November 2025: Towards NAM-based risk assessment for developmental neurotoxic effects, illustrated with chlorpyrifos
Next generation risk assessment based on NAMs require extrapolation steps to go from a NAM-based value, e.g. an in vitro effect concentration, to a point-of-departure that can be used for human risk assessment. RIVM researchers developed a novel QIVIVE framework outlining all the extrapolation steps to do this. Applying the framework to a theoretical risk assessment for chlorpyrifos and its metabolite highlighted the most important extrapolation steps and uncertainties that influence the dose estimation. Future improvements could be made in the selection and refinement of the in vitro endpoint and assay, and in the definition of the quantitative relationship between the selected key event and the adverse outcome.
October 2025: In vitro human brain barrier models for studying thyroid hormone transport
Early brain development depends on thyroid hormone (TH). Disruptions in TH levels during development are linked to cognitive and motor impairments in children. Traditional animal models used to study effects of TH system disruption on neurodevelopment have limited human relevance. This highlights the need for New Approach Methodologies (NAMs), for example human stem cell-based in vitro models. RIVM researchers performed a literature search to describe the in vitro models that are currently being developed for the human blood-brain- and blood-cerebrospinal fluid barrier. Also, the applicability of these in vitro models to study the inhibition of thyroid hormone transport to the brain by endocrine disrupting chemicals is described. More research is needed to evaluate relevance, predictiveness and reproducibility to evaluate these NAMs for regulatory purposes.
October 2025: In silico prediction of endocrine activity
Endocrine-disrupting chemicals (EDCs) can pose risks to both human health and the environment. Enhanced use of in silico approaches can contribute to earlier and increased detection of potential EDCs. Scientists from RIVM and other institutes wrote a review on advances in computational toxicology for early identification and prioritization of EDCs. The review explores recent advances in these in silico predictions, highlighting developments within both ligand-based (e.g. QSARs) and structure-based approaches. Additionally, it discusses their application in a regulatory setting and future prospects to advance the field.
September 2025: Pursuing societal impact in research projects on NAM development: An analysis of routes to impact
Despite extensive public investment, the societal impact of projects developing New Approach Methodologies (NAMs) for chemical and pharmaceutical risk assessment remains limited. Researchers from Utrecht University and RIVM investigated how sixteen large European research projects pursue impact and identified 25 types of activities across five main routes to impact: community and capacity building, changing policy and regulation, advancing scientific developments, commercialisation and industrial partnerships, and mobilising civil society. This analysis highlights key barriers and trade-offs between these routes and calls for more reflexive, co-productive, and inclusive approaches to enhance societal impact.
September 2025: An Alternative Safety Profiling Algorithm (ASPA) to transform next generation risk assessment into a structured and transparent process
Next-generation risk assessment (NGRA) strategies use animal-free new approach methodologies (NAMs) to generate information concerning chemical hazard, toxicokinetics (ADME), and exposure. There is general consensus on the required types of data. However, the processes for data collection, integration and reporting are poorly standardised. Researchers from the RISK-HUNT3R project, with a leading role for RIVM, therefore developed the Alternative Safety Profiling Algorithm (ASPA). This is a risk assessment workflow that allows documentation and integration of all types of information required for NGRA. To assist users ASPA is accompanied by an online dashboard, ASPA-assist, to formalise the reporting process in a reproducible and standardised fashion.
August 2025: Refinement of a workflow for human relevance assessment of adverse outcome pathways and associated new approach methodologies
To assess the suitability of new approach methodologies (NAMs) for human risk assessment, the human relevance of toxicological pathways can be evaluated. Following earlier work, RIVM researchers refined a workflow to evaluate this. They also developed a pragmatic guidance and templates that can be used to systematically assess human relevance. Additionally, they suggested an approach to integrate and weigh all the available evidence that can conclude on the human relevance of a toxicological pathway and its associated NAMs. This work serves as a next step towards a harmonized, structured and transparent approach for human relevance assessment.
August 2025: A Pragmatic Workflow for Human Relevance Assessment of Toxicological Pathways and Associated New Approach Methodologies
To ensure animal-free alternatives are suitable for human health risk assessment, RIVM researchers developed a new workflow to transparently evaluate the human relevance of toxicological pathways and new approach methodologies (NAMs). Human relevance is assessed through three main questions, related to the different components (steps) of the pathway, the pathology of human syndromes that have a similar adverse outcome, and quantitative aspects. A case study on birth defects caused by triazole exposure demonstrated the potential of this workflow, showing moderate to strong support for human relevance of both the various components of the AOP and its associated NAMs. This transparent, scientific evidence-based human relevance evaluation of NAMs and adverse outcome pathways supports the transition towards more human-relevant, animal-free chemical safety testing.
July 2025: Adverse Outcome Pathway (AOP) Coaching Program—How it functions and contributes to a more harmonized approach to AOP development and construction of AOP networks with regulatory utility
The AOP framework contributes to understanding how specific and measurable biological perturbations cause adverse effects on human and environmental health. Development of AOPs requires adherence to specific principles and considerations. To assist new AOP developers a coaching program was launched by the OECD, which aims to pair novices with experienced AOP developers. This paper, to which RIVM contributed, describes the AOP Coaching Program, which represents the latest international activity to ensure that AOPs are developed in a consistent manner that is designed to enhance their use for supporting public health decisions around the world.
July 2025: Unlocking liver physiology: comprehensive pathway maps for mechanistic understanding
Physiological maps help reveal biological processes in chemical-sensitive diseases like fatty liver disease. This study, to which RIVM contributed, introduces two detailed Liver Physiological Maps to enhance chemical safety assessment and support animal-free testing approaches. LiverLipidPM covers lipid metabolism pathways in hepatocytes, while LiverBilePM maps bile acid biosynthesis, transport, and secretion. Developed via extensive literature review and expert input, both maps were validated for cell-type specificity. They are interactive, machine-readable, and available online, allowing pathway exploration and data overlay. These Liver Physiological Maps fill knowledge gaps, refine Adverse Outcome Pathway networks, allow computational modeling, and support human-relevant toxicity testing.
July 2025: EU (European Union ) roadmap for phasing out animal testing for chemical safety assessments: Recommendations from a multi-stakeholder roundtable
The European Commission is committed to developing a roadmap to phase out animal testing for chemical safety assessments, with completion targeted for early 2026. In a multi-stakeholder roundtable, organised by five animal protection NGOs in June 2024, key elements, structures, and transition pathways for the roadmap were explored. RIVM supports this roadmap and participated in this roundtable. During the roundtable critical issues were discussed, including legislative revision, validation, coordination, education, data transparency, progress metrics, and funding. The workshop emphasised a multi-faceted, collaborative approach, integrating scientific, regulatory, and ethical dimensions and formulated recommendations to guide the Commission and similar global efforts.
July 2025: Including Genetic Susceptibility Towards Parkinson’s Disease in NAM-based Hazard and Risk Assessment of Pesticides: a Semi-systematic Review
Understanding how genetic differences influence the risk of developing pesticide-induced Parkinson’s disease is key for hazard and risk assessment of pesticides. RIVM researchers performed a literature search to identify potential gene-environment interactions. The resulting genes were related to toxicokinetics, mitochondrial functioning, neurotransmission and proteostasis that can impact the development of Parkinson. New Approach Methodologies (NAMs) offer the opportunity to include these genetic alterations in hazard and risk assessment of pesticides to ensure protection of particular vulnerable groups in the population. This study informs future NAM development and regulatory incorporation, supporting the transition towards more human-relevant, animal-free pesticide safety testing.
June 2025: Application of in vitro pulmonary models for hazard screening of silica particles
There is a growing need for new approach methodologies (NAMs) to assess nanomaterial safety as innovation accelerates. In vitro assays can help prioritise safer nanomaterials and reduce regulatory animal testing, but must accurately distinguish hazard potential. This study assessed four silica particle types in 24 in vitro assays that are relevant in the mechanism of action towards on lung inflammation. Results showed that hazard rankings depended on cell type, measurement and exposure method, complicating human risk assessment. Notably, assays for IL-1β secretion and hemolysis aligned best with animal data, showing promise as tests to assess silica particle and other particle hazards.
June 2025: A global AOP network for genotoxicity to drive the integration of NAMs
In recent years, numerous new approach methodologies (NAMs) for genotoxicity have emerged. To modernise genotoxicity assessment for next generation genotoxicity assessment, it is essential to develop Integrated Approaches for Testing and Assessment (IATAs) that incorporate these NAMs. RIVM scientists have contributed to an article that explores the key challenges and gaps within the current European regulatory frameworks for chemical compound genotoxicity assessment and discusses how an AOP-based IATA can address these issues. Additionally, the paper includes a global AOP network for permanent DNA damage, designed to guide IATA development and improve regulatory decision-making.
June 2025: A Common Adverse Outcome Pathway for Metal(loid)s Inducing Nephrotoxicity to Advance Next-Generation Risk Assessment of Chemical Mixtures
Chronic exposure to the metal(loid)s arsenic, cadmium, lead and mercury via e.g. contaminated food or drinking water may induce kidney toxicity. There is little consensus on the biological processes involved. Health risk assessment of these substances is further complicated by co-exposures which hampers causal interpretation of effects observed in population studies. To address these issues, RIVM researchers developed a common Adverse Outcome Pathway (AOP) describing how these metal(loid)s can induce kidney toxicity. Evaluation of (experimental) studies revealed a moderate to high confidence in this AOP. It enhances mechanistic understanding of metal(loid)-induced kidney toxicity. As such, this is an example how AOPs can facilitate next-generation risk assessment of combined exposure to different contaminants.
June 2025: Setting off on the right path: make your research regulatory relevant
The process of standardisation of scientifically well-established methods, such as animal-free innovations, is often seen as too complex or too resource consuming by the scientific community. This inhibits method developers from translating their scientific methods and protocols into standards or OECD Test Guidelines. Numerous incentives exist for scientists to be active in the standardisation process and allow regulation to keep up with new scientific developments. This paper, co-authored by RIVM, provides guidance on how to navigate successfully through the standards and OECD Test Guidelines development processes. Active participation of scientists in this entire process is key. Only then can their methods be expanded into a wider, regulatory application towards a safer world.
June 2025: Towards a quantitative adverse outcome pathway for liver carcinogenesis: From proliferation to prediction
Testing of chemicals for cancer risk largely relies on 2-year animal studies. The current study, by RIVM and Leiden University, demonstrates how available animal data from 90-day studies can contribute to cancer risk assessment through a quantitative adverse outcome pathway (qAOP). The researchers mathematically linked liver cell proliferation after 90 days of chemical exposure to the formation of liver tumors over 2 years. It was shown that 90-day proliferative lesions predict 2-year carcinogenicity with high specificity, therebyreducing the need for longer-term 2-year animal studies. This approach highlights how reusing and integrating data with computational modelling supports the reduction of animal testing in safety assessment.
June 2025: Altered cytokine release of airway epithelial cells in vitro by combinations of respiratory syncytial virus, Streptococcus pneumoniae, Printex 90 and diesel exhaust particles
Exposure to air pollution has been associated with an increased severity of respiratory infections. Insight into the cellular mechanisms involved in this could provide an understanding of this interaction. RIVM researchers studied the effects of such a combined exposure using human-relevant in vitro models. Exposures to bacteria and particulate matter, in addition to a viral infection, resulted in a larger increase in cytokine levels compared to the virus infection alone. This information may help to understand the cellular effects of combined exposure in an in vitro setting and the biological responses to these exposures in humans.
May 2025: Hazard screening of colloidal silica nanomaterials with varying degrees of silane surface functionalization: a safe-by-design case study
The Safe and Sustainable by Design (SSbD) concept facilitates the design of safer and more sustainable chemicals and materials. Together with international partners, RIVM researchers used in vitro assays to study the effects of silane surface modification on the toxicity of five colloidal silica nanoforms (SiO2-NFs). Increasing silane functionalisation decreased cytotoxicity, pro-inflammatory, and oxidative stress responses, but reduced particle deposition efficiency. This research highlights that in vitro toxicity assays provide valuable information for SSbD decision making, informing the sustainable production of safer nanomaterials and nano-enabled products.
May 2025: Uncertainties in the Extrapolation of In Vitro Data in Human Risk Assessment: A Case Study of qIVIVE for Imazalil Using the Monte Carlo Risk Assessment Platform
To aid regulatory acceptance of animal-free methods for hazard characterisation, RIVM researchers have developed a framework for quantitative in vitro-in vivo extrapolation (qIVIVE). This framework highlights the extrapolations and uncertainties related to the use of qIVIVE. qIVIVE is essential in the translation of in vitro data to a health-based guidance value. However, it introduces many uncertainties that are often not addressed and quantified, hampering development and acceptance of this approach. A case study on liver steatosis resulting from dietary exposure to imazalil demonstrates the application of qIVIVE. The presented framework calls for transparent reporting of qIVIVE for hazard characterisation purposes, facilitating the regulatory acceptance of NAMs.
May 2025: How to organise a successful toxicology workshop? A participant perspective on the Collaboration to Harmonise the Assessment of Next Generation Evidence (CHANGE) workshop in Oslo, 18-20 June 2024
The CHANGE project aimed at fostering cross-sector consensus on the challenging topic of effective use of NAMs. Therefore, three workshops have been planned to Collaborate and Harmonise the Assessment of Next Generation Evidence. This report, to which RIVM researchers contributed, reflects participant feedback from the first workshop in Oslo (June 2024). Participants were enthusiastic about CHANGE and believe the approach holds great promise in shaping the future effective use of NAMs, but also noted areas for improvement. These include broader representation across sectors/geographies and clearer explanations of novel approaches in workshop methodology. Enhancing how information gathering is structured and formatted for feedback was also recommended. Overall, the workshop was seen as promising in advancing NAMs for chemical safety assessments.
April 2025: Towards prospective identification of respiratory sensitizers: effects of piperazine, chloramine-T, and toluene-diisocyanate in an air–liquid interface model comprising human bronchial epithelial cells
Exposure to respiratory sensitising compounds is the major cause for occupational asthma. To date, no OECD-approved test guideline exists to study this endpoint. RIVM researchers have been working on an in vitro method using the bronchial epithelial cell line Calu-3 cultured at the air-liquid interface. The three known respiratory sensitising compounds affected different biological processes in this model, for example pro-inflammatory cytokine release and barrier function. These findings imply that these sensitisers exert their health effects through different mechanisms.
April 2025: Developmental neurotoxicity (DNT): A call for implementation of new approach methodologies for regulatory purposes: Summary of the 5th International Conference on DNT Testing
In April 2024, the 5th Congress on Developmental Neurotoxicity (DNT5) was held. This paper summarises the conference, which focused on integrating animal-free New Approach Methodologies (NAMs) into risk assessment, in particular the DNT in vitro test battery (DNT-IVB). Experts discussed the use of in vitro data to predict safe exposure levels for humans and the need for broad biological and chemical coverage by the DNT-IVB. Key innovations included embryonic zebrafish embryo methods, AI-driven models, and brain organoids, highlighting progress in human-relevant, animal-free DNT testing. The conference emphasised the importance of collaboration between academics, industry, and regulatory bodies to promote the development and implementation of safer and more predictive testing methods.
February 2025: Mechanisms of developmental neurotoxicity mediated by perturbed thyroid hormone homeostasis in the brain: an adverse outcome pathway network
Endocrine disrupting compounds can disrupt the thyroid hormone balance It is known that thyroid hormone homeostasis is essential for correct neurodevelopment. This highlights the need to better understand how chemical exposure can disrupt thyroid homeostasis in the developing brain, and how this leads to adversity. Adverse Outcome Pathways (AOPs) can aid in this understanding, providing a structure to map mechanisms of thyroid hormone regulation. In this manuscript, researchers from RIVM and Erasmus MC expanded existing AOP networks by incorporating the process of thyroid hormone homeostasis regulation in the brain. The newly proposed AOP framework provides a basis for selecting brain-specific in vitro and in silico test systems for an innovative human-based hazard testing strategy of endocrine disrupting compounds.
January 2025: The European Partnership PARC’s Role in Actively Promoting the Uptake of New Approach Methodologies and Next-Generation Risk Assessment into Regulatory Risk Assessment Practice
Next-Generation Risk Assessment (NGRA) using New Approach Methodologies (NAMs) is commonly regarded as the way forward for chemical safety testing. In this paper, BfR, RIVM, and ANSES describe how PARC (European Partnership for the Assessment of Risks from Chemicals) contributes to the development of NGRA approaches. PARC supports the development and evaluation of Adverse Outcome Pathway (AOP) networks, NAMs and testing strategies. It furthermore fosters collaboration between stakeholders from all sectors of the chemical risk assessment community to ensure that NGRA approaches adequately meet regulatory needs. This is pivotal for the launch of European and national strategies to reduce animal testing and implement NGRA strategies in regulatory practice.
January 2025: Developing a Global Education Hub for Animal-Free Innovation
The Global Education Hub for animal-free education, initiated by TPI Utrecht and Peta UK, advocates integration of animal-free methods into education curricula and courses from high school to academia and education for professionals. It aims to foster scientific excellence, collaboration, and global impact in the use of non-animal methods and addresses the ethical concerns and the limitations of the use of animals in teaching, research and testing. The hub does this through co-creating, sharing, and disseminating animal-free educational tools in various scientific disciplines, including veterinary and medical education, life sciences, regulatory testing. The efforts to initiate the Global Education Hub were recently described in a workshop report published in ALTEX, with RIVM as one of the authors/contributors.
January 2025: Mapping physiology: a systems biology approach for the development of alternative methods in toxicology
New approach methodologies (NAMs), such as in silico models and in vitro experiments, offer promising alternatives. However, the current NAMs often lack a detailed understanding of biological mechanisms. Physiological Maps (PMs) are in silico tools that can provide the necessary detail by visually representing biological processes within specific organs and cells. PMs use standardised formats and annotations, making them easy to share and understand. By providing insights into human biology, PMs can complement and enhance NAMs, aiming to improve predictions of chemical toxicity. This approach fosters collaboration between the toxicology and systems biology communities in order to advance human-relevant risk assessment methods.
December 2024: A computational dynamic systems model for in silico prediction of neural tube closure defects
Neural tube closure is a vital step in embryo development. It shapes the future brain and spinal cord. Small errors during this process, caused by disruptions in cellular processes, can lead to severe birth defects like spina bifida (open back). In collaboration with computational toxicology experts, RIVM researchers built a complex computer model that shows how genes and physical forces guide this closure. By simulating “what-if” scenarios for disruption of neural tube closure, they found neural tube defects that match clinical findings, such as open spinal cords and abnormal midline fusions. Future refinements to the model will boost its accuracy. This will allow for the prediction of chemical disruptions, paving the way for safer chemicals, healthier lives, and fewer animal studies.
December 2024: Young TPI: Empowering animal-free science among the next generation of scientists
Promotion and encouragement for use of animal-free innovations among the next generation of scientists are fundamental for advancing science and the welfare of lab animals. Young TPI, of which an RIVM researcher was co-founder, emerged as the-first-of-its-kind network comprising young professionals and students dedicated to revolutionizing scientific practices by catalysing the shift towards animal-free research. This manuscript describes the conception, establishment, and progress of Young TPI, detailing its strategy for communication, activities, funding mechanisms, and endeavours to enlist new members and forge strategic alliances in pursuit of its mission towards an animal-free future for science.
December 2024: Evaluation of the standard battery of in vitro genotoxicity tests to predict in vivo genotoxicity through mathematical modeling: A report from the 8th International Workshop on Genotoxicity Testing
Identification of genotoxicity hazard of a chemical or pharmaceutical usually starts with a combination of in vitro tests. Scientists at RIVM applied mathematical modelling to a database with publicly available in vitro and in vivo data for genotoxicity. The results indicate that a mammalian in vitro clastogenicity test and a mammalian cell gene mutation test together provide strong predictive weight-of-evidence for evaluating genotoxic hazard of a substance. Remarkably, adding the often used Ames test to this combination did not significantly change the predictions. Further research will provide more insights whether the composition of the standard in vitro genotoxicity battery should be reconsidered, improving in vitro prediction of genotoxicity.
December 2024: Causal, Predictive or Observational? Different Understandings of Key Event Relationships for Adverse Outcome Pathways and their implications on practice
Adverse Outcome Pathways (AOPs) are crucial in regulatory toxicology for extrapolating mechanistic evidence across biological levels and contribute to application of animal-free models. Key Event Relationships (KERs) within AOPs can be described as causal, observational, or predictive, but the implications of these terms are not always clear. A recently published paper to which RIVM contributed looked into the usage of KER terminology and found that more careful consideration of terminology could enhance interdisciplinary communication on AOPs. This can improve the robustness and reliability of AOP frameworks in toxicology and risk assessment.
November 2024: Models of Down Syndrome: the gold standard refuted and opportunities for cell-based models
Trisomy of human chromosome 21 (T21) gives rise to Down syndrome (DS). Despite successful preclinical treatment studies to improve neurocognition in mouse models of Down syndrome, translation to humans has failed. Two recent studies, to which the RIVM contributed, give insight into possible causes and suggest directions for improvement. In the first paper, the phenotype of a common mouse model for DS turns out to depend on genes that are non-orthologous to the human chromosome 21, which exemplifies the limitation of the use of these animal models. The second paper showed that in vitro models can help to capture the variation in transcriptomic and phenotypic characteristics in T21 cell types, which could pave the way towards development of more human-relevant and targeted therapies to improve outcomes for individuals with DS.
Read more:
- The Impact of Mmu17 Non-Hsa21 Orthologous Genes in the Ts65Dn Mouse Model of Down Syndrome: The Gold Standard Refuted
- Analysis of genotype effects and inter-individual variability in iPSC-derived trisomy 21 neural progenitor cells
October 2024: Animal-free safety assessment of chemicals: an innovation system perspective
The scientific and technical challenges relating to the transition to the use of New Approach Methodologies (NAMs) for chemical safety testing are well known. Additionally, there are broader societal, institutional, and regulatory shifts that can stir the transition. In this perspective paper, innovation scholars from Copernicus Institute and toxicologists from RIVM address these beyond pure technoscientific aspects necessary for NAM implementation with a comprehensive framework. The paper provides handholds to track progress and organise collective efforts of actors, such as universities, governments, large and small companies and NGOs, to guide the transition.
September 2024: The Virtual Human Platform for Safety Assessment (VHP4Safety) project: Next generation chemical safety assessment based on human data
The VHP4Safety project, started in 2021 and funded by the Dutch NWA-ORC, will help shape the transition to next generation safety assessment in which animal testing becomes redundant. In a recent publication, the VHP4Safety partners, including RIVM, presented their interdisciplinary project approach. Multiple academic, regulatory, industrial and societal partners collaborate on building, feeding and implementing the virtual human platform using three case studies representing regulatory questions for chemical and pharmaceutical safety assessment that cannot be addressed in animal studies. Co-creation through involvement of all project participants is facilitated through organisation of designathons and hackathons.
September 2024: Proposal for a qualification system for New Approach Methodologies (NAMs) in the food and feed sector: example of implementation for nanomaterial risk assessment
Although many New Approach Methodologies (NAMs) are developed in a research setting, only some find their way to regulatory implementation. The NAMs4Nano Consortium, in which RIVM participates, was tasked by EFSA to propose a generic framework for a qualification system to speed up the regulatory use of NAMs. This includes the overall process, evaluation criteria, test method description, and procedures to describe the set-up of the NAM, its application and evaluation phase. It also addresses the scientific validity: how to demonstrate its reliability and relevance for a specific context-of-use. This framework was designed to aid the uptake of NAMs for nanomaterial risk assessment in the food and feed sector, but will also be useful in other regulatory domains.
September 2024: Review of New Approach Methodologies for Application in Risk Assessment of Nanoparticles in the Food and Feed Sector: Status and Challenges
New Approach Methodologies (NAMs) are thought to be of great added value for the risk assessment of nanomaterials. The NAMs4Nano Consortium, of which RIVM is one of the partners, has written a review discussing NAMs that will be potentially useful for risk assessment of nanomaterials in the food and feed sector. The review begins with an overview on nano‐specific NAM‐frameworks, followed by a description of individual NAMs and their relevance to nanomaterial physicochemical characterisation, exposure and hazard assessment. Validation notwithstanding, NAMs could already prove relevant and reliable for risk assessment of nanomaterials, especially in integrated approaches.
September 2024: The use of weight-of-evidence approaches to characterise developmental toxicity risk for therapeutic monoclonal antibodies in humans without in vivo studies
Weight-of-evidence approaches (WoE) are increasingly becoming acceptable to evaluate safety risks of pharmaceuticals for humans. Researchers from CBG-MEB evaluated a WoE approach for developmental risk of monoclonal antibodies (mAbs). Reproductive toxicity studies of 65 mAbs were reviewed and compared to the WoE. The researchers found that this WoE approach could characterize presence and absence of developmental risk without animal studies. The current WoE could have reduced the need for developmental toxicity studies by 42% without loss of important patient information in the label, exemplifying the power of this approach to reduce animal testing.
August 2024: Application of Machine Learning in Nanotoxicology: A Critical Review and Perspective
Evaluating the adverse effects of nanomaterials by laboratory methods is expensive, time-consuming, uses animals, and often fails to keep pace with the invention of new materials. Therefore, in silico methods that utilise machine learning techniques to predict the toxicity of nanomaterials are a promising alternative approach. This review, by RIVM and other researchers, explores how to build better predictive models; the current state of the art is analysed via a statistical evaluation of literature, while the challenges faced, and future perspectives are summarised. Machine learning promises to be a powerful tool in unravelling the mechanisms of toxicity of nanomaterials and thereby also a powerful tool to reduce animal studies.
August 2024: The zebrafish embryo as a model for chemical-induced steatosis: a case study with three pesticides
The disease state steatosis, or “fatty liver”, is a well-known side effect of some pesticides and pharmaceuticals and may lead to detrimental health outcomes including liver cancer. RIVM researchers have assessed the use of zebrafish embryos as 3R model organism to test if pesticides can induce steatosis, instead of the long-term rodent studies generally required. The results demonstrate that the zebrafish embryo provides a promising model for assessment of chemical-induced steatosis. Moreover, the used method was able to reliably detect even rather subtle changes in hepatic lipid accumulation, important when considering the effects of chemical mixtures.
August 2024: Utilization of human stem cells to examine neurotoxic impacts on differentiation
Proper cell differentiation is one of the crucial processes for normal brain development and disruption by e.g. chemical substances can have lifelong effects. In this book chapter, RIVM researchers discuss the use of human stem cells as an animal-free and human-relevant model that can mimic cell differentiation in the brain for neurotoxicology research. They discuss how differentiation can be measured, list technical considerations for setting up differentiation protocols, and explore current and future applications of stem cell-based differentiation models, e.g. for regulatory testing and gene-environment interactions studies.
August 2024: Chemical respiratory sensitization—Current status of mechanistic understanding, knowledge gaps and possible identification methods of sensitizers
In some chemical processes, workers may be exposed to respiratory sensitising chemicals, which can result in occupational asthma. There are currently no validated test methods to identify respiratory sensitising chemicals. Due to a better understanding of the key events that lead to respiratory sensitisation, new approach methodologies (NAMs) can be developed that address these key events. In a review article by researchers from six countries, among which the RIVM, the current knowledge on respiratory sensitisation is summarised, using the concept of an Adverse Outcome Pathway, and potential NAMs are discussed.
August 2024: Evaluation of rat and rabbit embryofetal development studies with pharmaceuticals: the added value of a second species
Embryofetal development (EFD) studies for pharmaceutical safety testing are generally conducted in one rodent and one non-rodent species, commonly rats and rabbits. However, the added value of testing in two species is debatable. Researchers from CBG-MEB and RIVM studied the concordance between rat and rabbit outcomes of more than 350 registered pharmaceuticals. They found that in many cases the added value of conducting EFD studies in two species is limited. The findings of this study can be used identify scenarios or create a weight-of-evidence model to determine the need for (additional) EFD studies, reducing the number of animal experiments.
June 2024: Zebrafish and nematodes as whole organism models to measure developmental neurotoxicity
In line with the review written in May (see below), researchers from RIVM and the Vrije Universiteit have written a review about the added value of zebrafish embryos and nematodes for studying developmental neurotoxicity. They specifically outline the gaps that these whole organisms can fill in the current testing strategy for developmental neurotoxicity, such as network formation in the brain, motor function and learning and memory, contributing to the 3R (Refine, Reduce, Replace) principles. These models can provide the necessary whole organismcontext and be used to explore the impact of chemicals on brain function by linking molecular and/or cellular changes to behavioural readouts in one model system.
June 2024: How to optimize communication about animal and animal-free research methods
There is a long-standing debate about animal and animal-free methods in scientific research. In this essay, researchers that were selected by media on the topic as ‘opponents’ describe anecdotes including their initial rational thoughts and emotions after these media events. Common ground was found in the principles of good scientific practice and responsible research. The teaming up of the scientists with seemingly opposing positions was as such instrumental in improving communication on animal(-free) experimentation issues. The authors, among whom RIVM researchers, strongly believe that keeping in dialogue is essential for making well-informed decisions on what methods the scientific community is using now and will be using in the future.
May 2024: New approach methods to assess developmental and adult neurotoxicity for regulatory use: a PARC work package 5 project
In the European regulatory context, rodent studies are the main source of neurotoxicity information. There is a scientific and societal push toward the replacement of animal models with New Approach Methodologies (NAMs). This paper describes the upcoming activities to develop and evaluate NAMs for developmental neurotoxicity and adult neurotoxicity and to understand the applicability domain of specific NAMs for the detection of endocrine disruption and epigenetic perturbation. These activities will take place in the EU-funded Partnership for the Assessment of Risk from Chemicals (PARC) of which RIVM is part.
May 2024: How neurobehavior and brain development in alternative whole-organism models can contribute to prediction of developmental neurotoxicity
Developmental neurotoxicity (DNT) testing is currently mainly performed in mammals, which has ethical issues and scientific limitations. Researchers from RIVM, Vrije Universiteit and Swarthmore College have written a review about the added value of whole organism systems that qualify as 3R (Replace, Reduce and Refine) models, which are urgently needed to complement cell-based DNT testing. The nematode Caenorhabditis elegans, the planarian Dugesia japonica, and embryos of the zebrafish Danio rerio are all suited for high-throughput screening of DNT and each of these organisms have unique strengths. The researchers also discuss how combining these models in testing strategies that consider the complexity of the developing brain will contribute to better human-relevant risk assessment of chemicals.
April 2024: New approach methodologies to enhance human health risk assessment of immunotoxic properties of chemicals — a PARC project
Chemicals may adversely affect the immune system through various pathways, leading to immune suppression, hypersensitivity, or increased susceptibility to autoimmune diseases. Currently, the evaluation of immunotoxicity by regulatory bodies relies on rodent models and a limited set of OECD test guidelines. However these guidelines do not fully encompass the full spectrum of potential immunotoxic effects. This paper offers an overview of ongoing endeavours aimed at devising and standardising new methods, both in vivo as well as new approach methodologies (NAMs) for thoroughly characterising the immunotoxic impact of chemicals. This review was established in the EU-funded Partnership for the Assessment of Risk from Chemicals (PARC). of which RIVM is part.
April 2024: A roadmap towards a human-centric safety assessment of advanced therapy medicinal products (ATMPs)
ATMPs are among the most complex pharmaceuticals. Due to their high human specificity, the clinical relevance of data derived from animal studies is limited. Researchers from CBG and RIVM surveyed stakeholders involved in ATMP development about their perspective on the use of animal studies and New Approach Methodologies (NAMs), perceived hurdles and potential solutions towards non-clinical human-centric safety assessment of ATMPs. Based on these outcomes, a roadmap was established that displays key points to consider when deciding on the need for future in vivo studies and improving the implementation of NAM-based approaches for products within a well-known modality or technology platform.
April 2024: In Vitro Assessment of Translocation and Toxicological Effects of Nicotine and Ethyl Maltol from e-Cigarettes Using Air–Liquid Interface-Cultured Bronchial Epithelial Cells
The use of e-cigarettes increases rapidly. Still, only little information is available on the adverse effects of flavourings in e-cigarettes. Toxicity testing of these flavourings in animal studies is unethical, and therefore, animal-free methods are required. RIVM researchers used cell models of the lungs to determine the toxic effects of e-cigarette vapour with nicotine and ethyl maltol. Both compounds were found to induce an inflammatory response in lung cells. They were also able to cross the lung barrier, meaning that they could enter the bloodstream and reach other organs in the human body.
April 2024: New Approach Methodologies (NAMs) for ad hoc human health risk assessment of food and non-food products - Proceedings of a workshop
RIVM convened a workshop on the use of NAMs for ad hoc human health risk assessment. Two case studies were discussed: the botanical Tabernanthe iboga, which is associated with cardiotoxicity, and dermal creams containing female sex hormones, intended for use by perimenopausal women without medical supervision. The participants recognised that data from NAMs added valuable information for the ad hoc risk assessment of these products, although the available approaches were inadequate to derive health-based guidance values. Recommendations were provided on how to further enhance and implement NAM approaches in regulatory risk assessment, specifying scientific, technical aspects and stakeholder engagement aspects.
March 2024: Landscape New Approach Methodologies (NAMs) safety assessment pharmaceutical products
Regulatory safety testing of pharmaceuticals currently needs animal studies. In the last decades, increasing attention is being given to the use of New Approach Methodologies (NAMs) to reduce animal testing. Implementation of NAMs in pharmaceutical safety assessment involves different steps and requires engagement of different stakeholders. RIVM researchers, in collaboration with the Dutch Medical Evaluation Board, made an overview of steps to take to implement NAMs for regulatory safety testing. With this overview, the researchers envision to facilitate the uptake of NAMs in this field.
March 2024: The role of trust in the use of artificial intelligence for chemical risk assessment
Artificial intelligence/machine learning (AI/ML) models are New Approach Methodologies (NAMs) that have the potential to improve chemical risk assessment. Yet, trust in their predictions is lacking due to their perceived "black box" nature. RIVM researchers facilitated knowledge exchange between computational modellers and risk assessors using two AI/ML models: one on ecotoxicity and one on human toxicity. Results showed that trust, essential for regulatory acceptance, evolves over time and comprises technical and beyond-technical factors. The study underscores the importance of interdisciplinary collaboration for effectively integrating AI/ML models into regulation. Future activities should prioritise collaboration between modellers and assessors to optimize data and knowledge utilisation.
March 2024: Beyond Encapsulation: Exploring Macrophage-Fibroblast Crosstalk in Implant-Induced Fibrosis
Biomaterials are used to manufacture medical implants. Implantation is associated with a foreign body response, which may lead to implant failure. In this review, researchers from RIVM, TU Eindhoven and UMCG took a mechanism-based approach and proposed the use of Adverse Outcome Pathways to assess the causal link between material properties and the severity of the foreign body response. This approach can lead to more human-relevant and animal-free testing of biomaterials.
15 March 2024: Dutch National Growth Fund invests €124.5 million in transition to animal-free innovation
The Dutch National Growth Fund will invest 124.5 million euros in a new Centre for Animal-Free Biomedical Translation (CPBT) to accelerate the transition to animal-free research over the next decade. The centre aims to generate safer, more effective treatments while reducing animal suffering. RIVM, in collaboration with Utrecht University, UMC Utrecht and Utrecht University of Applied Sciences, is one of the initiators of the centre and is involved in strengthening the acceptance and use of animal-free biomedical innovations.
January 2024: Report of the First ONTOX Stakeholder Network Meeting: Digging Under the Surface of ONTOX Together With the Stakeholders
In March 2023, the first stakeholder meeting within the scope of the EU-funded project ONTOX was held. ONTOX stands for ontology-driven and artificial intelligence-based repeated dose toxicity testing of chemicals for next-generation risk assessment. The participants identified and discussed the drivers, barriers, and challenges of the implementation of new approach methodologies (NAMs) and probabilistic risk assessment with the ONTOX Hub platform as an example of an integrating platform. Specific topics, including hazard versus risk, the role of AI and machine learning, and regulatory requirements, were discussed in break-out groups.
Alternatives to Laboratory Animals 2024; online first January 18
November 2023: An integrated approach to testing and assessment (IATA) to support grouping and read-across of nanomaterials in aquatic systems
Many new materials are being developed, which have various nanoforms that can each have a different hazard profile potentially affecting the environment. It is challenging to test all these nanoforms, since this would require many tests to cover the range of hazard profiles. Some of these tests require experimentation with organisms like daphnia, worms and fish. In this paper by RIVM researchers and collaborators in the GRACIOUS project, an IATA to support grouping and read-across of nanomaterials in aquatic systems was developed. The tiered testing and grouping approach could lead to a reduction of the number of animal tests needed to assess the hazard profile of nanoforms.
November 2023: PhD defence: Identification of biomarkers of the retinoic acid signalling pathway in the zebrafish embryo model to predict human developmental toxicants
Laura Samrani of the RIVM, Utrecht University, and Université Paris-Saclay performed the research for this PhD thesis, which was funded by the CIFRE project. This thesis expands the understanding of developmental toxicity pathways by studying chemically induced gene expression changes related to the perturbation of the retinoic acid signalling pathway in a zebrafish embryo model. The optimal timing for studying the retinoic acid signalling pathway was found to be around 4 hours post-fertilisation. Exposure to teratogens revealed a number of potential gene biomarkers for developmental toxicity.
PhD thesis Laura Samrani. Utrecht University, 2023
November 2023: The importance of variations in in vitro dosimetry to support risk assessment of inhaled toxicants
Health effects of inhaled pollutants are determined by how low the pollutant is inhaled, how much we inhale and how often. Testing all these differences in animal studies is cumbersome, and the results may not reflect the impact on human health. In vitro models of the lung allow prediction of the health impact of many different exposure scenarios. RIVM researchers tested different models and exposure methods and listed criteria that can assist in experimental design and data interpretation. The authors recommend (i) determining what information is needed to plan the experiment, (ii) choosing an exposure method that is suitable for the pollutant of interest, (iii) determining the amount of pollutant that interacts with the lung (deposition), and (iv) considering the time between the exposure and measurement of the effect.
ALTEX, accepted manuscript, published October 12, 2023
November 2023: An adverse outcome pathway for chemical-induced Parkinson’s disease: Calcium is key
Exposure to pesticides is associated with an increased risk of developing Parkinson’ Disease later in life. Currently, rodent-based risk assessment studies cannot adequately capture neurodegenerative effects of pesticides. To improve risk assessment guidelines, a mechanism-based, human-relevant approach is needed. Researchers from RIVM and the Insitute of Risk Assessment Sciences (IRAS) of the Utrecht University developed an adverse outcome pathway (AOP) network for chemically induced Parkinson’s disease. This AOP can serve as the biological basis for this approach. Developing a testing strategy based on this AOP by combining in vitro and in silico systems, can facilitate hazard assessment of neurodegeneration, without the use of animals.
NeuroToxicology 2023: 99: 226-243
August 2023: Variability of in vivo potency assays of whole-cell pertussis, inactivated polio, and meningococcal B vaccines
For some vaccines, batch-release testing to determine potency or safety involves animal experiments. These in vivo potency assays show large inherent variability. In a study by CBG-MEB, Medicines and Healthcare Products Regulatory Agency (UK), Utrecht University and RIVM, this variability in the outcome of measurements of the potency of three vaccines was evaluated. Results showed, for example, that antibody responses to four meningococcal B antigens in >300 mice varied strongly and showed no correlation in individual mice. The large differences between individual animals likely contribute considerably to the inherent variability of in vivo potency assays. The low precision makes in vivo assays less suitable for monitoring manufacturing consistency and supports the notion of moving away from these assays for batch release testing.
Vaccine 2023; 41(38):5603-5613
August 2023: New approach methodologies in human regulatory toxicology – Not if, but how and when!
New Approach Methodologies (NAMs) have the potential to transform approaches to regulatory hazard and risk assessment fundamentally. Yet, major challenges need to be overcome for NAMs towards implementation into current legal frameworks. This manuscript describes a conceptual perspective that is grounded on the main findings and conclusions from a symposium held in Berlin in November 2021, at which RIVM was one of the experts. Experts address the need for developing a strategic framework for making legal frameworks “NAM-ready” step by step. Furthermore, they suggest the establishment of working groups to evaluate and re-describe regulatory testing requirements. A tentative timeline is presented to involve all relevant stakeholders in these activities and to make the transition to NAM-based risk assessment happen.
Environment Int 2023; 178:108082
August 2023: New approach methodologies to facilitate and improve the hazard assessment of non-genotoxic carcinogens—a PARC project
Non-genotoxic carcinogenic chemicals operate via various modes of action, thus requiring a suite of New Approach Methodologies (NAMs) for detecting this type of chemicals. RIVM is contributing to this integrative project that is performed within the context of the European Partnership for the Assessment of Risks from Chemicals (PARC). The aim is to identify suitable NAMs for the detection of carcinogenic potential of chemical substances. Ultimately, this work will contribute to an efficient and improved, internationally recognised hazard assessment of non-genotoxic carcinogens.
Front Toxicol 2023; 5
July 2023: Evaluation of non-clinical toxicity studies of COVID-19 vaccines
The safety of new vaccines is non-clinically tested in animal studies. Researchers from Leiden University and the Dutch Medicine Evaluation Board evaluated the outcomes of non-clinical toxicity studies of various SARS-CoV-2 vaccines. From a 3R perspective, they evaluated whether additional animal studies are needed or whether previous historical studies are already sufficient to inform the vaccine assessment process. The authors concluded that only product-specific safety studies, combined with a justified package of supportive historical studies that used the same product carrier, are considered sufficient to support clinical development. This reduces the number of animals required for nonclinical development of vaccines.
Regulatory Toxicology and Pharmacology 2023; 142:105438
July 2023: Retinoic acid signalling pathway perturbation impacts mesodermal-tissue development in the zebrafish embryo: Biomarker candidate identification using transcriptomics
In an effort to further optimize a NAM for developmental toxicity, RIVM researchers used a whole transcriptome approach to identify teratogenicity biomarker genes in the zebrafish embryo model. There were 248 genes regulated by the teratogens tested (all-trans retinoic acid and valproic acid), which were not regulated by the non-teratogenic compound (folic acid). A sub selection of the genes related to the retinoic acid pathway were identified as potential gene expression biomarkers of mesodermal tissue and organ malformation in early vertebrate embryos. To determine their robustness as biomarkers, these genes should be more broadly studied using different in vitro models and chemical exposures.
Reproductive Toxicology 2023: 119:108404
June 2023: Transferability and reproducibility of exposed air-liquid interface co-culture lung models
Lung models that can be exposed via the air, by means of an air-liquid interface, are promising in vitro models for evaluating the safety of chemicals and nanomaterials after inhalation exposure. Together with six other laboratories, RIVM researchers performed an interlaboratory comparison study to evaluate the transferability and reproducibility of a monoculture and coculture model. The lung models were exposed to nanomaterials at physiologically relevant dose levels. Interlaboratory variation for cell viability and barrier integrity were acceptable, whereas variation in production of inflammatory proteins was relatively high. The authors recommend further optimisation of the model and testing higher doses to improve reproducibility.
NanoImpact 2023; 31:100466
April 2023: An adverse outcome pathway network for chemically-induced oxidative stress leading to (non-)genotoxic carcinogenesis
Currently, carcinogenicity assessment of chemicals primarily relies on genotoxicity testing. Since non-genotoxic carcinogens do not induce direct DNA damage, these chemicals may remain undetected. To enhance the predictivity of carcinogenicity assessment testing strategies, a shift towards mechanism-based, human-relevant approaches is required. Researchers from RIVM and Leiden University have developed an adverse outcome pathway network for chemically-induced oxidative stress leading to carcinogenesis. This mechanism-based, human-relevant approach can aid in reducing the number of laboratory animals for carcinogenicity assessment.
Chem Res Toxicol 2023; online ahead of print
April 2023: Rational arguments for regulatory acceptance of consistency testing: benefits of non-animal testing over in vivo release testing of vaccines
Some vaccine batch release testing involves animal experiments that determine the potency or safety of the batch. In this perspective article, international regulatory bodies, including the CBG-MEB, call for the replacement of these animal tests by advanced in vitro methods, not only from an ethical perspective. They describe the hurdles and solutions to substitute in vivo assays and the advantages of in vitro methods, both as a superior monitoring tool for the quality of vaccines and as a more practical, economical, and ethical method. Government incentives and supportive regulatory authorities are deemed necessary to implement these novel animal-free tests.
Expert Review of Vaccines 2023; 22(1):369-77
April 2023: ZonMw TPI Knowledge Agenda for animal-free innovations in biomedical research
While the number of animal-free innovations that are being developed is increasing, they are not yet widely used in fundamental and translational biomedical science. ZonMw, the Dutch funding organisation of the programme “More Knowledge with Fewer Animals (Dutch acronym: MKMD) published the TPI Knowledge Agenda, in which they identify the obstacles, potential solutions and recommendations to stimulate the transition towards animal-free science (Dutch acronym: TPI). The TPI Knowledge Agenda is based on broad consultation of the biomedical research field in the Netherlands and written with the help of RIVM researchers. TPI Knowledge Agenda (news only in Dutch)
March 2023: PhD defence: Paving the road towards the Next Generation Risk Assessment - Quantitative in vitro to in vivo extrapolations for the pre-diction of human toxicity based on alternative testing
The research for this PhD thesis was performed by Stella Fragki of the RIVM and Utrecht University, and was funded by the European human biomonitoring project HBM4EU. Her thesis describes the extrapolation of concentration-response relations observed in in vitro assays to dose-response relations in human. For example, she showed that including data from a cell culture system mimicking the placental barrier improves extrapolation significantly. This approach, called quantitative in vitro to in vivo extrapolation (QIVIVE) is an important element in innovative hazard and risk assessment of chemicals and pharmaceuticals.
February 2023: 3Rs Working Party (3RsWP) plenary meeting - Public session on the 2023 work plan
Join the public session of the 3RsWP of the European Medicine Agency (EMA) on 28 February, 09:00–09:45. In this session the 3RsWP will present their work plan and priorities for 2023 and give an opportunity to stakeholders to comment and provide their views on the working party's activities. This public session is part of this newly established working party's two-day plenary meeting to showcase EMA's commitment to ensuring the implementation of 3Rs principles in the development and evaluation of human and veterinary medicinal products.
3Rs Working Party (3RsWP) plenary meeting
February 2023: Innovating human chemical hazard and risk assessment through a holistic approach
Regulatory toxicology has relied on animal studies as the gold standard for over half a century. It has increasingly been realised that the detour of the animal study for human risk assessment comes with drawbacks, both scientifically as well as ethically. In this publication, RIVM researchers pose that innovative New Approach Methodologies provide molecular and mechanistic data that can be integrated in computational models to predict adverse outcome at the level of the intact human. It is expected that in due course, current significant advances in computational toxicology and machine learning already effective in clinical practice, will be able to advance into a human-focused, animal-free risk assessment strategy. This strategy will be fine-tuned to the human situation and will therefore lack the scientific and ethical concerns of current classical regulatory animal testing for human chemical risk assessment.
Current Opinion in Toxicology 2023; available online 10 February 2023, 100386
February 2023: Prolonged differentiation of neuron-astrocyte co-cultures results in emergence of dopaminergic neurons
Epidemiological and experimental data suggest that pesticides affect dopaminergic cells in the brain. An effect that has been related to human neurodegenerative diseases such as Parkinson’s disease. To allow for human-relevant assessment of (developmental) neurotoxicity related to dopamine disruption, researchers from RIVM have developed a human stem cell-based differentiation protocol for dopaminergic neurons. Further characterisation and chemical testing are needed to define the role of the current model in a testing strategy to test how pesticides or other chemicals might affect the dopaminergic system.
Int J Mol Sci 2023; 24(4): 3608
February 2023: The State of the Art and Challenges of In Vitro Methods for Human Hazard Assessment of Nanomaterials in the Context of Safe-by-Design
The Safe-by-Design (SbD) concept aims to facilitate the development of safer materials/products throughout their life cycle. Early hazard screening is therefore essential. Partners of the SAbyNA consortium reviewed commonly used in vitro tests for their suitability for SbD hazard testing of nanomaterials. Some simple in vitro assays are capable of accurately predicting hazard warnings and hazard rankings for some but not all toxicological endpoints. The authors recommend that joint efforts of innovators, scientists, and regulators are needed to improve hazard testing for SbD approaches.
Nanomaterials 2023; 13(3): 472
January 2023: Smoking-associated exposure of human primary bronchial epithelial cells to aldehydes: impact on molecular mechanisms controlling mitochondrial content and function
Respiratory toxic aldehydes are generated during smoking of tobacco. To study the role of aldehydes on the molecular regulation of mitochondrial content/function, Maastricht University and RIVM used a differentiated in vitro model of human primary bronchial epithelial cells exposed to cigarette smoke following a realistic exposure protocol. Cigarette smoke caused disruption of molecular mechanisms controlling mitochondrial content and quality. However, exposure to a mixture of three aldehydes only caused a dysregulation of markers associated with autophagy and mitophagy. This suggests that other compounds then aldehydes contribute to cigarette smoke-induced dysregulation of mitochondrial content/function.
Cells 2022; 11(21): 3481
January 2023: Re-evaluating the need for chronic toxicity studies with therapeutic monoclonal antibodies, using a weight of evidence approach
For the registration of monoclonal antibodies (mAbs) for chronic indications, 6-month as well as 3-month toxicity studies in rodents are conducted. However, experience has shown that mAbs are generally well tolerated, thereby questioning the relevance to assess human safety in long term rodent toxicity studies. Researchers of the CBG-MEB studied whether a 3-month study is already sufficient to assess the long-term safety of mAbs. For the majority of the 111 assessed mAbs novel toxicity is rarely detected in animal studies of longer than 3 months. This may allow for a case-by-case approach to reduce animal use by conducting a 3- instead of 6-month study.
Regul Toxicol Pharmacol 2023; 138:105329
January 2023: The use of recovery animals in nonclinical safety assessment studies with monoclonal antibodies: further 3Rs opportunities remain
When performing nonclinical safety testing of a monoclonal antibody (mAb) in animals, it is sometimes required to include a recovery group. According to the guidelines, this is only needed in one of the nonclinical studies. A working group, including participants from CBG-MEB, reviewed 51 mAb toxicity studies and found that often recovery groups are included in multiple nonclinical studies over the course of testing a mAb. The working group concluded that by limiting inclusion of a recovery group to a single nonclinical toxicology study provides opportunities to decrease animal use for this drug class. Regul Toxicol Pharmacol 2023; 138:105339
December 2022: Towards achieving a modern science-based paradigm for agrochemical carcinogenicity assessment
The rodent cancer bioassay has been the standard approach to test chemicals for human carcinogenic potential. However, there are known limitations to this assay, leading to a search for more human-relevant approaches providing mechanistic information on chemical carcinogenesis. In this paper the authors outlined the challenges and opportunities that authorities should consider when building a roadmap that should lead to global acceptance and incorporation of fit-for-purpose new approaches for carcinogenicity assessment of agrochemicals. New approaches include shorter-term tests, including in vitro and in silico approaches. To the publication.
December 2022: Application of AOPs to assist regulatory assessment of chemical risks – Case studies, needs and recommendations
The Adverse Outcome Pathway (AOP) framework is an important tool in moving towards next generation risk assessment (NGRA). A workshop organised within the European Union partnerships HBM4EU and PARC was held to address issues related to using AOPs in a regulatory context. AOPs have shown useful in e.g. hazard characterisation and in identification and validation of biomarkers of effect. However, e.g. lack of quantitative information on response-response relationships holds AOP application back. The paper discusses suggestions, initiatives and tools that may help to better implement AOPs in the NGRA. To the publication.
December 2022: Neurodegeneration in a Regulatory Context: The Need for Speed
There is increasing evidence that pesticides can induce or accelerate neurodegenerative diseases. Current regulatory guidelines for safety assessment cannot fully capture the neurodegenerative effects of chemical substances. In this publication, researchers from RIVM propose a tiered approach to update the regulatory guidelines. In the short term, adaptations to current guideline studies can be made. In the long term transition of chemical safety assessment to a strategy based on human biology using in vitro and in silico methods should be adopted. To the publication.
December 2022: Applicability of generic PBK modelling in chemical hazard assessment: A case study with IndusChemFate
Various physiologically based kinetic (PBK) models exist that may all vary in their chemical applicability domain. In this study, the generic physiologically based kinetic model IndusChemFate was evaluated and compared to the PBK “TNO Model”. Both models were evaluated with a broad span of chemicals varying in physicochemical properties. Results showed that the simpler IndusChemFate model performed better as a first tier for simulating toxicokinetics based on QSARs and in vitro parameters. Findings were illustrated in HepaRG cells exposed to acetaminophen. To the publication.
December 2022: Dynamic regulation of gene expression and morphogenesis in the zebrafish embryo test after exposure to all-trans retinoic acid
This publication evaluated the Zebrafish Embryo Test (ZET) for the prediction of developmental toxicity. The effect of exposure duration of all-trans retinoic acid on the magnitude of gene expression changes was investigated. Results showed that gene expression regulation indicative of developmental toxicity could be observed already at early time points after exposure. Considering early time point measurements of gene expression changes could help optimise the sensitivity of the ZET in future studies. To the publication.
18 November 2022: Hugo van Poelgeest Award won by Niels Harlaar
The Hugo van Poelgeest Award is a scientific prize for excellent, animal-free research in the life sciences. Niels Harlaar (LUMC) won the award for his research on transformed human heart cells. Valentina Palacio-Castañeda (Radboudumc) won the audience award, and Victoria de Leeuw (RIVM) was nominated. The prize is awarded every other year by stichting Proefdiervrij and stichting Bouwstenen voor Dierenwelzijn. Read more (in Dutch).
November 2022: The Current Status and Work of Three Rs Centres and Platforms in Europe
Over recent years, an increasing number of 3R centres and platforms have been established in Europe. Together with representatives of European 3R initiatives, RIVM contributed to a publication that provides an overview of the current activities of the 3R centres: the financial and organisational structures of the, what their Three Rs focus and core activities are (dissemination, education, implementation, scientific quality/translatability, ethics), and their areas of responsibility and projects. See the publication.
22 October 2022: Analyses of Transcriptomics Cell Signalling for Pre-Screening Applications in the Integrated Approach for Testing and Assessment of Non-Genotoxic Carcinogens
Mechanistic understanding of biological processes that lead to carcinogenesis is expanding. Transcriptomics can inform on non-genotoxic mechanisms of cancer caused by chemicals. An OECD working group, in which RIVM participates, reviewed the application of transcriptomics for regulatory applications. Specifically, they evaluated the potential of gene expression analyses to identify the most relevant approaches that could be utilised as (pre-)screening tools. Challenges related to the integration of gene panels and transcriptomic assays into an Integrated Approach to the Testing and Assessment of non-genotoxic carcinogens is also discussed. To the publication.
11 October 2022: Renewal of the EURL ECVAM Scientific Advisory Committee (ESAC) and open call for application subgroups
The Joint Research Centre (JRC) formally appointed nine experts as the new member of the ESAC on 11 October 2022. Anne Kienhuis, senior scientist of the RIVM is one of the new members. ESAC provides EURL ECVAM with independent scientific advice on the adequacy and outcome of formal validation studies carried out to assess the reliability and relevance of NAMs in the context of regulatory safety assessment. Read more in this news item.
New videos on TPI.tv
Working towards animal-free research strategies is an effort crossing international and interdisciplinary borders. TPI.tv is designed to facilitate this transition through connecting people and ideas via video messages. On TPI.tv, short video messages (‘pitches’) provide the medium for researchers to display their findings and request input if needed. New videos on TPI.tv:
In3 | Helpathon #6 | Helpathon #7 | In vitro atrial fibrillation | In vitro neurodevelopmental toxicity assessment | Tumour-on-chip
October 2022: 3Rs concepts to improve the quality of biomedical science COST-Action IMPROVE
Improving the quality of biomedical science by using 3Rs concepts is the main aim of the new COST-Action IMPROVE. In this initiative, a network of researchers will be established to refine, harmonise and promote 3Rs concepts, data and documents, in order to improve the quality of biomedical science. Researchers interested in contributing can apply for working groups on the (1) Quality and Translatability of Science, (2) Implementation, (3) Dissemination, and (4) Education. The consortium is co-led by Utrecht University and RIVM and is funded by the Dutch National Research Agenda. Read more about IMPROVE.
September 2022: Prioritisation of chemicals in food for risk assessment by integrating exposure estimates and new approach methodologies: A next generation risk assessment case study
Next generation risk assessment (NGRA) is a knowledge-driven system that allows for cost-efficient, animal-free chemical risk assessment. In this case study, researchers at RIVM showed how NGRA can facilitate prioritisation of chemical substances in food. They explored how exposure data combined with the Threshold of Toxicological Concern concept could be used to prioritise chemicals for toxicity testing. A selected set of compounds from this analysis was screened for toxicological potential using high-throughput screening. This study is a proof-of-principle that this approach can be a supportive element in NGRA. To the publication.
September 2022: Development of a cell line-based in vitro assay for assessment of Diphtheria, Tetanus and acellular Pertussis (DTaP)-induced inflammasome activation
Safety and potency assessment for batch-release testing of established vaccines still partly relies on animal tests. As part of the VAC2VAC project, researchers at RIVM developed an in vitro testing method for adjuvant biological activity that can contribute to animal-free potency testing of vaccines. Specifically, they developed an NLRP3 inflammasome activation test for aluminium-adjuvanted vaccines (the DTaP vaccine). The results indicated that inflammasome activation may be used to measure adjuvant biological activity as an important quality attribute for control or characterisation of the adjuvant. More information in the publication Development of a cell line-based in vitro assay for assessment of Diphtheria, Tetanus and acellular Pertussis (DTaP)-induced inflammasome activation.
September 2022: Realistic in vitro exposure of neuronal cells to e-cigarette vapour
Chemical exposure through the airways may affect the airways themselves, but can also cause secondary effects on, for example the brain. Researchers at RIVM and Utrecht University studied an in vitro approach to test the effects of e-cigarette vapour, using a combination of a lung cell model and a neuronal cell model. Components of the e-cigarette vapour could cross the lung cell barrier and subsequent exposure on the neurons modulated their spontaneous electrical activity. This approach, when optimised, can improve realistic exposure in vitro, decreasing the need for animal studies. Read more Neuromodulatory and neurotoxic effects of e-cigarette vapor using a realistic exposure method.
August 2022: Biomarkers for quality control of Weil’s disease vaccines
Potency testing of Leptospira (Weil's disease) vaccine batches relies partially on laboratory animals. Cell-based assays could replace in vivo tests for vaccine quality control if biomarkers indicative of Leptospira vaccine potency are identified. RIVM contributed to a study that used a canine monocyte/macrophage cell line to investigate innate immune responsiveness by Leptospira species as well as Leptospira vaccines from two different manufacturers. Gene and protein expression analysis led to the identification of four proteins for the development of biomarker-based assays. Read more Transcriptome and proteome analysis of innate immune responses to inactivated Leptospira and bivalent Leptospira vaccines in canine 030-D cells.
August 2022: Animal-free assays for endocrine disruption – from science to regulatory acceptance
Researchers from, amongst others, Utrecht University and RIVM will assess how to improve the regulatory acceptance and use of animal-free assays for endocrine disruption. The consortium receives funding worth 1 million euros from the Dutch Research Agenda for this purpose. The ultimate aim of the project is to achieve regulatory and societal acceptance towards the implementation of existing animal-free assays for endocrine disruption in regulatory frameworks. In this project, a multi-disciplinary consortium with experts in toxicology, social sciences and legislation will combine their efforts to investigate and accelerate the transition towards the use of animal-free assays for endocrine disruption. Read more (in Dutch).
July 2022: Identification of proteome markers for drug-induced liver injury in zebrafish embryos
The zebrafish embryo is a promising alternative non-rodent model in toxicology. Scientists at RIVM and Leiden University exposed zebrafish embryos to nine hepatotoxicants (targeted at induction of steatosis, cholestasis and necrosis). Next, they determined protein expression changes using LC-MS/MS and identified differentially expressed proteins and pathways. Overlap between these responses resulted in a set of ten common protein markers for the detection of adverse liver responses. This may allow for a reduction in rodent use for hepatotoxicity assessment. Read more Identification of proteome markers for drug-induced liver injury in zebrafish embryos.
June 2022: Roadmap for Action on New Approach Methodologies in Risk Assessment
The European Food Safety Authority set out a call to develop a roadmap for the implementation of New Approach Methodologies (NAMs) in food risk assessment. A team of experts, from Fraunhofer ITEM, RIVM, Vrije Universiteit Amsterdam, Technical University of Denmark, and EURA AG defined key barriers and opportunities as well as activity proposals for implementation of NAMs in food risk assessment in seven areas: development of additional adverse outcome pathways (AOPs)/AOP networks, advanced cell culture models including organ-on-chip, toxicokinetic assessment with a focus on physiological based kinetic modelling, use of exposome data, integration of human susceptibility, data integration and new concepts in human risk assessment for NAM implementation. Furthermore, a Forum is proposed to facilitate interaction between stakeholders for the implementation of NAMs in risk assessment. Read more.
May 2022: Update Landscape NAMs
We have published an update of the Landscape NAMs for the safety assessment of chemical substances. The overview provides information on each step in the implementation curve. With this Landscape, we facilitate the uptake of NAMs in regulatory safety testing. Go to the Landscape NAMs.