Return on investment in science: twenty years of European Commission funded research in Alzheimer's dementia, breast cancer and prostate cancer.

Alzheimer's disease (AD), breast cancer (BC) and prostate cancer (PC) continue to be high in the research and innovation agenda of the European Commission (EC). This is due to their exceptionally large burden to the national health systems, the profound economic effects of opportunity costs attributable to decreased working ability, premature mortality and the ever-increasing demand for both hospital and home-based medical care. Over the last two decades, the EC has been steadily increasing both the number of proposals being funded and the amounts of financial resources being allocated to these fields of research. This trend has continued throughout four consecutive science funding cycles, namely framework programme (FP)5, FP6, FP7 and Horizon 2020 (H2020). We performed a retrospective assessment of the outputs and outcomes of EC funding in AD, BC and PC research over the 1999-2019 period by means of selected indicators. These indicators were assessed for their ability to screen the past, present and future for an array of causal relationships and long-term trends in clinical, epidemiological and public health sphere, while considering also the broader socioeconomic impact of funded research on the society at large. This analysis shows that public-private partnerships with large industry and university-based consortia have led to some of the most impactful proposals being funded over the analysed time period. New pharmaceuticals, small molecules and monoclonal antibodies alike, along with screening and prevention, have been the most prominent sources of innovation in BC and PC, extending patients' survival and enhancing their quality of life. Unlike oncology, dementia drug development has been way less successful, with only minor improvements related to the quality of supportive medical care for symptoms and more sensitive diagnostics, without any ground-breaking disease-modifying treatment(s). Significant progresses in imaging diagnostics and nanotechnology have been largely driven by the participation of medical device industry multinational companies. Clinical trials funded by the EC were conducted, leading to the development of brand-new drug molecules featuring novel mechanisms of action. Some prominent cases of breakthrough discoveries serve as evidence for the European capability to generate cutting-edge technological innovation in biomedicine. Less productive areas of research may be reconsidered as priorities when shaping the new agenda for forthcoming science funding programmes.

Recommendations on fit-for-purpose criteria to establish quality management for microphysiological systems and for monitoring their reproducibility.

Cell culture technology has evolved, moving from single-cell and monolayer methods to 3D models like reaggregates, spheroids, and organoids, improved with bioengineering like microfabrication and bioprinting. These advancements, termed microphysiological systems (MPSs), closely replicate tissue environments and human physiology, enhancing research and biomedical uses. However, MPS complexity introduces standardization challenges, impacting reproducibility and trust. We offer guidelines for quality management and control criteria specific to MPSs, facilitating reliable outcomes without stifling innovation. Our fit-for-purpose recommendations provide actionable advice for achieving consistent MPS performance.

Transitioning biomedical research toward human-centric methodologies: systems-based strategies.

Human-centric methodologies like microphysiological systems and in silico methods have shown promise in addressing the limitations of animal models in understanding human biology and responding to public health priorities. However, the prevailing paradigm based on animal research persists. The article proposes a systemic thinking approach, endorsed by the OECD and the EU, as a tool to leverage innovation to reframe the issue and achieve transformative policies. By identifying the complex factors shaping method selection in basic and biomedical research, a simplified model is presented to illuminate the systemic nature of this decision-making process. The goal is not to prescribe solutions but to offer policymakers a new framework for more-effective strategies, emphasizing collaboration among stakeholders and the need for robust data.

Taking the leap toward human-specific nonanimal methodologies: The need for harmonizing global policies for microphysiological systems.

With a recent amendment, India joined other countries that have removed the legislative barrier toward the use of human-relevant methods in drug development. Here, global stakeholders weigh in on the urgent need to globally harmonize the guidelines toward the standardization of microphysiological systems. We discuss a possible framework for establishing scientific confidence and regulatory approval of these methods.

Gauging innovation and health impact from biomedical research: survey results and interviews with recipients of EU-funding in the fields of Alzheimer's disease, breast cancer and prostate cancer.

Biomedical research on Alzheimer's disease (AD), breast cancer (BC) and prostate cancer (PC) has globally improved our understanding of the etiopathological mechanisms underlying the onset of these diseases, often with the goal to identify associated genetic and environmental risk factors and develop new medicines. However, the prevalence of these diseases and failure rate in drug development remain high. Being able to retrospectively monitor the major scientific breakthroughs and impact of such investment endeavors is important to re-address funding strategies if and when needed. The EU has supported research into those diseases via its successive framework programmes for research, technological development and innovation. The European Commission (EC) has already undertaken several activities to monitor research impact. As an additional contribution, the EC Joint Research Centre (JRC) launched in 2020 a survey addressed to former and current participants of EU-funded research projects in the fields of AD, BC and PC, with the aim to understand how EU-funded research has contributed to scientific innovation and societal impact, and how the selection of the experimental models may have underpinned the advances made. Further feedback was also gathered through in-depth interviews with some selected survey participants representative of the diverse pre-clinical models used in the EU-funded projects. A comprehensive analysis of survey replies, complemented with the information derived from the interviews, has recently been published in a Synopsis report. Here we discuss the main findings of this analysis and propose a set of priority actions that could be considered to help improving the translation of scientific innovation of biomedical research into societal impact.

Prediction of in vivo prenatal chlorpyrifos exposure leading to developmental neurotoxicity in humans based on in vitro toxicity data by quantitative in vitro-in vivo extrapolation.

Introduction: Epidemiological studies in children suggested that in utero exposure to chlorpyrifos (CPF), an organophosphate insecticide, may cause developmental neurotoxicity (DNT). We applied quantitative in vitro-in vivo extrapolation (QIVIVE) based on in vitro concentration and non-choline esterase-dependent effects data combined with Benchmark dose (BMD) modelling to predict oral maternal CPF exposure during pregnancy leading to fetal brain effect concentration. By comparing the results with data from epidemiological studies, we evaluated the contribution of the in vitro endpoints to the mode of action (MoA) for CPF-induced DNT. Methods: A maternal-fetal PBK model built in PK-Sim® was used to perform QIVIVE predicting CPF concentrations in a pregnant women population at 15 weeks of gestation from cell lysate concentrations obtained in human induced pluripotent stem cell-derived neural stem cells undergoing differentiation towards neurons and glia exposed to CPF for 14 days. The in vitro concentration and effect data were used to perform BMD modelling. Results: The upper BMD was converted into maternal doses which ranged from 3.21 to 271 mg/kg bw/day. Maternal CPF blood levels from epidemiological studies reporting DNT findings in their children were used to estimate oral CPF exposure during pregnancy using the PBK model. It ranged from 0.11 to 140 µg/kg bw/day. Discussion: The effective daily intake doses predicted from the in vitro model were several orders of magnitude higher than exposures estimated from epidemiological studies to induce developmental non-cholinergic neurotoxic responses, which were captured by the analyzed in vitro test battery. These were also higher than the in vivo LOEC for cholinergic effects. Therefore, the quantitative predictive value of the investigated non-choline esterase-dependent effects, although possibly relevant for other chemicals, may not adequately represent potential key events in the MoA for CPF-associated DNT.

Assessment of neurotransmitter release in human iPSC-derived neuronal/glial cells: a missing in vitro assay for regulatory developmental neurotoxicity testing.

Human induced pluripotent stem cell (hiPSC)-derived neural stem cells (NSCs) and their differentiated neuronal/glial derivatives have been recently considered suitable to assess in vitro developmental neurotoxicity (DNT) triggered by exposure to environmental chemicals. The use of human-relevant test systems combined with in vitro assays specific for different neurodevelopmental events, enables a mechanistic understanding of the possible impact of environmental chemicals on the developing brain, avoiding extrapolation uncertainties associated with in vivo studies. Currently proposed in vitro battery for regulatory DNT testing accounts for several assays suitable to study key neurodevelopmental processes, including NSC proliferation and apoptosis, differentiation into neurons and glia, neuronal migration, synaptogenesis, and neuronal network formation. However, assays suitable to measure interference of compounds with neurotransmitter release or clearance are at present not included, which represents a clear gap of the biological applicability domain of such a testing battery. Here we applied a HPLC-based methodology to measure the release of neurotransmitters in a previously characterized hiPSC-derived NSC model undergoing differentiation towards neurons and glia. Glutamate release was assessed in control cultures and upon depolarization, as well as in cultures repeatedly exposed to some known neurotoxicants (BDE47 and lead) and chemical mixtures. Obtained data indicate that these cells have the ability to release glutamate in a vesicular manner, and that both glutamate clearance and vesicular release concur in the maintenance of extracellular glutamate levels. In conclusion, analysis of neurotransmitter release is a sensitive readout that should be included in the envisioned battery of in vitro assays for DNT testing.

Poor Translatability of Biomedical Research Using Animals - A Narrative Review.

The failure rate for the translation of drugs from animal testing to human treatments remains at over 92%, where it has been for the past few decades. The majority of these failures are due to unexpected toxicity - that is, safety issues revealed in human trials that were not apparent in animal tests - or lack of efficacy. However, the use of more innovative tools, such as organs-on-chips, in the preclinical pipeline for drug testing, has revealed that these tools are more able to predict unexpected safety events prior to clinical trials and so can be used for this, as well as for efficacy testing. Here, we review several disease areas, and consider how the use of animal models has failed to offer effective new treatments. We also make some suggestions as to how the more human-relevant new approach methodologies might be applied to address this.

The Adverse Outcome Pathway Framework Applied to Neurological Symptoms of COVID-19.

Several reports have shown that the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has the potential to also be neurotropic. However, the mechanisms by which SARS-CoV-2 induces neurologic injury, including neurological and/or psychological symptoms, remain unclear. In this review, the available knowledge on the neurobiological mechanisms underlying COVID-19 was organized using the AOP framework. Four AOPs leading to neurological adverse outcomes (AO), anosmia, encephalitis, stroke, and seizure, were developed. Biological key events (KEs) identified to induce these AOs included binding to ACE2, blood-brain barrier (BBB) disruption, hypoxia, neuroinflammation, and oxidative stress. The modularity of AOPs allows the construction of AOP networks to visualize core pathways and recognize neuroinflammation and BBB disruption as shared mechanisms. Furthermore, the impact on the neurological AOPs of COVID-19 by modulating and multiscale factors such as age, psychological stress, nutrition, poverty, and food insecurity was discussed. Organizing the existing knowledge along an AOP framework can represent a valuable tool to understand disease mechanisms and identify data gaps and potentially contribute to treatment, and prevention. This AOP-aligned approach also facilitates synergy between experts from different backgrounds, while the fast-evolving and disruptive nature of COVID-19 emphasizes the need for interdisciplinarity and cross-community research.

A worldwide survey on the use of animal-derived materials and reagents in scientific experimentation.

The use of cell and tissue-based methods in basic, applied and regulatory science has been increasing exponentially. Animal-derived components, including serum, coating materials, growth factors and antibodies are routinely used in cell/tissue cultures and in general laboratory practices. In addition to ethical issues, the use and production of animal-derived materials and reagents raises many scientific concerns, generally associated with presence of undefined components and batch-to-batch variability, which may compromise experimental reproducibility. On the other hand, non-animal materials and reagents, such as human cells, alternatives to animal sera or non-animal recombinant antibodies, are becoming increasingly available, and their use is encouraged by the EU Directive 2010/63 and the Guidance Document on Good In vitro Method Practices (GIVIMP), published by the Organization for Economic Cooperation and Development (OECD). In an effort to map the current state of use of animal-derived reagents across different sectors and to identify the obstacles possibly hampering the implementation of non-animal derived alternatives, a global online survey addressed to scientists working on in vivo, in vitro, in silico methods, in academia as well as pharmaceutical or cosmetic companies, was conducted with the goal to understand: 1) the most commonly used animal-derived materials and reagents, 2) the main issues associated with the production and use of animal-derived materials and reagents, 3) the current level of knowledge on available non-animal alternative materials and reagents, and 4) what educational and information sources could be most useful or impactful to disseminate knowledge on non-animal alternatives. This paper provides an overview of the survey replies and discusses possible proposals to increase awareness, acceptance and use of non-animal ingredients.

In Vitro-In Vivo Extrapolation by Physiologically Based Kinetic Modeling: Experience With Three Case Studies and Lessons Learned.

Physiologically based kinetic (PBK) modeling has been increasingly used since the beginning of the 21st century to support dose selection to be used in preclinical and clinical safety studies in the pharmaceutical sector. For chemical safety assessment, the use of PBK has also found interest, however, to a smaller extent, although an internationally agreed document was published already in 2010 (IPCS/WHO), but at that time, PBK modeling was based mostly on in vivo data as the example in the IPCS/WHO document indicates. Recently, the OECD has published a guidance document which set standards on how to characterize, validate, and report PBK models for regulatory purposes. In the past few years, we gained experience on using in vitro data for performing quantitative in vitro-in vivo extrapolation (QIVIVE), in which biokinetic data play a crucial role to obtain a realistic estimation of human exposure. In addition, pharmaco-/toxicodynamic aspects have been introduced into the approach. Here, three examples with different drugs/chemicals are described, in which different approaches have been applied. The lessons we learned from the exercise are as follows: 1) in vitro conditions should be considered and compared to the in vivo situation, particularly for protein binding; 2) in vitro inhibition of metabolizing enzymes by the formed metabolites should be taken into consideration; and 3) it is important to extrapolate from the in vitro measured intracellular concentration and not from the nominal concentration to the tissue/organ concentration to come up with an appropriate QIVIVE for the relevant adverse effects.

Developmental neurotoxicity of acrylamide and its metabolite glycidamide in a human mixed culture of neurons and astrocytes undergoing differentiation in concentrations relevant for human exposure.

Neural stem cells (NSCs) derived from human induced pluripotent stem cells were used to investigate effects of exposure to the food contaminant acrylamide (AA) and its main metabolite glycidamide (GA) on key neurodevelopmental processes. Diet is an important source of human AA exposure for pregnant women, and AA is known to pass the placenta and the newborn may also be exposed through breast feeding after birth. The NSCs were exposed to AA and GA (1 ×10-8 - 3 ×10-3 M) under 7 days of proliferation and up to 28 days of differentiation towards a mixed culture of neurons and astrocytes. Effects on cell viability was measured using Alamar Blue™ cell viability assay, alterations in gene expression were assessed using real time PCR and RNA sequencing, and protein levels were quantified using immunocytochemistry and high content imaging. Effects of AA and GA on neurodevelopmental processes were evaluated using endpoints linked to common key events identified in the existing developmental neurotoxicity adverse outcome pathways (AOPs). Our results suggest that AA and GA at low concentrations (1 ×10-7 - 1 ×10-8 M) increased cell viability and markers of proliferation both in proliferating NSCs (7 days) and in maturing neurons after 14-28 days of differentiation. IC50 for cell death of AA and GA was 5.2 × 10-3 M and 5.8 × 10-4 M, respectively, showing about ten times higher potency for GA. Increased expression of brain derived neurotrophic factor (BDNF) concomitant with decreased synaptogenesis were observed for GA exposure (10-7 M) only at later differentiation stages, and an increased number of astrocytes (up to 3-fold) at 14 and 21 days of differentiation. Also, AA exposure gave tendency towards decreased differentiation (increased percent Nestin positive cells). After 28 days, neurite branch points and number of neurites per neuron measured by microtubule-associated protein 2 (Map2) staining decreased, while the same neurite features measured by ßIII-Tubulin increased, indicating perturbation of neuronal differentiation and maturation.

Quality criteria for in vitro human pluripotent stem cell-derived models of tissue-based cells.

The advent of the technology to isolate or generate human pluripotent stem cells provided the potential to develop a wide range of human models that could enhance understanding of mechanisms underlying human development and disease. These systems are now beginning to mature and provide the basis for the development of in vitro assays suitable to understand the biological processes involved in the multi-organ systems of the human body, and will improve strategies for diagnosis, prevention, therapies and precision medicine. Induced pluripotent stem cell lines are prone to phenotypic and genotypic changes and donor/clone dependent variability, which means that it is important to identify the most appropriate characterization markers and quality control measures when sourcing new cell lines and assessing differentiated cell and tissue culture preparations for experimental work. This paper considers those core quality control measures for human pluripotent stem cell lines and evaluates the state of play in the development of key functional markers for their differentiated cell derivatives to promote assurance of reproducibility of scientific data derived from pluripotent stem cell-based systems.

Effects of spike protein and toxin-like peptides found in COVID-19 patients on human 3D neuronal/glial model undergoing differentiation: Possible implications for SARS-CoV-2 impact on brain development.

The possible neurodevelopmental consequences of SARS-CoV-2 infection are presently unknown. In utero exposure to SARS-CoV-2 has been hypothesized to affect the developing brain, possibly disrupting neurodevelopment of children. Spike protein interactors, such as ACE2, have been found expressed in the fetal brain, and could play a role in potential SARS-CoV-2 fetal brain pathogenesis. Apart from the possible direct involvement of SARS-CoV-2 or its specific viral components in the occurrence of neurological and neurodevelopmental manifestations, we recently reported the presence of toxin-like peptides in plasma, urine and fecal samples specifically from COVID-19 patients. In this study, we investigated the possible neurotoxic effects elicited upon 72-hour exposure to human relevant levels of recombinant spike protein, toxin-like peptides found in COVID-19 patients, as well as a combination of both in 3D human iPSC-derived neural stem cells differentiated for either 2 weeks (short-term) or 8 weeks (long-term, 2 weeks in suspension + 6 weeks on MEA) towards neurons/glia. Whole transcriptome and qPCR analysis revealed that spike protein and toxin-like peptides at non-cytotoxic concentrations differentially perturb the expression of SPHK1, ELN, GASK1B, HEY1, UTS2, ACE2 and some neuronal-, glia- and NSC-related genes critical during brain development. Additionally, exposure to spike protein caused a decrease of spontaneous electrical activity after two days in long-term differentiated cultures. The perturbations of these neurodevelopmental endpoints are discussed in the context of recent knowledge about the key events described in Adverse Outcome Pathways relevant to COVID-19, gathered in the context of the CIAO project (https://www.ciao-covid.net/).

Upscaling biological complexity to boost neuronal and oligodendroglia maturation and improve in vitro developmental neurotoxicity (DNT) evaluation.

Human induced pluripotent stem cell (iPSC)-derived neuronal and glial cell models are suitable to assess the effects of environmental chemicals on the developing brain. Such test systems can recapitulate several key neurodevelopmental features, such as neural stem cell formation and differentiation towards different neuronal subtypes and astrocytes, neurite outgrowth, synapse formation and neuronal network formation and function, which are crucial for brain development. While monolayer, two-dimensional (2D) cultures of human iPSC-neuronal or glial derivatives are generally suited for high-throughput testing, they also show some limitations. In particular, differentiation towards myelinating oligodendrocytes can only be achieved after extended periods in differentiation. In recent years, the implementation of three-dimensional (3D) neuronal and glial models obtained from human iPSCs has been shown to compensate for such limitations, enabling robust differentiation towards both neuronal and glial cell populations, myelination and formation of more mature neuronal network activity. Here we compared the differentiation capacity of human iPSC-derived neural stem cells cultured either as 2D monolayer or as 3D neurospheres, and assessed chlorpyrifos (CPF) effects. Data indicate that 3D neurospheres differentiate towards neurons and oligodendroglia more rapidly than 2D cultures; however, the 2D model is more suitable to assess neuronal functionality by analysis of spontaneous electrical activity using multielectrode array. Moreover, 2D and 3D test systems are diversely susceptible to CPF treatment. In conclusion, the selection of the most suitable in vitro test system (either 2D or 3D) should take into account the context of use and intended research goals ('fit for purpose' principle).

Sporadic Alzheimer's Disease- and Neurotoxicity-Related microRNAs Affecting Key Events of Tau-Driven Adverse Outcome Pathway Toward Memory Loss.

BACKGROUND: A complex network of aging-related homeostatic pathways that are sensitive to further deterioration in the presence of genetic, systemic, and environmental risk factors, and lifestyle, is implicated in the pathogenesis of progressive neurodegenerative diseases, such as sporadic (late-onset) Alzheimer's disease (sAD). OBJECTIVE: Since sAD pathology and neurotoxicity share microRNAs (miRs) regulating common as well as overlapping pathological processes, environmental neurotoxic compounds are hypothesized to exert a risk for sAD initiation and progression. METHODS: Literature search for miRs associated with human sAD and environmental neurotoxic compounds was conducted. Functional miR analysis using PathDip was performed to create miR-target interaction networks. RESULTS: The identified miRs were successfully linked to the hypothetical starting point and key events of the earlier proposed tau-driven adverse outcome pathway toward memory loss. Functional miR analysis confirmed most of the findings retrieved from literature and revealed some interesting findings. The analysis identified 40 miRs involved in both sAD and neurotoxicity that dysregulated processes governing the plausible adverse outcome pathway for memory loss. CONCLUSION: Creating miR-target interaction networks related to pathological processes involved in sAD initiation and progression, and environmental chemical-induced neurotoxicity, respectively, provided overlapping miR-target interaction networks. This overlap offered an opportunity to create an alternative picture of the mechanisms underlying sAD initiation and early progression. Looking at initiation and progression of sAD from this new angle may open for new biomarkers and novel drug targets for sAD before the appearance of the first clinical symptoms.

COVID-19 through Adverse Outcome Pathways: Building networks to better understand the disease - 3rd CIAO AOP Design Workshop.

On April 28-29, 2021, 50 scientists from different fields of expertise met for the 3rd online CIAO workshop. The CIAO project "Modelling the Pathogenesis of COVID-19 using the Adverse Outcome Pathway (AOP) framework" aims at building a holistic assembly of the available scientific knowledge on COVID-19 using the AOP framework. An individual AOP depicts the disease progression from the initial contact with the SARS-CoV-2 virus through biological key events (KE) toward an adverse outcome such as respiratory distress, anosmia or multiorgan failure. Assembling the individual AOPs into a network highlights shared KEs as central biological nodes involved in multiple outcomes observed in COVID-19 patients. During the workshop, the KEs and AOPs established so far by the CIAO members were presented and posi­tioned on a timeline of the disease course. Modulating factors influencing the progression and severity of the disease were also addressed as well as factors beyond purely biological phenomena. CIAO relies on an interdisciplinary crowd­sourcing effort, therefore, approaches to expand the CIAO network by widening the crowd and reaching stakeholders were also discussed. To conclude the workshop, it was decided that the AOPs/KEs will be further consolidated, inte­grating virus variants and long COVID when relevant, while an outreach campaign will be launched to broaden the CIAO scientific crowd.

Guidance document on Good Cell and Tissue Culture Practice 2.0 (GCCP 2.0).

Good Cell and Tissue Culture Practice (GCCP) 2.0 is an updated guidance document from GCCP 1.0 (published by ECVAM in 2005), which was developed for practical use in the laboratory to assure the reproducibility of in vitro (cell-based) work. The update in the guidance was essential as cell models have advanced dramatically to more complex culture systems and need more comprehensive quality management to ensure reproducibility and high-quality scientific data. This document describes six main principles to consider when performing cell culture including characterization and maintenance of essential characteristics, quality management, documentation and reporting, safety, education and training, and ethics. The document does not intend to impose detailed procedures but to describe potential quality issues. It is foreseen that the document will require further updates as the science and technologies evolve over time.

Combining in vitro assays and mathematical modelling to study developmental neurotoxicity induced by chemical mixtures.

Prenatal and postnatal co-exposure to multiple chemicals at the same time may have deleterious effects on the developing nervous system. We previously showed that chemicals acting through similar mode of action (MoA) and grouped based on perturbation of brain derived neurotrophic factor (BDNF), induced greater neurotoxic effects on human induced pluripotent stem cell (hiPSC)-derived neurons and astrocytes compared to chemicals with dissimilar MoA. Here we assessed the effects of repeated dose (14 days) treatments with mixtures containing the six chemicals tested in our previous study (Bisphenol A, Chlorpyrifos, Lead(II) chloride, Methylmercury chloride, PCB138 and Valproic acid) along with 2,2'4,4'-tetrabromodiphenyl ether (BDE47), Ethanol, Vinclozolin and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)), on hiPSC-derived neural stem cells undergoing differentiation toward mixed neurons/astrocytes up to 21 days. Similar MoA chemicals in mixtures caused an increase of BDNF levels and neurite outgrowth, and a decrease of synapse formation, which led to inhibition of electrical activity. Perturbations of these endpoints are described as common key events in adverse outcome pathways (AOPs) specific for DNT. When compared with mixtures tested in our previous study, adding similarly acting chemicals (BDE47 and EtOH) to the mixture resulted in a stronger downregulation of synapses. A synergistic effect on some synaptogenesis-related features (PSD95 in particular) was hypothesized upon treatment with tested mixtures, as indicated by mathematical modelling. Our findings confirm that the use of human iPSC-derived mixed neuronal/glial models applied to a battery of in vitro assays anchored to key events in DNT AOP networks, combined with mathematical modelling, is a suitable testing strategy to assess in vitro DNT induced by chemical mixtures.