For almost fifteen years, the availability and regulatory acceptance of new approach methodologies (NAMs) to assess the absorption, distribution, metabolism and excretion (ADME/biokinetics) in ...chemical risk evaluations are a bottleneck. To enhance the field, a team of 24 experts from science, industry, and regulatory bodies, including new generation toxicologists, met at the Lorentz Centre in Leiden, The Netherlands. A range of possibilities for the use of NAMs for biokinetics in risk evaluations were formulated (for example to define species differences and human variation or to perform quantitative in vitro-in vivo extrapolations). To increase the regulatory use and acceptance of NAMs for biokinetics for these ADME considerations within risk evaluations, the development of test guidelines (protocols) and of overarching guidance documents is considered a critical step. To this end, a need for an expert group on biokinetics within the Organisation of Economic Cooperation and Development (OECD) to supervise this process was formulated. The workshop discussions revealed that method development is still required, particularly to adequately capture transporter mediated processes as well as to obtain cell models that reflect the physiology and kinetic characteristics of relevant organs. Developments in the fields of stem cells, organoids and organ-on-a-chip models provide promising tools to meet these research needs in the future.
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.
•We propose a mechanism for a liver immune response involving alkaline phosphatase.•The system requires transcytosis of alkaline phosphatase in an IgG immune complex.•This is triggered by inflammation and the asialoglycoprotein receptor is the carrier.•This mechanism can explain the anti-inflammatory effects of alkaline phosphatase.•We support the hypothesis with a solid literature foundation.
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.
Methods and approaches that can be used in toxicology and safety assessment are changing at a faster pace than ever. Members of the International Life Sciences Institute (ILSI) Europe have formed an ...expert group to review possibilities, opportunities and challenges for the potential use of non-animal testing strategies in food safety and nutrition research, which can ultimately be used in support of regulatory submissions for pre-market authorisation.
For the different areas of food improvement agents, genetically modified foods and novel foods, the acceptability of non-animal strategies is evaluated in comparison to legislative requirements in Europe. Current hazard and risk assessment tools that do not require additional animal testing are reviewed and emerging tools and methodologies considered, covering advanced in vitro methods, in silico and system biology approaches and high-throughput methods for mode-of-action assessment.
The paper highlights the great potential for research strategies to be developed that reduce or avoid the use of animal tests, with the generation of more human-relevant data from multiple sources. It also shows the discordance in current legislation: on one hand saying non-animal strategies should be used, but on the other hand not providing sufficient guidance, leading in practice to lack of use of these non-animal testing strategies. This emphasizes the need for scientific developments and acceptability to be more reflected in legislation (e.g. guidance). What are we waiting for?
•It is urgent to apply existing non-animal strategies in food safety assessment.•There is great potential for research strategies that reduce the use of animal tests.•Action is required by all stakeholders to be more challenging in applying non-animal approaches.•Acceptability of non-animal approaches needs to be more reflected in food safety legislation.
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).
Carcinogenicity is a crucial endpoint for the safety assessment of chemicals and products. During the last few decades, the development of quantitative structure-activity relationship ((Q)SAR) models ...has gained importance for regulatory use, in combination with in vitro testing or expert-based reasoning. Several classification models can now predict both human and rat carcinogenicity, but there are few models to quantitatively assess carcinogenicity in humans. To our knowledge, slope factor (SF), a parameter describing carcinogenicity potential used especially for human risk assessment of contaminated sites, has never been modeled for both inhalation and oral exposures. In this study, we developed classification and regression models for inhalation and oral SFs using data from the Risk Assessment Information System (RAIS) and different machine learning approaches. The models performed well in classification, with accuracies for the external set of 0.76 and 0.74 for oral and inhalation exposure, respectively, and r
values of 0.57 and 0.65 in the regression models for oral and inhalation SFs in external validation. These models might therefore support regulators in (de)prioritizing substances for regulatory action and in weighing evidence in the context of chemical safety assessments. Moreover, these models are implemented on the VEGA platform and are now freely downloadable online.
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.
Investigative Toxicology describes the de-risking and mechanistic elucidation of toxicities, supporting early safety decisions in the pharmaceutical industry. Recently, Investigative Toxicology has ...contributed to a shift in pharmaceutical toxicology, from a descriptive to an evidence-based, mechanistic discipline. This was triggered by high costs and low throughput of Good Laboratory Practice in vivo studies, and increasing demands for adhering to the 3R (Replacement, Reduction and Refinement) principles of animal welfare. Outside the boundaries of regulatory toxicology, Investigative Toxicology has the flexibility to embrace new technologies, enhancing translational steps from in silico, in vitro to in vivo mechanistic understanding to eventually predict human response. One major goal of Investigative Toxicology is improving preclinical decisions, which coincides with the concept of animal-free safety testing. Currently, compounds under preclinical development are being discarded due to the use of inappropriate animal models. Progress in Investigative Toxicology could lead to humanized in vitro test systems and the development of medicines less reliant on animal tests. To advance this field a group of 14 European-based leaders from the pharmaceutical industry founded the Investigative Toxicology Leaders Forum (ITLF), an open, non-exclusive and pre-competitive group that shares knowledge and experience. The ITLF collaborated with the Centre for Alternatives to Animal Testing Europe (CAAT-Europe) to organize an "Investigative Toxicology Think-Tank", which aimed to enhance the interaction with experts from academia and regulatory bodies in the field. Summarizing the topics and discussion of the workshop, this article highlights Investigative Toxicology's position by identifying key challenges and perspectives.
There is a need for long‐lived hepatic in vitro models to better predict drug induced liver injury (DILI). Human liver‐derived epithelial organoids are a promising cell source for advanced in vitro ...models. Here, organoid technology is combined with biofabrication techniques, which holds great potential for the design of in vitro models with complex and customizable architectures. Here, porous constructs with human hepatocyte‐like cells derived from organoids are generated using extrusion‐based printing technology. Cell viability of bioprinted organoids remains stable for up to ten days (88–107% cell viability compared to the day of printing). The expression of hepatic markers, transporters, and phase I enzymes increased compared to undifferentiated controls, and is comparable to non‐printed controls. Exposure to acetaminophen, a well‐known hepatotoxic compound, decreases cell viability of bioprinted liver organoids to 21–51% (p < 0.05) compared to the start of exposure, and elevated levels of damage marker miR‐122 are observed in the culture medium, indicating the potential use of the bioprinted constructs for toxicity testing. In conclusion, human liver‐derived epithelial organoids can be combined with a biofabrication approach, thereby paving the way to create perfusable, complex constructs which can be used as toxicology‐ and disease‐models.
This study shows that human‐liver derived organoids can be combined with extrusion‐based bioprinting, thereby showing the first step in the development of a more complex, and hence more physiologically relevant, hepatic in vitro model system. It provides the basis of a humanized testing platform for personalized medicine and/or drug screening based on the creation of liver constructs through bioprinting.
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