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.

Progress towards an OECD reporting framework for transcriptomics and metabolomics in regulatory toxicology.

Omics methodologies are widely used in toxicological research to understand modes and mechanisms of toxicity. Increasingly, these methodologies are being applied to questions of regulatory interest such as molecular point-of-departure derivation and chemical grouping/read-across. Despite its value, widespread regulatory acceptance of omics data has not yet occurred. Barriers to the routine application of omics data in regulatory decision making have been: 1) lack of transparency for data processing methods used to convert raw data into an interpretable list of observations; and 2) lack of standardization in reporting to ensure that omics data, associated metadata and the methodologies used to generate results are available for review by stakeholders, including regulators. Thus, in 2017, the Organisation for Economic Co-operation and Development (OECD) Extended Advisory Group on Molecular Screening and Toxicogenomics (EAGMST) launched a project to develop guidance for the reporting of omics data aimed at fostering further regulatory use. Here, we report on the ongoing development of the first formal reporting framework describing the processing and analysis of both transcriptomic and metabolomic data for regulatory toxicology. We introduce the modular structure, content, harmonization and strategy for trialling this reporting framework prior to its publication by the OECD.

Artificial Intelligence for chemical risk assessment.

As the basis for managing the risks of chemical exposure, the Chemical Risk Assessment (CRA) process can impact a substantial part of the economy, the health of hundreds of millions of people, and the condition of the environment. However, the number of properly assessed chemicals falls short of societal needs due to a lack of experts for evaluation, interference of third party interests, and the sheer volume of potentially relevant information on the chemicals from disparate sources. In order to explore ways in which computational methods may help overcome this discrepancy between the number of chemical risk assessments required on the one hand and the number and adequateness of assessments actually being conducted on the other, the European Commission's Joint Research Centre organised a workshop on Artificial Intelligence for Chemical Risk Assessment (AI4CRA). The workshop identified a number of areas where Artificial Intelligence could potentially increase the number and quality of regulatory risk management decisions based on CRA, involving process simulation, supporting evaluation, identifying problems, facilitating collaboration, finding experts, evidence gathering, systematic review, knowledge discovery, and building cognitive models. Although these are interconnected, they are organised and discussed under two main themes: scientific-technical process and social aspects and the decision making process.

Carcinogenicity assessment: Addressing the challenges of cancer and chemicals in the environment.

Cancer is a key public health concern, being the second leading cause of worldwide morbidity and mortality after cardiovascular diseases. At the global level, cancer prevalence, incidence and mortality rates are increasing. These trends are not fully explained by a growing and ageing population: with marked regional and socioeconomic disparities, lifestyle factors, the resources dedicated to preventive medicine, and the occupational and environmental control of hazardous chemicals all playing a role. While it is difficult to establish the contribution of chemical exposure to the societal burden of cancer, a number of measures can be taken to better assess the carcinogenic properties of chemicals and manage their risks. This paper discusses how these measures can be informed not only by the traditional data streams of regulatory toxicology, but also by using new toxicological assessment methods, along with indicators of public health status based on biomonitoring. These diverse evidence streams have the potential to form the basis of an integrated and more effective approach to cancer prevention.

Advancing human health risk assessment.

The current/traditional human health risk assessment paradigm is challenged by recent scientific and technical advances, and ethical demands. The current approach is considered too resource intensive, is not always reliable, can raise issues of reproducibility, is mostly animal based and does not necessarily provide an understanding of the underlying mechanisms of toxicity. From an ethical and scientific viewpoint, a paradigm shift is required to deliver testing strategies that enable reliable, animal-free hazard and risk assessments, which are based on a mechanistic understanding of chemical toxicity and make use of exposure science and epidemiological data. This shift will require a new philosophy, new data, multidisciplinary expertise and more flexible regulations. Re-engineering of available data is also deemed necessary as data should be accessible, readable, interpretable and usable. Dedicated training to build the capacity in terms of expertise is necessary, together with practical resources allocated to education. The dialogue between risk assessors, risk managers, academia and stakeholders should be promoted further to understand scientific and societal needs. Genuine interest in taking risk assessment forward should drive the change and should be supported by flexible funding. This publication builds upon presentations made and discussions held during the break-out session 'Advancing risk assessment science - Human health' at EFSA's third Scientific Conference 'Science, Food and Society' (Parma, Italy, 18-21 September 2018).

Harvesting the promise of AOPs: An assessment and recommendations.

The Adverse Outcome Pathway (AOP) concept is a knowledge assembly and communication tool to facilitate the transparent translation of mechanistic information into outcomes meaningful to the regulatory assessment of chemicals. The AOP framework and associated knowledgebases (KBs) have received significant attention and use in the regulatory toxicology community. However, it is increasingly apparent that the potential stakeholder community for the AOP concept and AOP KBs is broader than scientists and regulators directly involved in chemical safety assessment. In this paper we identify and describe those stakeholders who currently-or in the future-could benefit from the application of the AOP framework and knowledge to specific problems. We also summarize the challenges faced in implementing pathway-based approaches such as the AOP framework in biological sciences, and provide a series of recommendations to meet critical needs to ensure further progression of the framework as a useful, sustainable and dependable tool supporting assessments of both human health and the environment. Although the AOP concept has the potential to significantly impact the organization and interpretation of biological information in a variety of disciplines/applications, this promise can only be fully realized through the active engagement of, and input from multiple stakeholders, requiring multi-pronged substantive long-term planning and strategies.

Introduction.

Alternative approaches to animal testing are gaining momentum with an increasing number of test methods obtaining international acceptance, thanks in large part to the validation efforts conducted on these assays. The principles and process of validation were first established in the 1990s in Europe and USA, and further gained international recognition ensuring the broader acceptance of alternative test methods at a regulatory level. If these principles were successful in pioneering the regulatory acceptance of alternative methods for less complex endpoints, an evolution of concepts is needed to embrace emerging technologies and the increased complexity of endpoints. Innovative concepts and approaches of scientific validation can help to ensure the continued regulatory and international acceptance of novel alternative methods and technologies for toxicity testing such as human-based in vitro models derived from induced pluripotent stem cells and significant advances in bioengineering. This chapter provides a historical overview of the establishment and evolution of the principles of the scientific validation of alternative methods for toxicity testing as well as the challenges and opportunities for adapting those principles to keep pace with scientific progress whilst ensuring human safety and best serve the needs of society.

Evolving the Principles and Practice of Validation for New Alternative Approaches to Toxicity Testing.

Validation is essential for the translation of newly developed alternative approaches to animal testing into tools and solutions suitable for regulatory applications. Formal approaches to validation have emerged over the past 20 years or so and although they have helped greatly to progress the field, it is essential that the principles and practice underpinning validation continue to evolve to keep pace with scientific progress. The modular approach to validation should be exploited to encourage more innovation and flexibility in study design and to increase efficiency in filling data gaps. With the focus now on integrated approaches to testing and assessment that are based on toxicological knowledge captured as adverse outcome pathways, and which incorporate the latest in vitro and computational methods, validation needs to adapt to ensure it adds value rather than hinders progress. Validation needs to be pursued both at the method level, to characterise the performance of in vitro methods in relation their ability to detect any association of a chemical with a particular pathway or key toxicological event, and at the methodological level, to assess how integrated approaches can predict toxicological endpoints relevant for regulatory decision making. To facilitate this, more emphasis needs to be given to the development of performance standards that can be applied to classes of methods and integrated approaches that provide similar information. Moreover, the challenge of selecting the right reference chemicals to support validation needs to be addressed more systematically, consistently and in a manner that better reflects the state of the science. Above all however, validation requires true partnership between the development and user communities of alternative methods and the appropriate investment of resources.

The Next Generation of Risk Assessment Multi-Year Study-Highlights of Findings, Applications to Risk Assessment, and Future Directions.

BACKGROUND: The Next Generation (NexGen) of Risk Assessment effort is a multi-year collaboration among several organizations evaluating new, potentially more efficient molecular, computational, and systems biology approaches to risk assessment. This article summarizes our findings, suggests applications to risk assessment, and identifies strategic research directions. OBJECTIVE: Our specific objectives were to test whether advanced biological data and methods could better inform our understanding of public health risks posed by environmental exposures. METHODS: New data and methods were applied and evaluated for use in hazard identification and dose-response assessment. Biomarkers of exposure and effect, and risk characterization were also examined. Consideration was given to various decision contexts with increasing regulatory and public health impacts. Data types included transcriptomics, genomics, and proteomics. Methods included molecular epidemiology and clinical studies, bioinformatic knowledge mining, pathway and network analyses, short-duration in vivo and in vitro bioassays, and quantitative structure activity relationship modeling. DISCUSSION: NexGen has advanced our ability to apply new science by more rapidly identifying chemicals and exposures of potential concern, helping characterize mechanisms of action that influence conclusions about causality, exposure-response relationships, susceptibility and cumulative risk, and by elucidating new biomarkers of exposure and effects. Additionally, NexGen has fostered extensive discussion among risk scientists and managers and improved confidence in interpreting and applying new data streams. CONCLUSIONS: While considerable uncertainties remain, thoughtful application of new knowledge to risk assessment appears reasonable for augmenting major scope assessments, forming the basis for or augmenting limited scope assessments, and for prioritization and screening of very data limited chemicals. Citation: Cote I, Andersen ME, Ankley GT, Barone S, Birnbaum LS, Boekelheide K, Bois FY, Burgoon LD, Chiu WA, Crawford-Brown D, Crofton KM, DeVito M, Devlin RB, Edwards SW, Guyton KZ, Hattis D, Judson RS, Knight D, Krewski D, Lambert J, Maull EA, Mendrick D, Paoli GM, Patel CJ, Perkins EJ, Poje G, Portier CJ, Rusyn I, Schulte PA, Simeonov A, Smith MT, Thayer KA, Thomas RS, Thomas R, Tice RR, Vandenberg JJ, Villeneuve DL, Wesselkamper S, Whelan M, Whittaker C, White R, Xia M, Yauk C, Zeise L, Zhao J, DeWoskin RS. 2016. The Next Generation of Risk Assessment multiyear study-highlights of findings, applications to risk assessment, and future directions. Environ Health Perspect 124:1671-1682; http://dx.doi.org/10.1289/EHP233.

Adverse Outcome Pathways can drive non-animal approaches for safety assessment.

Adverse Outcome Pathways (AOPs) provide an opportunity to develop new and more accurate safety assessment processes for drugs and other chemicals, and may ultimately play an important role in regulatory decision making. Not only can the development and application of AOPs pave the way for the development of improved evidence-based approaches for hazard and risk assessment, there is also the promise of a significant impact on animal welfare, with a reduced reliance on animal-based methods. The establishment of a useable and coherent knowledge framework under which AOPs will be developed and applied has been a first critical step towards realizing this opportunity. This article explores how the development of AOPs under this framework, and their application in practice, could benefit the science and practice of safety assessment, while in parallel stimulating a move away from traditional methods towards an increased acceptance of non-animal approaches. We discuss here the key areas where current, and future initiatives should be focused to enable the translation of AOPs into routine chemical safety assessment, and lasting 3Rs benefits.

The SEURAT-1 approach towards animal free human safety assessment.

SEURAT-1 is a European public-private research consortium that is working towards animal-free testing of chemical compounds and the highest level of consumer protection. A research strategy was formulated based on the guiding principle to adopt a toxicological mode-of-action framework to describe how any substance may adversely affect human health.The proof of the initiative will be in demonstrating the applicability of the concepts on which SEURAT-1 is built on three levels:(i) Theoretical prototypes for adverse outcome pathways are formulated based on knowledge already available in the scientific literature on investigating the toxicological mode-of-actions leading to adverse outcomes (addressing mainly liver toxicity);(ii)adverse outcome pathway descriptions are used as a guide for the formulation of case studies to further elucidate the theoretical model and to develop integrated testing strategies for the prediction of certain toxicological effects (i.e., those related to the adverse outcome pathway descriptions);(iii) further case studies target the application of knowledge gained within SEURAT-1 in the context of safety assessment. The ultimate goal would be to perform ab initio predictions based on a complete understanding of toxicological mechanisms. In the near-term, it is more realistic that data from innovative testing methods will support read-across arguments. Both scenarios are addressed with case studies for improved safety assessment. A conceptual framework for a rational integrated assessment strategy emerged from designing the case studies and is discussed in the context of international developments focusing on alternative approaches for evaluating chemicals using the new 21st century tools for toxicity testing.

Systems toxicology: from basic research to risk assessment.

Systems Toxicology is the integration of classical toxicology with quantitative analysis of large networks of molecular and functional changes occurring across multiple levels of biological organization. Society demands increasingly close scrutiny of the potential health risks associated with exposure to chemicals present in our everyday life, leading to an increasing need for more predictive and accurate risk-assessment approaches. Developing such approaches requires a detailed mechanistic understanding of the ways in which xenobiotic substances perturb biological systems and lead to adverse outcomes. Thus, Systems Toxicology approaches offer modern strategies for gaining such mechanistic knowledge by combining advanced analytical and computational tools. Furthermore, Systems Toxicology is a means for the identification and application of biomarkers for improved safety assessments. In Systems Toxicology, quantitative systems-wide molecular changes in the context of an exposure are measured, and a causal chain of molecular events linking exposures with adverse outcomes (i.e., functional and apical end points) is deciphered. Mathematical models are then built to describe these processes in a quantitative manner. The integrated data analysis leads to the identification of how biological networks are perturbed by the exposure and enables the development of predictive mathematical models of toxicological processes. This perspective integrates current knowledge regarding bioanalytical approaches, computational analysis, and the potential for improved risk assessment.

Incorporating new technologies into toxicity testing and risk assessment: moving from 21st century vision to a data-driven framework.

Based on existing data and previous work, a series of studies is proposed as a basis toward a pragmatic early step in transforming toxicity testing. These studies were assembled into a data-driven framework that invokes successive tiers of testing with margin of exposure (MOE) as the primary metric. The first tier of the framework integrates data from high-throughput in vitro assays, in vitro-to-in vivo extrapolation (IVIVE) pharmacokinetic modeling, and exposure modeling. The in vitro assays are used to separate chemicals based on their relative selectivity in interacting with biological targets and identify the concentration at which these interactions occur. The IVIVE modeling converts in vitro concentrations into external dose for calculation of the point of departure (POD) and comparisons to human exposure estimates to yield a MOE. The second tier involves short-term in vivo studies, expanded pharmacokinetic evaluations, and refined human exposure estimates. The results from the second tier studies provide more accurate estimates of the POD and the MOE. The third tier contains the traditional animal studies currently used to assess chemical safety. In each tier, the POD for selective chemicals is based primarily on endpoints associated with a proposed mode of action, whereas the POD for nonselective chemicals is based on potential biological perturbation. Based on the MOE, a significant percentage of chemicals evaluated in the first 2 tiers could be eliminated from further testing. The framework provides a risk-based and animal-sparing approach to evaluate chemical safety, drawing broadly from previous experience but incorporating technological advances to increase efficiency.

Adverse outcome pathway-based screening strategies for an animal-free safety assessment of chemicals.

Currently, the assessment of risk to human health from exposure to manufactured chemicals is mainly based on experiments performed on living animals (in vivo). Substantial efforts are being undertaken to develop alternative solutions to in vivo toxicity testing. This new paradigm, based on the Mode-of-Action (MoA) framework, postulates that any adverse human health effect caused by exposure to an exogenous substance can be described by a series of causally-linked biochemical or biological key events with measurable parameters. The elaboration of mechanistic knowledge through literature research is necessary for a MoA-driven design of integrated testing strategies using in vitro methods for in vivo predictions. The objective of our ongoing research is to demonstrate the feasibility of an integrated approach to predict human toxicity following the Adverse Outcome Pathway (AOP) framework. In our previous work on MoA with the HepaRG cell model, we developed a strategy to identify chemicals that were hepatotoxic. This pioneered an innovative way of using data from in vitro experiments to group chemicals based on their MoA, which is likely to be an important step in a toxicity testing strategy.

Assessment of the predictive capacity of the 3T3 Neutral Red Uptake cytotoxicity test method to identify substances not classified for acute oral toxicity (LD50>2000 mg/kg): results of an ECVAM validation study.

Assessing chemicals for acute oral toxicity is a standard information requirement of regulatory testing. However, animal testing is now prohibited in the cosmetics sector in Europe, and strongly discouraged for industrial chemicals. Building on the results of a previous international validation study, a follow up study was organised to assess if the 3T3 Neutral Red Uptake cytotoxicity assay could identify substances not requiring classification as acute oral toxicants under the EU regulations. Fifty-six coded industrial chemicals were tested in three laboratories, each using one of the following protocols: the previously validated protocol, an abbreviated version of the protocol and the protocol adapted for an automation platform. Predictions were very similar among the three laboratories. The assay exhibited high sensitivity (92-96%) but relatively low specificity (40-44%). Three chemicals were under predicted. Assuming that most industrial chemicals are not likely to be acutely toxic, this test method could prove a valuable component of an integrated testing strategy, a read-across argument, or weight-of-evidence approach to identify non toxic chemicals (LD50>2000 mg/kg). However, it is likely to under predict chemicals acting via specific mechanisms of action not captured by the 3T3 test system, or which first require biotransformation in vivo.

Assessment of an automated in vitro basal cytotoxicity test system based on metabolically-competent cells.

When in vitro test systems are evaluated for assessment of the toxicity of chemical compounds, particular efforts are made to mimic the in vivo reality as close as possible. Cellular models with appropriate metabolic competence, i.e. with the potency to biotransform chemical compounds, are considered crucial since some metabolites have a different toxicity than their parent compounds. In this study a cell based in vitro test system is proposed to investigate the basal cytotoxicity of several reference chemicals. Both metabolic competent HepaRG cells and cells with no or low hepatic enzyme activity (undifferentiated HepaRG and proliferating HepG2) were used. The classic Neutral Red Uptake (NRU) assay proved to be robust and reliable to be applied as viability assay. The test was performed on a robotic platform, which enabled fully automated and simultaneous screening of the compounds. The outcome of these tests grouped the tested compounds in three categories following their detoxification effect (benzo(a)pyrene, valproic acid), their bio-activation effect (aflatoxin B1) and their specific effect on inhibition of cell proliferation (cycloheximide, sodium lauryl sulphate, atropine sulphate monohydrate, acetylsalicylic acid).

Feasibility Assessment of Micro-Electrode Chip Assay as a Method of Detecting Neurotoxicity in vitro.

Detection and characterization of chemically induced toxic effects in the nervous system represent a challenge for the hazard assessment of chemicals. In vivo, neurotoxicological assessments exploit the fact that the activity of neurons in the central and peripheral nervous system has functional consequences. And so far, no in vitro method for evaluating the neurotoxic hazard has yet been validated and accepted for regulatory purpose. The micro-electrode array (MEA) assay consists of a culture chamber into which an integrated array of micro-electrodes is capable of measuring extracellular electrophysiology (spikes and bursts) from electro-active tissues. A wide variety of electrically excitable biological tissues may be placed onto the chips including primary cultures of nervous system tissue. Recordings from this type of in vitro cultured system are non-invasive, give label free evaluations and provide a higher throughput than conventional electrophysiological techniques. In this paper, 20 substances were tested in a blinded study for their toxicity and dose-response curves were obtained from fetal rat cortical neuronal networks coupled to MEAs. The experimental procedure consisted of evaluating the firing activity (spiking rate) and modification/reduction in response to chemical administration. Native/reference activity, 30 min of activity recording per dilution, plus the recovery points (after 24 h) were recorded. The preliminary data, using a set of chemicals with different mode-of-actions (13 known to be neurotoxic, 2 non-neuroactive and not toxic, and 5 non-neuroactive but toxic) show good predictivity (sensitivity: 0.77; specificity: 0.86; accuracy: 0.85). Thus, the MEA with a neuronal network has the potency to become an effective tool to evaluate the neurotoxicity of substances in vitro.

Performance assessment of photon migration instruments: the MEDPHOT protocol.

We propose a comprehensive protocol for the performance assessment of photon migration instruments. The protocol has been developed within the European Thematic Network MEDPHOT (optical methods for medical diagnosis and monitoring of diseases) and is based on five criteria: accuracy, linearity, noise, stability, and reproducibility. This protocol was applied to a total of 8 instruments with a set of 32 phantoms, covering a wide range of optical properties.