no toxicological data are available. This manuscript provides a description and validation of a regression-based QSAR model to predict, in a quantitative manner, acute lethal toxicity of aromatic chemicals in tadpoles of the Japanese brown frog (Rana japonica). QSAR models for acute median lethal molar concentrations (LC50-12 h) of waterborne chemicals using the Monte Carlo method were developed. The statistical characteristics of the QSARs were described as average values obtained from five random distributions into training and validation sets. Predictions from the model gave satisfactory results for the overall training set (R2 = 0.72 and RMSE = 0.33) and were even more robust for the validation set (R2 = 0.96 and RMSE = 0.11). Further development of QSAR models in amphibians, particularly for other life stages and species, are discussed.