New approach methodologies (NAMs) are increasingly recognised as central to modernising safety assessment in medicines development, offering the potential for improved human relevance, mechanistic insight, and reduced reliance on animal testing. Despite substantial scientific progress and growing regulatory encouragement in their use, the routine integration of NAMs into regulatory decision-making remains inconsistent, even though they are widely used internally across discovery and development. This article brings together several lines of evidence and opinions to explore why uptake continues to lag behind capability, including insights from a cross-sector workshop convened by the Medicines and Healthcare products Regulatory Agency (MHRA), the Association of the British Pharmaceutical Industry (ABPI) and the UK National Centre for the Replacement, Refinement and Reduction of Animals in Research (NC3Rs). For many applications, the key constraints are no longer scientific, but relate to confidence, clarity of expectations, and implementation, although important scientific challenges remain for complex endpoints such as chronic toxicity. Regulatory frameworks are increasingly supportive, with growing evidence of confidence and alignment on weight-of-evidence approaches incorporating NAMs, yet residual uncertainty and limited practical experience in their application means that questions regarding context-of-use, validation, and regulatory acceptability persist. The next phase requires a shift from technology development to practical implementation, including clearer guidance and increased transparency through the sharing of data and case-studies. Building consensus and confidence across the scientific community will be critical to normalising the use of NAMs and realising their potential in medicines development.
The kidney proximal tubule (PT) is a principal site of nutrient and electrolyte reabsorption, metabolism, and clearance of drugs and toxins. However, owing to its high mitochondrial content, exposure to concentrated filtrate, and dense expression of broad transporters, such as organic cation tranporters, organic anion tranporters, multidrug and toxin extrusion proteins, and ATP-binding cassette superfamily members, the PT is particularly vulnerable to nephrotoxic injury arising from xenobiotic-induced damage. Accurate models are essential for predicting nephrotoxicity and drug–drug interactions. However, traditional animal models and 2D PT cell cultures have faced barriers such as limited human-relevance, dedifferentiation, and PT segment–specific identity, presenting challenges for their translational utility. Recent advances in 3D culture systems, perfusion, kidney-on-chip platforms, and pluripotent stem cell technologies have sought to restore physiological relevance and native architecture, improving functional transporter expression and nephrotoxic injury responses. Emerging stem cell–derived kidney organoid strategies to bias engineered tissue towards distinct PT cell types has strengthened this approach. Despite this progress, challenges such as incomplete maturation, variability, throughput, and limited vascular and immune components present ongoing hurdles. This review explores recent developments in PT-specific modelling systems, evaluating their predictive performance for drug-induced injury compared to traditional models, and discusses future directions towards physiologically relevant and scalable platforms for nephrotoxicity assessment.
Membrane transporters, expressed across major organs, are essential for evaluating the pharmacokinetics of xenobiotics. Among them, P-glycoprotein (P-gp/MDR1/ABCB1), a major apical efflux transporter of the ATP-binding cassette (ABC) superfamily, plays a central protective role at the intestine, liver, kidney, and blood–brain barrier (BBB) by limiting the intracellular accumulation of xenobiotics and facilitating their excretion. While P-gp inhibition has been widely studied for overcoming multidrug resistance in cancer, emerging evidence suggests that positive modulation of P-gp— either through transcriptional induction (a delayed, nuclear-receptor-mediated increase in P-gp expression) or through direct activation (a rapid, post-translational enhancement of the intrinsic efflux activity of membrane-bound P-gp)—may enhance detoxification and mitigate drug-induced toxicity. In enterocytes, hepatocytes, proximal tubular epithelial cells, and brain endothelial cells, compounds that increase P-gp expression (inducers) or activity (activators) reduce substrate bioavailability, systemic exposure, or CNS penetration, thereby offering therapeutic opportunities for toxicity management.This review synthesizes current knowledge on compound-mediated positive P-gp modulation, highlighting experimental evidence of its capacity to reduce xenobiotic-induced toxicity. Collectively, these insights position P-gp as a promising pharmacological target for enhancing drug safety and fostering more effective and individualized therapies. Harnessing P-gp modulation thus offers a promising avenue for safer therapeutic strategies across diverse organ systems. Importantly, the available evidence supporting positive P-gp modulation as a detoxification strategy currently derives from preclinical in vitro and in vivo studies; future research should focus on its translation into human pharmacotherapy.
The cosmetic safety evaluation has undergone a paradigm shift driven by animal testing bans now implemented across countries representing one-third of the global population and nearly half of worldwide cosmetics sales. The legislative mandates have accelerated the development and adoption of next generation risk assessment (NGRA), an innovative framework that proposes the making of context-specific safety decisions using exposure-driven, mechanism-based approaches based on non-animal test methods. Significant progress has been achieved in replacing animal tests for specific endpoints, including skin sensitization, irritation, and phototoxicity through validated new approach methodologies (NAMs). However, major gaps remain for the one-for-one replacement of systemic toxicity, reproductive and developmental toxicity, and carcinogenicity animal tests. Recent advances demonstrate that NGRA can successfully address many of these complex endpoints through integrated approaches combining physiologically based kinetic (PBK) modeling, high-throughput transcriptomics and in vitro phenotyping to derive bioactivity-exposure ratios (BERs) for risk characterization. Numerous case studies across diverse chemical types, including coumarin, benzophenone-4, caffeine, and benzyl salicylate, have demonstrated NGRA's utility for making protective safety decisions without animal data. These examples highlight critical considerations that need to be addressed in NGRA, including metabolite assessment, dose metric selection, and quantitative uncertainty characterization. While a like-for-like replacement of all animal tests remains unachievable, NGRA provides a scientifically robust pathway for protective assessments where the absence of systemic effects is the primary goal, particularly for cosmetic ingredients. The challenge now lies in harmonizing methodologies, building regulatory confidence in these approaches, and evolving institutional structures to enable routine NGRA implementation across global regulatory regimes.
An IARC-sponsored Workshop in July 2023 deliberated on the interpretation of endpoints and relevance of such evidence to inform carcinogenic hazards. Here we summarize the discussions regarding the informative endpoints associated with three of the 10 Key Characteristics (KCs) of Carcinogens. KC1 (electrophilicity) may include data on electrophilic metabolites, DNA adducts, or skin sensitivity. KC2 (genotoxicity) may include all mutagenicity data, including in silico predictions, and DNA damage endpoints other than DNA adducts, such as DNA strand breaks, micronuclei, and chromosomal aberrations, measured in vitro or in vivo. KC3 (alters DNA repair or causes genomic instability) may include assays of DNA repair capacity or genome stability in vitro or in vivo.
Thyroid hormones (THs) regulate key developmental processes, including neurogenesis, growth, and metabolism. They are essential for brain and skeletal development, as well as for the differentiation of multiple organs, acting primarily through nuclear TH receptors that modulate gene transcription. THs also influence testis development, primarily by regulating the proliferation and differentiation of Sertoli and Leydig cells. During early postnatal life, THs promote the cessation of Sertoli cell proliferation and their maturation, ultimately determining testis size. Despite important functions of TH signaling in other organs, regulatory testing for TH-system-disrupting chemicals has predominantly focused on neurodevelopment. With TH signaling influencing reproductive development, effects on male reproductive endpoints mediated by TH disruption may currently be underrepresented in regulatory frameworks for identifying endocrine-disrupting chemicals. Few mechanistic adverse outcome pathways, biomarkers, or sensitive testing windows have been established for these potential toxicity pathways, a gap that could increase the risk of not detecting chemicals that impair male reproductive health and fertility via TH-mediated mechanisms. Addressing these deficiencies is important to fully assess the reproductive risks posed by TH-system-disrupting compounds.
The cosmetic safety evaluation has undergone a paradigm shift driven by animal testing bans now implemented across countries representing one-third of the global population and nearly half of worldwide cosmetics sales. The legislative mandates have accelerated the development and adoption of Next Generation Risk Assessment (NGRA), an innovative framework that proposes making of context-specific safety decisions using exposure-driven, mechanism-based approaches based on non-animal test methods. Significant progress has been achieved in replacing animal tests for specific endpoints including skin sensitization, irritation, and phototoxicity through validated New Approach Methodologies (NAMs). However, major gaps remain for one-for-one replacement of systemic toxicity, reproductive and developmental toxicity, and carcinogenicity animal tests. Recent advances demonstrate that NGRA can successfully address many of these complex endpoints through integrated approaches combining physiologically-based kinetic (PBK) modeling, high-throughput transcriptomics and in vitro phenotyping to derive Bioactivity-Exposure Ratios (BERs) for risk characterization. Numerous case studies across diverse chemical types, including coumarin, benzophenone-4, caffeine, and benzyl salicylate, have demonstrated NGRA's utility for making protective safety decisions without animal data. These examples highlight critical considerations that need to be addressed in NGRA, including metabolite assessment, dose metric selection, and quantitative uncertainty characterization. While like-for-like replacement of all animal tests remains unachievable, NGRA provides a scientifically robust pathway for protective assessments where an absence of systemic effects is the primary goal, particularly for cosmetic ingredients. The challenge now lies in harmonizing methodologies, building regulatory confidence in these approaches, and evolving institutional structures to enable routine NGRA implementation across global regulatory regimes.
Drug transporters belong to the solute carrier (SLC) and ATP-binding cassette (ABC) superfamilies of proteins. They play a central role in the absorption, distribution, and elimination of many drugs along the enterohepatic axis. This review summarizes how bile acids, acting as both competitive substrates and signaling molecules, regulate the activity of these transporters in the liver and intestine, thereby influencing drug disposition in health and disease. Bile acids affect the expression and post-translational events, including epigenetic modifications and membrane insertion dynamics, of hepatocyte basolateral uptake transporters (NTCP, OATP1B1, OATP1B3, OATP2B1, OCT1, and OAT2), and canalicular or basolateral efflux pumps (BSEP, MRP2, MDR3, MDR1, MRP3, MRP4, and BCRP), as well as enterocyte apical and basolateral SLC transporters (PEPT1, OCTN1/2, OATP2B1, MCT1, ASBT, and OST alpha/beta), and ABC efflux pumps (MDR1, MRP2, BCRP, and MRP3). Regulatory mechanisms, including plasma membrane (TGR5) and nuclear receptors (FXR, PXR, PPAR alpha, and VDR), detect changes in bile acid pool and adjust transporter expression and membrane insertion/retrieval accordingly. This controls bile acid metabolism but also markedly influences drug disposition. Under pathological conditions such as cholestasis, chronic liver disease, inflammatory bowel disease, or altered intestinal bile acid levels, major shifts in bile acid signaling induce changes in transporter networks. These changes can alter the pharmacokinetics, efficacy, and toxicity of many drugs, as well as their interactions with other drugs and endogenous substances. Therefore, understanding how bile acids regulate drug transporters is essential for optimizing pharmacological treatments and forecasting differences in drug responses in patients with hepatobiliary and intestinal disorders.
The increasing number of chemicals (including those found cosmetics and food) and pharmaceutical modalities challenges traditional animal-based approaches for hazard identification and risk assessment. New approach methodologies (NAMs) offer promising alternatives to improve efficiency, reduce animal use, and enhance human relevance in regulatory toxicology. This manuscript synthesizes insights from expert discussions and recent literature to identify gaps and opportunities for NAMs adoption. We outline the evolution of NAMs, emphasizing the importance of context of use, mechanistic understanding, and reproducibility. Current NAMs applications include skin and eye irritation, sensitization, genotoxicity, and endocrine activity, while systemic, reproductive, and developmental toxicity remain poorly addressed. Obstacles to broader implementation include technical limitations in replicating complex physiology, lack of standardized protocols, and challenges in quantitative extrapolation to human outcomes. Emerging technologies such as micro-physiological systems (MPS), organ-on-chip models, and artificial intelligence (AI) frameworks show potential to bridge these gaps but require rigorous quality control and harmonized validation standards. Regulatory acceptance is hindered by inconsistent terminology, limited confidence in predictive power, and resource constraints for qualification studies. We propose a unified framework focusing on context of use, performance standards, biological variability, and reproducibility to facilitate integration of NAMs into regulatory decision making. Collaborative efforts among developers, regulators, and stakeholders are essential to advance NAMs as reliable tools for next-generation risk assessment, ultimately supporting global initiatives to reduce animal testing while ensuring human (and animal) safety.
Zebrafish (Danio rerio) have emerged as a cornerstone of modern toxicology, providing unique advantages including strong genetic similarity to humans, experimental accessibility, and well-characterized behavioral assays. A defining feature of zebrafish biology is their remarkable regenerative capacity, which enables repair of fins, retina, spinal cord, liver, heart, and even brain regions. This regenerative ability supports important discoveries in developmental and regenerative biology, yet it also presents a limitation for toxicology. Tissue repair and functional recovery may mask persistent damage, obscure toxicant-induced molecular changes, or create the impression of resilience where concealed injury remains. As a result, zebrafish studies may underestimate the long-term consequences of chemical exposures when extrapolated to humans, who possess much more limited regenerative potential. At the same time, insights from zebrafish regeneration can reveal adaptive pathways that inform protective responses and therapeutic development. Recognizing regeneration as both a strength and a limitation is essential. By integrating complementary endpoints and comparative models, the use of zebrafish will continue to provide valuable and contextually grounded insights into toxicological mechanisms and human health risks.
Read-across is a well-established New Approach Methodology (NAM). Its approach and methods are mature and widely used, especially for data-gap filling relating to in vivo tests. It is supported by guidance and reporting templates, as well as computational tools. However, greater expertise and clarity are required to gain further acceptance. Challenges and needs focus on improving the acceptability of read-across predictions. These include: 1) understanding how uncertainties are quantified and how the overall (tolerable) uncertainty can be defined, 2) assessing similarity between target and source molecules, especially beyond strict 2D structural similarity, whilst avoiding activity cliffs, 3) key similarity challenges exist in metabolism, including the ability to confirm the principle pathways and biotransformations leading to toxicologically significant metabolites, as well as the rates of their formation, 4) the lack of suitable data to support read-across; thus, efforts to justify using non-standard data should continue to be developed, and 5) the use of artificial intelligence (AI) in read-across presenting its own set of challenges, which are, however, outweighed by the opportunities and gains. The role of AI in assessing similarity and, indeed, all aspects of read-across will grow, but at this time, how and at what speed it will grow is unknown. Read-across also has the potential to assist in the integration of non-animal chemical safety assessments and Next Generation Risk Assessment (NGRA).
The widespread use of phytochemicals, often perceived as safe, raises increasing concern regarding their potential to interfere with drug disposition. Among these interactions, the modulation of drug transporters plays a pivotal yet underexplored role in determining drug bioavailability and toxicity. This review focuses on the interplay between dietary phytochemicals, particularly phytoestrogens, and key efflux transporters, including MDR1/ABCB1/P-glycoprotein, BCRP/ABCG2, MRP2/ABCC2, and MRP3/ABCC3. These ATP-binding cassette (ABC) transporters are essential for maintaining cellular homeostasis by mediating drug and xenobiotic efflux in the intestine, liver, and other organs. Evidence suggests that certain phytoestrogens can directly inhibit transporter activity or regulate their expression through nuclear receptor pathways such as PXR, CAR, or ERα/β. Such modulation can alter drug exposure, contribute to hepatotoxicity, or compromise therapeutic efficacy. Understanding phytochemical–transporter interactions is therefore critical to improve risk assessment and guide safe co-administration of phytochemicals with pharmacological treatments. We highlight current knowledge gaps, mechanistic insights, and future directions for predictive toxicology and personalized medicine.
Substance use disorder (SUD) has surged over the past cocaine, and opioids being some of the most misused substances. Chronic substance use impairs brain function and behavior; thus, SUD is a serious public health concern with wide-ranging effects on families, public safety, and the economy. Medication-assisted treatment (MAT) may help manage withdrawal symptoms and reduce cravings to support longterm recovery for those affected by a SUD. However, aside from opioid use disorder (OUD), there are no approved medications to treat disorders related to the most misused drugs, despite the need. Here, we briefly summarize research progress on SUD and MAT for cannabis, psychostimulants, and opioids and highlight the challenges that remain.
Psychoactive natural products-including cannabis, psychedelics, and traditional and indigenous medicines such as kratom and peyote-have gained an emerging interest among the general public and health providers as therapies for a variety of health conditions. These medicinal substances are not part of the regulated agri-food systems and lack the established production guidelines and safety standards of most agricultural commodities. Additionally, many of these therapeutics were made illegal in the United Nations Single Convention on Narcotic Drugs in 1961 [1] and during the 'War on Drugs' movement in the U.S. in the 1970s [2]. The complex safety and policy issues related to these therapeutics are often beyond the scope of conventional risk assessment, as exemplified by the challenges faced by the state-level cannabis legalization in the U.S. [3]. In this editorial, we would like to highlight 14 articles in the special issue 'Psychedelics and Cannabis: Toxicological Perspectives and Public Health Challenges' and discuss the unique safety challenges of emerging therapeutics.
Substance use disorder (SUD) has surged over the past decade, with cannabis, amphetamine, methamphetamine, cocaine, and opioids being some of the most misused substances. Chronic drug use impairs brain function and behavior; thus, SUD is a serious public health concern with wide-ranging effects on families, public safety, and the economy. Medication-assisted treatment (MAT) may help manage withdrawal symptoms and reduce cravings to support long-term recovery for those affected by a SUD. However, aside from opioid use disorder (OUD), there are no approved medications to treat disorders related to the most misused drugs, despite the need. Here, we briefly summarize research progress on SUD and MAT for cannabis, psychostimulants, and opioids and highlight the challenges that remain.
Minimizing exposure to endocrine disruptors (EDs) is a priority within the European Union's political agenda. These chemical substances interfere with the endocrine system, leading to adverse effects on human health and the environment. Despite extensive research, significant uncertainties remain regarding safe levels of exposure to EDs. This paper highlights the need for cautious risk assessment due to substantial knowledge gaps, limitations in testing and unresolved questions about thresholds, non-monotonic dose-responses and low-dose effects. On this basis, we recommend a precautionary approach to risk assessment of EDs and, in general, support initiatives to reduce exposure by phasing out use and production. In cases where EDs are found in, for instance, food and drinking water, we propose the use of an additional uncertainty factor of 10 (as default) for the nature of ED effects to ensure more protective risk assessments. Our recommendations aim to contribute to the ongoing discussion on how to effectively manage the risks associated with EDs.
Assessing human health risks of micro- and nanoplastics (MNPs) demands a paradigm shift in toxicology. Traditional in vitro and in vivo models fail to capture the complexity of human exposure and physiology. Organ-on-chip (OoC) technology offers a promising alternative—not as a universal solution, but as a precision tool for mechanistic questions inaccessible to conventional systems. This review highlights the emerging potential of OoCs to advance understanding in three critical domains : (1) inter-organ communication (e.g., liver-heart metabolic crosstalk), (2) disease-state vulnerability (e.g., enhanced intestinal uptake in Crohn's disease), and (3) dynamic barrier penetration (e.g., size-dependent blood-brain transfer). Despite their potential, OoCs face limitations from material artifacts, low throughput, and lack of validation with human biomarker data. We propose a tiered hybrid framework, where computational and high-throughput assays first identify high-risk MNPs, followed by targeted OoC studies to uncover human-specific mechanisms, balancing scalability with physiological relevance and supporting regulatory evaluation of chronic, low-dose exposures.
