Probabilistic risk assessment of residential exposure to electric arc furnace steel slag using Bayesian model of relative bioavailability and PBPK modeling of manganese.

Electric arc furnace (EAF) slag is a coproduct of steel production used primarily for construction purposes. Some applications of EAF slag result in residential exposures by incidental ingestion and inhalation of airborne dust. To evaluate potential health risks, an EAF slag characterization program was conducted to measure concentrations of metals and leaching potential (including oral bioaccessibility) in 38 EAF slag samples. Arsenic, hexavalent chromium, iron, vanadium, and manganese (Mn) were identified as constituents of interest (COIs). Using a probabilistic risk assessment (PRA) approach, estimated distributions of dose for COIs were assessed, and increased cancer risks and noncancer hazard quotients (HQs) at the 50th and 95th percentiles were calculated. For the residents near slag-covered roads, cancer risk and noncancer HQs were <1E - 6 and 1, respectively. For residential driveway or landscape exposure, at the 95th percentile, cancer risks were 1E - 6 and 7E - 07 based on oral exposure to arsenic and hexavalent chromium, respectively. HQs ranged from 0.07 to 2 with the upper bound due to ingestion of Mn among children. To expand the analysis, a previously published physiologically based pharmacokinetic (PBPK) model was used to estimate Mn levels in the globus pallidus for both exposure scenarios and further evaluate the potential for Mn neurotoxicity. The PBPK model estimated slightly increased Mn in the globus pallidus at the 95th percentile of exposure, but concentrations did not exceed no-observed-adverse-effect levels for neurological effects. Overall, the assessment found that the application of EAF slag in residential areas is unlikely to pose a health hazard or increased cancer risk.

Assessment of the in vivo genotoxic potential of three smoke flavoring primary product mixtures.

Smoke flavorings are mixtures generated from wood pyrolysis that are filtered to remove tar and are often considered healthier alternatives to conventional smoking processes. While the latter is mostly unregulated, smoke-flavoring primary products (SFPPs) are undergoing the 10-year required re-evaluation in the European Union (EU). To comply with recent smoke flavor guidance, in vivo micronucleus studies in rats and transgenic rodent (TGR) mutation assays in Muta™Mice were conducted on three SFPPs. For most studies, typical limit doses were exceeded to comply with regulatory requests. Exposure to SFPPs by oral gavage did not result in significant increases in bone marrow micronucleus formation. Except for one group, exposure to SFPPs via feed for 28 days did not result in significant increases in mutant frequency (MF) in the glandular stomach or liver. One group exposed to a maximal feasible dietary dose of 50,000 ppm (>10,000 mg/kg bodyweight per day) exhibited a statistically significant increase in liver MF; however, the MF in all mice in this group were within the historical vehicle control 95% quantile confidence intervals and therefore not considered biologically relevant. Based on estimates of human dietary exposure to each SFPP, the margin of exposure (MOE) values in the TGR assays exceed 10,000. The MOE for one unintentionally present constituent, 2,5(H)-furanone, also exceeds 10,000. Collectively, these data indicate that these SFPPs pose no genotoxic risk and are safe alternatives to conventional smoking.

Use of physiologically based pharmacokinetic modeling to support development of an acute (24-hour) health-based inhalation guideline for manganese.

The toxicokinetics of manganese (Mn) are controlled through homeostasis because Mn is an essential element. However, at elevated doses, Mn is also neurotoxic and has been associated with respiratory, reproductive, and developmental effects. While health-based criteria have been developed for chronic inhalation exposure to ambient Mn, guidelines for short-term (24-h) environmental exposure are also needed. We reviewed US state, federal, and international health-based inhalation toxicity criteria, and conducted a literature search of recent publications. The studies deemed most appropriate to derive a 24-h guideline have a LOAEL of 1500 µg/m3 for inflammatory airway changes and biochemical measures of oxidative stress in the brain following 90 total hours of exposure in monkeys. We applied a cumulative uncertainty factor of 300 to this point of departure, resulting in a 24-h guideline of 5 µg/m3. To address uncertainty regarding potential neurotoxicity, we used a previously published physiologically based pharmacokinetic model for Mn to predict levels of Mn in the brain target tissue (i.e., globus pallidus) for exposure at 5 µg/m3 for two short-term human exposure scenarios. The PBPK model predictions support a short-term guideline of 5 µg/m3 as protective of both respiratory effects and neurotoxicity, including exposures of infants and children.

A multi-tiered hierarchical Bayesian approach to derive toxic equivalency factors for dioxin-like compounds.

In 2005, the World Health Organization (WHO) re-evaluated Toxic Equivalency factors (TEFs) developed for dioxin-like compounds believed to act through the Ah receptor based on an updated database of relative estimated potency (REP)(REP2004 database). This re-evalution identified the need to develop a consistent approach for dose-response modeling. Further, the WHO Panel discussed the significant heterogeneity of experimental datasets and dataset quality underlying the REPs in the database. There is a critical need to develop a quantitative, and quality weighted approach to characterize the TEF for each congener. To address this, a multi-tiered approach that combines Bayesian dose-response fitting and meta-regression with a machine learning model to predict REPS' quality categorizations was developed to predict the most likely relationship between each congener and its reference and derive model-predicted TEF uncertainty distributions. As a proof of concept, this 'Best-Estimate TEF workflow' was applied to the REP2004 database to derive TEF point-estimates and characterizations of uncertainty for all congeners. Model-TEFs were similar to the 2005 WHO TEFs, with the data-poor congeners having larger levels of uncertainty. This transparent and reproducible computational workflow incorporates WHO expert panel recommendations and represents a substantial improvement in the TEF methodology.

Manganese relative oral bioavailability in electric arc furnace steel slag is influenced by high iron content and low bioaccessibility.

Electric arc furnace (EAF) slag is a rock-like aggregate produced with carbon steel and used for construction, including residential ground cover. It is enriched with manganese (Mn) and other metals, including iron (Fe), but because metals are bound in mineral matrices, in vitro bioaccessibility (BA) is limited. We conducted a relative bioavailability (RBA) study using F344 rats to assess Mn RBA from EAF slag ingestion, compared with Mn in diet. Mn and Fe were measured in liver, and Mn in lung and striatum, the target tissue of the brain. Mn levels in each tissue were fit by dose-to-tissue concentration (D-TC) curves. The D-TC relationship was the most highly significant for the linear model using liver Mn, and the RBA was 48%. The D-TC relationship in lung showed a positive slope for chow, but that for EAF slag was slightly negative, and the RBA was 14%. In comparison, the striatum D-TC remained relatively constant, supporting that homeostasis was maintained. Increased Fe was observed in the liver of EAF slag-dosed groups, suggesting that Mn absorption was inhibited by the high Fe content of slag. Lung and striatum D-TC curves demonstrated that systemic delivery of Mn from EAF slag ingestion is limited and support an RBA of 14% for risk assessment. Although Mn levels in slag are elevated compared with health-based screening guidelines, this study supports that incidental ingestion of Mn in EAF slag is unlikely to pose a neurotoxicity hazard due to homeostatic controls, low BA, and high Fe content.

A tiered approach to population-based in vitro testing for cardiotoxicity: Balancing estimates of potency and variability.

Population-wide in vitro studies for characterization of cardiotoxicity hazard, risk, and population variability show that human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) are a powerful and high-throughput testing platform for drugs and environmental chemicals alike. However, studies in multiple donor-derived hiPSC-CMs, across large libraries of chemicals tested in concentration-response are technically complex, and study design optimization is needed to determine sufficient and fit-for-purpose population size considerations. Therefore, we tested a hypothesis that a computational down-sampling analysis based on the data from hiPSC-CM screening of 136 diverse compounds in a population of 43 non-diseased donors, including multiple replicates of the "standard" donor hiPSC-CMs, will inform optimal study designs depending on the decision context (hazard, risk and/or inter-individual variability in cardiotoxicity). Through 50 independent random subsamples of 5, 10, or 20 donors, we estimated accuracy and precision for quantifying potency, inter-individual variability, and QT prolongation risk; the results were compared to the full 43-donor cohort. We found that for potency and clinical risk of QT prolongation, a cohort of 5 randomly-selected unique donors provides accurate and precise estimates. Larger cohort sizes afforded marginal improvements, and 5 replicates of a single donor performed worse. For estimating inter-individual variability, cohorts of at least 20 donors are needed, with smaller populations on average showing bias towards underestimation in population variance. Collectively, this study shows that a variable-size hiPSC-CM-based population-wide in vitro model can be used in a number of decision scenarios for identifying cardiotoxic hazards of drugs and environmental chemicals in the population context.

Cardiotoxicity Hazard and Risk Characterization of ToxCast Chemicals Using Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes from Multiple Donors.

Heart disease remains a significant human health burden worldwide with a significant fraction of morbidity attributable to environmental exposures. However, the extent to which the thousands of chemicals in commerce and the environment may contribute to heart disease morbidity is largely unknown, because in contrast to pharmaceuticals, environmental chemicals are seldom tested for potential cardiotoxicity. Human induced pluripotent stem cell (iPSC)-derived cardiomyocytes have become an informative in vitro model for cardiotoxicity testing of drugs with the availability of cells from multiple individuals allowing in vitro testing of population variability. In this study, we hypothesized that a panel of iPSC-derived cardiomyocytes from healthy human donors can be used to screen for the potential cardiotoxicity hazard and risk of environmental chemicals. We conducted concentration-response testing of 1029 chemicals (drugs, pesticides, flame retardants, polycyclic aromatic hydrocarbons (PAHs), plasticizers, industrial chemicals, food/flavor/fragrance agents, etc.) in iPSC-derived cardiomyocytes from 5 donors. We used kinetic calcium flux and high-content imaging to derive quantitative measures as inputs into Bayesian population concentration-response modeling of the effects of each chemical. We found that many environmental chemicals pose a hazard to human cardiomyocytes in vitro with more than half of all chemicals eliciting positive or negative chronotropic or arrhythmogenic effects. However, most of the tested environmental chemicals for which human exposure and high-throughput toxicokinetics data were available had wide margins of exposure and, thus, do not appear to pose a significant human health risk in a general population. Still, relatively narrow margins of exposure (<100) were estimated for some perfuoroalkyl substances and phthalates, raising concerns that cumulative exposures may pose a cardiotoxicity risk. Collectively, this study demonstrated the value of using a population-based human in vitro model for rapid, high-throughput hazard and risk characterization of chemicals for which little to no cardiotoxicity data are available from guideline studies in animals.

Human induced pluripotent stem cell (iPSC)-derived cardiomyocytes as an in vitro model in toxicology: strengths and weaknesses for hazard identification and risk characterization.

INTRODUCTION: Human induced pluripotent stem cell (iPSC)-derived cardiomyocytes is one of the most widely used cell-based models that resulted from the discovery of how non-embryonic stem cells can be differentiated into multiple cell types. In just one decade, iPSC-derived cardiomyocytes went from a research lab to widespread use in biomedical research and preclinical safety evaluation for drugs and other chemicals. AREAS COVERED: This manuscript reviews data on toxicology applications of human iPSC-derived cardiomyocytes. We detail the outcome of a systematic literature search on their use (i) in hazard assessment for cardiotoxicity liabilities, (ii) for risk characterization, (iii) as models for population variability, and (iv) in studies of personalized medicine and disease. EXPERT OPINION: iPSC-derived cardiomyocytes are useful to increase the accuracy, precision, and efficiency of cardiotoxicity hazard identification for both drugs and non-pharmaceuticals, with recent efforts beginning to demonstrate their utility for risk characterization. Notable limitations include the needs to improve the maturation of cells in culture, to better understand their potential use identifying structural cardiotoxicity, and for additional case studies involving population-wide and disease-specific risk characterization. Ultimately, the greatest future benefits are likely for non-pharmaceutical chemicals, filling a critical gap where no routine testing for cardiotoxicity is currently performed.

Relationships between constituents of energy drinks and beating parameters in human induced pluripotent stem cell (iPSC)-Derived cardiomyocytes.

Consumption of energy drinks has been associated with adverse cardiovascular effects; however, little is known about the ingredients that may contribute to these effects. We therefore characterized the chemical profiles and in vitro effects of energy drinks and their ingredients on human induced pluripotent stem cell (iPSC)-derived cardiomyocytes, and identified the putative active ingredients using a multivariate prediction model. Energy drinks from 17 widely-available over-the-counter brands were evaluated in this study. The concentrations of six common ingredients (caffeine, taurine, riboflavin, pantothenic acid, adenine, and L-methionine) were quantified by coupling liquid chromatography with a triple quadrupole mass spectrometer for the acquisition of LC-MS/MS spectra. In addition, untargeted analyses for each beverage were performed with a platform combining LC, ion mobility spectrometry and mass spectrometry (LC-IMS-MS) measurements. Approximately 300 features were observed across samples in the untargeted studies, and of these ~100 were identified. In vitro effects of energy drinks and some of their ingredients were then tested in iPSC-derived cardiomyocytes. Data on the beat rate (positive and negative chronotropy), ion channel function (QT prolongation), and cytotoxicity were collected in a dilution series. We found that some of the energy drinks elicited adverse effects on the cardiomyocytes with the most common being an increase in the beat rate, while QT prolongation was also observed at the lowest concentrations. Finally, concentration addition modeling using quantitative data from the 6 common ingredients and multivariate prediction modeling was used to determine potential ingredients responsible for the adverse effects on the cardiomyocytes. These analyses suggested theophylline, adenine, and azelate as possibly contributing to the in vitro effects of energy drinks on QT prolongation in cardiomyocytes.

A Bayesian Method for Population-wide Cardiotoxicity Hazard and Risk Characterization Using an In Vitro Human Model.

Human induced pluripotent stem cell (iPSC)-derived cardiomyocytes are an established model for testing potential chemical hazards. Interindividual variability in toxicodynamic sensitivity has also been demonstrated in vitro; however, quantitative characterization of the population-wide variability has not been fully explored. We sought to develop a method to address this gap by combining a population-based iPSC-derived cardiomyocyte model with Bayesian concentration-response modeling. A total of 136 compounds, including 54 pharmaceuticals and 82 environmental chemicals, were tested in iPSC-derived cardiomyocytes from 43 nondiseased humans. Hierarchical Bayesian population concentration-response modeling was conducted for 5 phenotypes reflecting cardiomyocyte function or viability. Toxicodynamic variability was quantified through the derivation of chemical- and phenotype-specific variability factors. Toxicokinetic modeling was used for probabilistic in vitro-to-in vivo extrapolation to derive population-wide margins of safety for pharmaceuticals and margins of exposure for environmental chemicals. Pharmaceuticals were found to be active across all phenotypes. Over half of tested environmental chemicals showed activity in at least one phenotype, most commonly positive chronotropy. Toxicodynamic variability factor estimates for the functional phenotypes were greater than those for cell viability, usually exceeding the generally assumed default of approximately 3. Population variability-based margins of safety for pharmaceuticals were correctly predicted to be relatively narrow, including some below 10; however, margins of exposure for environmental chemicals, based on population exposure estimates, generally exceeded 1000, suggesting they pose little risk at current general population exposures even to sensitive subpopulations. Overall, this study demonstrates how a high-throughput, human population-based, in vitro-in silico model can be used to characterize toxicodynamic population variability in cardiotoxic risk.

Population-based toxicity screening in human induced pluripotent stem cell-derived cardiomyocytes.

The potential for cardiotoxicity is carefully evaluated for pharmaceuticals, as it is a major safety liability. However, environmental chemicals are seldom tested for their cardiotoxic potential. Moreover, there is a large variability in both baseline and drug-induced cardiovascular risk in humans, but data are lacking on the degree to which susceptibility to chemically-induced cardiotoxicity may also vary. Human induced pluripotent stem cell (iPSC)-derived cardiomyocytes have become an important in vitro model for drug screening. Thus, we hypothesized that a population-based model of iPSC-derived cardiomyocytes from a diverse set of individuals can be used to assess potential hazard and inter-individual variability in chemical effects on these cells. We conducted concentration-response screening of 134 chemicals (pharmaceuticals, industrial and environmental chemicals and food constituents) in iPSC-derived cardiomyocytes from 43 individuals, comprising both sexes and diverse ancestry. We measured kinetic calcium flux and conducted high-content imaging following chemical exposure, and utilized a panel of functional and cytotoxicity parameters in concentration-response for each chemical and donor. We show reproducible inter-individual variability in both baseline and chemical-induced effects on iPSC-derived cardiomyocytes. Further, chemical-specific variability in potency and degree of population variability were quantified. This study shows the feasibility of using an organotypic population-based human in vitro model to quantitatively assess chemicals for which little cardiotoxicity information is available. Ultimately, these results advance in vitro toxicity testing methodologies by providing an innovative tool for population-based cardiotoxicity screening, contributing to the paradigm shift from traditional animal models of toxicity to in vitro toxicity testing methods.

Thorough QT/QTc in a Dish: An In Vitro Human Model That Accurately Predicts Clinical Concentration-QTc Relationships.

"Thorough QT/corrected QT (QTc)" (TQT) studies are cornerstones of clinical cardiovascular safety assessment. However, TQT studies are resource intensive, and preclinical models predictive of the threshold of regulatory concern are lacking. We hypothesized that an in vitro model using induced pluripotent stem cell (iPSC)-derived cardiomyocytes from a diverse sample of human subjects can serve as a "TQT study in a dish." For 10 positive and 3 negative control drugs, in vitro concentration-QTc, computed using a population Bayesian model, accurately predicted known in vivo concentration-QTc. Moreover, predictions of the percent confidence that the regulatory threshold of 10 ms QTc prolongation would be breached were also consistent with in vivo evidence. This "TQT study in a dish," consisting of a population-based iPSC-derived cardiomyocyte model and Bayesian concentration-QTc modeling, has several advantages over existing in vitro platforms, including higher throughput, lower cost, and the ability to accurately predict the in vivo concentration range below the threshold of regulatory concern.

A human population-based organotypic in vitro model for cardiotoxicity screening.

Assessing inter-individual variability in responses to xenobiotics remains a substantial challenge, both in drug development with respect to pharmaceuticals and in public health with respect to environmental chemicals. Although approaches exist to characterize pharmacokinetic variability, there are no methods to routinely address pharmacodynamic variability. In this study, we aimed to demonstrate the feasibility of characterizing inter-individual variability in a human in vitro model. Specifically, we hypothesized that genetic variability across a population of iPSC-derived cardiomyocytes translates into reproducible variability in both baseline phenotypes and drug responses. We measured baseline and drug-related effects in iPSC-derived cardiomyocytes from 27 healthy donors on kinetic Ca2+ flux and high-content live cell imaging. Cells were treated in concentration-response with cardiotoxic drugs: isoproterenol (ß-adrenergic receptor agonist/positive inotrope), propranolol (ß-adrenergic receptor antagonist/negative inotrope), and cisapride (hERG channel inhibitor/QT prolongation). Cells from four of the 27 donors were further evaluated in terms of baseline and treatment-related gene expression. Reproducibility of phenotypic responses was evaluated across batches and time. iPSC-derived cardiomyocytes exhibited reproducible donor-specific differences in baseline function and drug-induced effects. We demonstrate the feasibility of using a panel of population-based organotypic cells from healthy donors as an animal replacement experimental model. This model can be used to rapidly screen drugs and chemicals for inter-individual variability in cardiotoxicity. This approach demonstrates the feasibility of quantifying inter-individual variability in xenobiotic responses, and can be expanded to other cell types for which in vitro populations can be derived from iPSCs.