Carcinogenic chemicals, or their metabolites, can be classified as genotoxic or non-genotoxic carcinogens (NGTxCs). Genotoxic compounds induce DNA damage, which can be detected by an established in... Show moreCarcinogenic chemicals, or their metabolites, can be classified as genotoxic or non-genotoxic carcinogens (NGTxCs). Genotoxic compounds induce DNA damage, which can be detected by an established in vitro and in vivo battery of genotoxicity assays. For NGTxCs, DNA is not the primary target, and the possible modes of action (MoA) of NGTxCs are much more diverse than those of genotoxic compounds, and there is no specific in vitro assay for detecting NGTxCs. Therefore, the evaluation of the carcinogenic potential is still dependent on long-term studies in rodents. This 2-year bioassay, mainly applied for testing agrochemicals and pharmaceuticals, is time-consuming, costly and requires very high numbers of animals. More importantly, its relevance for human risk assessment is questionable due to the limited predictivity for human cancer risk, especially with regard to NGTxCs. Thus, there is an urgent need for a transition to new approach methodologies (NAMs), integrating human-relevant in vitro assays and in silico tools that better exploit the current knowledge of the multiple processes involved in carcinogenesis into a modern safety assessment toolbox. Here, we describe an integrative project that aims to use a variety of novel approaches to detect the carcinogenic potential of NGTxCs based on different mechanisms and pathways involved in carcinogenesis. The aim of this project is to contribute suitable assays for the safety assessment toolbox for an efficient and improved, internationally recognized hazard assessment of NGTxCs, and ultimately to contribute to reliable mechanism-based next-generation risk assessment for chemical carcinogens. Show less
Schmeisser, S.; Miccoli, A.; Bergen, M. von; Berggren, E.; Braeuning, A.; Busch, W.; ... ; Tralau, T. 2023
The predominantly animal-centric approach of chemical safety assessment has increasingly come under pressure. Society is questioning overall performance, sustainability, continued relevance for... Show moreThe predominantly animal-centric approach of chemical safety assessment has increasingly come under pressure. Society is questioning overall performance, sustainability, continued relevance for human health risk assessment and ethics of this system, demanding a change of paradigm. At the same time, the scientific toolbox used for risk assessment is continuously enriched by the development of "New Approach Methodologies" (NAMs). While this term does not define the age or the state of readiness of the innovation, it covers a wide range of methods, including quantitative structure-activity relationship (QSAR) predictions, high-throughput screening (HTS) bioassays, omics applications, cell cultures, organoids, microphysiological systems (MPS), machine learning models and artificial intelligence (AI). In addition to promising faster and more efficient toxicity testing, NAMs have the potential to fundamentally transform today's regulatory work by allowing more human-relevant decision-making in terms of both hazard and exposure assessment. Yet, several obstacles hamper a broader application of NAMs in current regulatory risk assessment. Constraints in addressing repeated-dose toxicity, with particular reference to the chronic toxicity, and hesitance from relevant stakeholders, are major challenges for the implementation of NAMs in a broader context. Moreover, issues regarding predictivity, reproducibility and quantification need to be addressed and regulatory and legislative frameworks need to be adapted to NAMs. The conceptual perspective presented here has its focus on hazard assessment and is grounded on the main findings and conclusions from a symposium and workshop held in Berlin in November 2021. It intends to provide further insights into how NAMs can be gradually integrated into chemical risk assessment aimed at protection of human health, until eventually the current paradigm is replaced by an animal-free "Next Generation Risk Assessment" (NGRA). Show less
Veltman, C.H.J.; Pennings, J.L.A.; Water, B. van de; Luijten, M. 2023
Nongenotoxic (NGTX) carcinogens induce cancer via other mechanisms than direct DNA damage. A recognized mode of action for NGTX carcinogens is induction of oxidative stress, a state in which the... Show moreNongenotoxic (NGTX) carcinogens induce cancer via other mechanisms than direct DNA damage. A recognized mode of action for NGTX carcinogens is induction of oxidative stress, a state in which the amount of oxidants in a cell exceeds its antioxidant capacity, leading to regenerative proliferation. Currently, carcinogenicity assessment of environmental chemicals primarily relies on genetic toxicity end points. Since NGTX carcinogens lack genotoxic potential, these chemicals may remain undetected in such evaluations. To enhance the predictivity of test strategies for carcinogenicity assessment, a shift toward mechanism-based approaches is required. Here, we present an adverse outcome pathway (AOP) network for chemically induced oxidative stress leading to (NGTX) carcinogenesis. To develop this AOP network, we first investigated the role of oxidative stress in the various cancer hallmarks. Next, possible mechanisms for chemical induction of oxidative stress and the biological effects of oxidative damage to macromolecules were considered. This resulted in an AOP network, of which associated uncertainties were explored. Ultimately, development of AOP networks relevant for carcinogenesis in humans will aid the transition to a mechanism-based, human relevant carcinogenicity assessment that involves a substantially lower number of laboratory animals. Show less
While human regulatory risk assessment (RA) still largely relies on animal studies, new approach methodologies (NAMs) based on in vitro, in silico or non-mammalian alternative models are... Show moreWhile human regulatory risk assessment (RA) still largely relies on animal studies, new approach methodologies (NAMs) based on in vitro, in silico or non-mammalian alternative models are increasingly used to evaluate chemical hazards. Moreover, human epidemiological studies with biomarkers of effect (BoE) also play an invaluable role in identifying health effects associated with chemical exposures. To move towards the next generation risk assessment (NGRA), it is therefore crucial to establish bridges between NAMs and standard approaches, and to establish processes for increasing mechanistically-based biological plausibility in human studies. The Adverse Outcome Pathway (AOP) framework constitutes an important tool to address these needs but, despite a significant increase in knowledge and awareness, the use of AOPs in chemical RA remains limited. The objective of this paper is to address issues related to using AOPs in a regulatory context from various perspectives as it was discussed in a workshop organized within the European Union partnerships HBM4EU and PARC in spring 2022. The paper presents examples where the AOP framework has been proven useful for the human RA process, particularly in hazard prioritization and characterization, in integrated approaches to testing and assessment (IATA), and in the identification and validation of BoE in epidemiological studies. Nevertheless, several limitations were identified that hinder the optimal usability and acceptance of AOPs by the regulatory community including the lack of quantitative information on response-response relationships and of efficient ways to map chemical data (exposure and toxicity) onto AOPs. The paper summarizes suggestions, ongoing initiatives and third-party tools that may help to overcome these obstacles and thus assure better implementation of AOPs in the NGRA. Show less
Pallocca, G.; Moné, M.J.; Kamp, H.; Luijten, M.; Water, B. van de; Leist, M. 2022
In many industrial sectors, there is a need for reliable ways to evaluate the safety of chemicals with methods anchored to human biology and pathology. For this purpose, many animal-free new... Show moreIn many industrial sectors, there is a need for reliable ways to evaluate the safety of chemicals with methods anchored to human biology and pathology. For this purpose, many animal-free new approach methods (NAMs) have been developed and implemented in various stages of risk assessment. Now it is time to assemble individual NAMs into a comprehensive next-generation risk assessment (NGRA) strategy. The European Horizon 2020 RISK-HUNT3R project ("Risk assessment of chemicals integrating human-centric next-generation testing strategies promoting the 3Rs") has been designed to promote a combination of computational toxicology, in vitro toxicology, and systems biology. It is assumed that this approach will lead to faster and more accurate risk assessment procedures. The RISK-HUNT3R NGRA strategy will be developed to address the implementation of a comprehensive NAM toolbox into the regulatory framework. Critical conceptual approaches of the project include i) the integration of human-relevant data on biotransformation and elimination, ii) the translation of high-content mode-of-action datasets into predictions of adverse outcomes, iii) development of quantitative adverse outcome pathways (qAOPs), and iv) quantification of uncertainties associated with the predictions based on NGRA strategies. Many of the project steps will be used iteratively to generate datasets with sufficient quality and certainty for NGRA. Scientists and regulators will work together on case studies to evaluate NAMs' practical applicability and the strategies to combine information therefrom. Here we delineate how the strategy will be deployed to establish an overall NGRA framework for chemicals, pesticides, food additives, and drugs. Show less
Associations between per- and polyfluoroalkyl substances (PFASs) and increased blood lipids have been repeatedly observed in humans, but a causal relation has been debated. Rodent studies show... Show moreAssociations between per- and polyfluoroalkyl substances (PFASs) and increased blood lipids have been repeatedly observed in humans, but a causal relation has been debated. Rodent studies show reverse effects, i.e. decreased blood cholesterol and triglycerides, occurring however at PFAS serum levels at least 100-fold higher than those in humans. This paper aims to present the main issues regarding the modulation of lipid homeostasis by the two most common PFASs, PFOS and PFOA, with emphasis on the underlying mechanisms relevant for humans. Overall, the apparent contrast between human and animal data may be an artifact of dose, with different molecular pathways coming into play upon exposure to PFASs at very low versus high levels. Altogether, the interpretation of existing rodent data on PFOS/PFOA-induced lipid perturbations with respect to the human situation is complex. From a mechanistic perspective, research on human liver cells shows that PFOS/PFOA activate the PPAR alpha pathway, whereas studies on the involvement of other nuclear receptors, like PXR, are less conclusive. Other data indicate that suppression of the nuclear receptor HNF4 alpha signaling pathway, as well as perturbations of bile acid metabolism and transport might be important cellular events that require further investigation. Future studies with human-relevant test systems would help to obtain more insight into the mechanistic pathways pertinent for humans. These studies shall be designed with a careful consideration of appropriate dosing and toxicokinetics, so as to enable biologically plausible quantitative extrapolations. Such research will increase the understanding of possible perturbed lipid homeostasis related to PFOS/ PFOA exposure and the potential implications for human health. Show less
Luijten, M.; Terwee, C.; Oers, H. van; Joosten, M.; Berg, M. van den; Schonenberg-Meinema, D.; ... ; Haverman, L. 2019
Alternative methods to detect non-genotoxic carcinogens are urgently needed, as this class of carcinogens goes undetected in the current testing strategy for carcinogenicity under REACH. A... Show moreAlternative methods to detect non-genotoxic carcinogens are urgently needed, as this class of carcinogens goes undetected in the current testing strategy for carcinogenicity under REACH. A complicating factor is that non-genotoxic carcinogens act through several distinctive modes of action, which makes prediction of their carcinogenic property difficult. We have recently demonstrated that gene expression profiling in primary mouse hepatocytes is a useful approach to categorize non-genotoxic carcinogens according to their modes of action. In the current study, we improved the methods used for analysis and added mouse embryonic stem cells as a second in vitro test system, because of their features complementary to hepatocytes. Our approach involved an unsupervised analysis based on the 30 most significantly up- and down-regulated genes per chemical. Mouse embryonic stem cells and primary mouse hepatocytes were exposed to a selected set of chemicals and subsequently subjected to gene expression profiling. We focused on non-genotoxic carcinogens, but also included genotoxic carcinogens and non-carcinogens to test the robustness of this approach. Application of the optimized comparison approach resulted in improved categorization of non-genotoxic carcinogens. Mouse embryonic stem cells were a useful addition, especially for genotoxic substances, but also for detection of non-genotoxic carcinogens that went undetected by primary hepatocytes. The approach presented here is an important step forward to categorize chemicals, especially those that are carcinogenic. Show less
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