Establishing scientific confidence in a two-chamber co-culture system to evaluate androgenic response in the presence of hepatic metabolism.

For the in vitro determination of toxicity on target organs in the presence of physiologically relevant human metabolism, we recently developed a two-chamber liver-target organ co-culture system in a medium-throughput 96-well format. Our proof-of-concept study using human HepaRG microtissues cultured in three-dimension (3D) and AR-CALUX reporter cells demonstrated the significantly reduced testosterone (T)-mediated androgen receptor (AR) responses in the presence of human liver metabolism. The present study further increased the scientific confidence in this two-chamber co-culture system as a flexible and robust tool to capture androgen-mediated responses by incorporating alternate AR reporter cell systems as the target and examining additional androgenic compounds.

An advancement in developmental and reproductive toxicity (DART) risk assessment: evaluation of a bioactivity and exposure-based NAM toolbox.

Traditional chemical safety assessment involves identifying the lowest level of a chemical that impacts endpoints measured in standardized animal studies to establish human exposure limits. assays have shown promise in providing points of departure that can be protective of human health when combined with exposure predictions into a bioactivity:exposure ratio (BER). Using a combination of broad screening tools and DART-targeted assays, we previously demonstrated high biological coverage of this NAM toolbox against a list of DART-relevant genes and pathways.

Advancing systemic toxicity risk assessment: Evaluation of a NAM-based toolbox approach.

For many years, a method that allowed systemic toxicity safety assessments to be conducted without generating new animal test data, seemed out of reach. However, several different research groups and regulatory authorities are beginning to use a variety of in silico, in chemico, and in vitro techniques to inform safety decisions. To manage this transition to animal-free safety assessments responsibly, it is important to ensure that the level of protection offered by a safety assessment based on new approach methodologies (NAMs), is at least as high as that provided by a safety assessment based on traditional animal studies.

Development of a human liver microphysiological coculture system for higher throughput chemical safety assessment.

Chemicals in the systemic circulation can undergo hepatic xenobiotic metabolism, generate metabolites, and exhibit altered toxicity compared with their parent compounds. This article describes a 2-chamber liver-organ coculture model in a higher-throughput 96-well format for the determination of toxicity on target tissues in the presence of physiologically relevant human liver metabolism. This 2-chamber system is a hydrogel formed within each well consisting of a central well (target tissue) and an outer ring-shaped trough (human liver tissue).

The phosphoproteome is a first responder in tiered cellular adaptation to chemical stress followed by proteomics and transcriptomics alteration.

Next-generation risk assessment (NGRA) for environmental chemicals involves a weight of evidence (WoE) framework integrating a suite of new approach methodologies (NAMs) based on points of departure (PoD) obtained from in vitro assays. Among existing NAMs, the omic-based technologies are of particular importance based on the premise that any apical endpoint change indicative of impaired health must be underpinned by some alterations at the omics level, such as transcriptome, proteome, metabolome, epigenome and genome. Transcriptomic assay plays a leading role in providing relatively conservative PoDs compared with apical endpoints.

Analysis of reproducibility and robustness of a renal proximal tubule microphysiological system OrganoPlate 3-lane 40 for in vitro studies of drug transport and toxicity.

Microphysiological systems are an emerging area of in vitro drug development, and their independent evaluation is important for wide adoption and use. The primary goal of this study was to test reproducibility and robustness of a renal proximal tubule microphysiological system, OrganoPlate 3-lane 40, as an in vitro model for drug transport and toxicity studies. This microfluidic model was compared with static multiwell cultures and tested using several human renal proximal tubule epithelial cell (RPTEC) types.

Next generation risk assessment of human exposure to estrogens using safe comparator compound values based on in vitro bioactivity assays.

In next generation risk assessment (NGRA), the Dietary Comparator Ratio (DCR) can be used to assess the safety of chemical exposures to humans in a 3R compliant approach. The DCR compares the Exposure Activity Ratio (EAR) for exposure to a compound of interest (EAR) to the EAR for an established safe exposure level to a comparator compound (EAR), acting by the same mode of action. It can be concluded that the exposure to a test compound is safe at a corresponding DCR ≤ 1.

Using transcriptomics, proteomics and phosphoproteomics as new approach methodology (NAM) to define biological responses for chemical safety assessment.

Omic-based technologies are of particular interest and importance for hazard identification and health risk characterization of chemicals. Their application in the new approach methodologies (NAMs) anchored on cellular toxicity pathways is based on the premise that any apical health endpoint change must be underpinned by some alterations at the omic levels. In the present study we examined the cellular responses to two chemicals, caffeine and coumarin, by generating and integrating multi-omic data from multi-dose and multi-time point transcriptomic, proteomic and phosphoproteomic experiments.

Are Non-animal Systemic Safety Assessments Protective? A Toolbox and Workflow.

An important question in toxicological risk assessment is whether non-animal new approach methodologies (NAMs) can be used to make safety decisions that are protective of human health, without being overly conservative. In this work, we propose a core NAM toolbox and workflow for conducting systemic safety assessments for adult consumers. We also present an approach for evaluating how protective and useful the toolbox and workflow are by benchmarking against historical safety decisions.

Ready for regulatory use: NAMs and NGRA for chemical safety assurance.

New approach methodologies (NAMs) that do not use experimental animals are, in certain settings, entirely appropriate for assuring the safety of chemical ingredients, although regulatory adoption has been slow. In this opinion article we discuss how scientific advances that utilize NAMs to certify systemic safety are available now and merit broader acceptance within the framework of next generation risk assessments (NGRA).

Next Generation Risk Assessment of the Anti-Androgen Flutamide Including the Contribution of Its Active Metabolite Hydroxyflutamide.

In next generation risk assessment (NGRA), non-animal approaches are used to quantify the chemical concentrations required to trigger bioactivity responses, in order to assure safe levels of human exposure. A limitation of many bioactivity assays, which are used in an NGRA context as new approach methodologies (NAMs), is that toxicokinetics, including biotransformation, are not adequately captured. The present study aimed to include, as a proof of principle, the bioactivity of the metabolite hydroxyflutamide (HF) in an NGRA approach to evaluate the safety of the anti-androgen flutamide (FLU), using the AR-CALUX assay to derive the NAM point of departure (PoD).

Microphysiological Systems Evaluation: Experience of TEX-VAL Tissue Chip Testing Consortium.

Much has been written and said about the promise and excitement of microphysiological systems, miniature devices that aim to recreate aspects of human physiology on a chip. The rapid explosion of the offerings and persistent publicity placed high expectations on both product manufacturers and regulatory agencies to adopt the data. Inevitably, discussions of where this technology fits in chemical testing paradigms are ongoing.

Quantitative phosphoproteomics reveal cellular responses from caffeine, coumarin and quercetin in treated HepG2 cells.

Protein phosphorylation is the most common type of post-translational modification where serine, threonine or tyrosine are reversibly bound to the phosphate group of ATP in a reaction catalyzed by protein kinases. Phosphorylation plays an important role in regulation of cell homeostasis, including but not limited to signal perception and transduction, gene expression and function of proteins. Protein phosphorylation happens on a fast time scale and represents an energy-efficient way for the cell to adapt to exposure to chemical stressors.

Beyond AOPs: A Mechanistic Evaluation of NAMs in DART Testing.

New Approach Methodologies (NAMs) promise to offer a unique opportunity to enable human-relevant safety decisions to be made without the need for animal testing in the context of exposure-driven Next Generation Risk Assessment (NGRA). Protecting human health against the potential effects a chemical may have on embryo-foetal development and/or aspects of reproductive biology using NGRA is particularly challenging. These are not single endpoint or health effects and risk assessments have traditionally relied on data from Developmental and Reproductive Toxicity (DART) tests in animals.

Implementing organ-on-chip in a next-generation risk assessment of chemicals: a review.

Organ-on-chip (OoC) technology is full of engineering and biological challenges, but it has the potential to revolutionize the Next-Generation Risk Assessment of novel ingredients for consumer products and chemicals. A successful incorporation of OoC technology into the Next-Generation Risk Assessment toolbox depends on the robustness of the microfluidic devices and the organ tissue models used. Recent advances in standardized device manufacturing, organ tissue cultivation and growth protocols offer the ability to bridge the gaps towards the implementation of organ-on-chip technology.

Predictive performance of next generation human physiologically based kinetic (PBK) models based on in vitro and in silico input data.

The goal of the present study was to assess the predictive performance of a generic human physiologically based kinetic (PBK) model based on in vitro and in silico input data and the effect of using different input approaches for chemical parameterization on those predictions. For this purpose, a dataset was created of 38,772 Cmax predictions for 44 compounds by applying different combinations of in vitro and in silico approaches for chemical parameterization, and these predicted Cmax values were compared to reported in vivo data. Best results were achieved when the hepatic clearance was parameterized based on in vitro (i.

Next generation risk assessment (NGRA): Bridging in vitro points-of-departure to human safety assessment using physiologically-based kinetic (PBK) modelling - A case study of doxorubicin with dose metrics considerations.

Using the chemical doxorubicin (DOX), the objective of the present study was to evaluate the impact of dose metrics selection in the new approach method of integrating physiologically-based kinetic (PBK) modelling and relevant human cell-based assays to inform a priori the point of departure for human health risk. We reviewed the literature on the clinical consequences of DOX treatment to identify dosing scenarios with no or mild cardiotoxicity observed. Key concentrations of DOX that induced cardiomyocyte toxicity in vitro were derived from studies of our own and others.

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

Next Generation Risk Assessment (NGRA) can use the so-called Dietary Comparator Ratio (DCR) to evaluate the safety of a defined exposure to a compound of interest. The DCR compares the Exposure Activity Ratio (EAR) for the compound of interest, to the EAR of an established safe level of human exposure to a comparator compound with the same putative mode of action. A DCR ≤ 1 indicates the exposure evaluated is safe.

The Alginate Immobilization of Metabolic Enzymes Platform Retrofits an Estrogen Receptor Transactivation Assay With Metabolic Competence.

The U.S. EPA Endocrine Disruptor Screening Program utilizes data across the ToxCast/Tox21 high-throughput screening (HTS) programs to evaluate the biological effects of potential endocrine active substances.

Identifying and Characterizing Stress Pathways of Concern for Consumer Safety in Next-Generation Risk Assessment.

Many substances for which consumer safety risk assessments need to be conducted are not associated with specific toxicity modes of action, but rather exhibit nonspecific toxicity leading to cell stress. In this work, a cellular stress panel is described, consisting of 36 biomarkers representing mitochondrial toxicity, cell stress, and cell health, measured predominantly using high content imaging. To evaluate the panel, data were generated for 13 substances at exposures consistent with typical use-case scenarios.

Integration of in vitro data from three dimensionally cultured HepaRG cells and physiologically based pharmacokinetic modeling for assessment of acetaminophen hepatotoxicity.

Selection of appropriate fit-for-purpose in vitro and in silico models is critical for non-animal safety assessment of chemical-induced hepatoxicity. The present study evaluated the feasibility of integrating in vitro data from three-dimensionally (3D)-cultured HepaRG cells and physiologically based pharmacokinetic (PBPK) modeling to predict chemical-induced liver toxicity. A 3D organoid culture system was established using an ultralow attachment method.

A Next-Generation Risk Assessment Case Study for Coumarin in Cosmetic Products.

Next-Generation Risk Assessment is defined as an exposure-led, hypothesis-driven risk assessment approach that integrates new approach methodologies (NAMs) to assure safety without the use of animal testing. These principles were applied to a hypothetical safety assessment of 0.1% coumarin in face cream and body lotion.

The way forward for assessing the human health safety of cosmetics in the EU - Workshop proceedings.

Although the need for non-animal alternatives has been well recognised for the human health hazard assessment of chemicals in general, it has become especially pressing for cosmetic ingredients due to the full implementation of testing and marketing bans on animal testing under the European Cosmetics Regulation. This means that for the safety assessment of cosmetics, the necessary safety data for both the ingredients and the finished product can be drawn from validated (or scientifically-valid), so-called "Replacement methods". In view of the challenges for safety assessment without recourse to animal test data, the Methodology Working Group of the Scientific Committee on Consumer Safety organised a workshop in February 2019 to discuss the key issues in regard to the use of animal-free alternative methods for the safety evaluation of cosmetic ingredients.

Integrating in vitro testing and physiologically-based pharmacokinetic (PBPK) modelling for chemical liver toxicity assessment-A case study of troglitazone.

In vitro to in vivo extrapolation (IVIVE) for next-generation risk assessment (NGRA) of chemicals requires computational modeling and faces unique challenges. Using mitochondria-related toxicity data of troglitazone (TGZ), a prototype drug known for liver toxicity, from HepaRG, HepG2, HC-04, and primary human hepatocytes, we explored inherent uncertainties in IVIVE, including cell models, cellular response endpoints, and dose metrics. A human population physiologically-based pharmacokinetic (PBPK) model for TGZ was developed to predict in vivo doses from in vitro point-of-departure (POD) concentrations.