JRC Summer School on Non-animal Approaches in Science,May 2021

Background

“The JRC Summer School on Non-animal Approaches in Science — a flair of holidays and fun. Learning and socialising, intellectual challenge and playful competition.” That was our tempting vision, back in 2016, for installing such a training course at EURL ECVAM — and we decided to go for it. We already had some experience in this respect, as every year students from the Karolinska Toxicology Master course programme visited the EURL ECVAM laboratories to learn more about non-animal methods and regulatory toxicology, and to have a closer look on the life in an international research centre such as the Joint Research Centre (JRC).

Our aim was to share knowledge and experience on the latest non-animal approaches in science with young scientists and professionals, to give them new insights and to enable networking and exchange among all participants — students as well as invited experts and ourselves at the JRC. We wanted the experience to be as interactive as possible for the students, hopefully eliciting input from every participant.

With the approval of our hierarchy, a rather small group of brave people started to tackle the preparatory work for the inaugural summer school, which turned out to be quite extensive. Besides the elaboration of the programme itself, there was also a broad range of logistical challenges. Increasingly more people became involved, beyond the staff of our unit — for example, technical, catering, cleaning, transport and security services were all required. Many people had to be transported around (local public transport is, to put it mildly, suboptimal), accommodated and fed. In order to reach as many students as possible to inform them about the event, a range of different communication channels were explored. To limit the participation to our targeted audience, i.e. students or early-career scientists (maximum of four years post-masters or PhD) in relevant fields, and to ensure a good level of existing scientific education, we asked for a motivation letter, a CV and a poster abstract to be submitted along with the application. Based on these pre-defined criteria, these documents were used for the selection of participants.

The inaugural JRC Summer School

In May 2017, our first Summer School — ‘Alternative Approaches for Risk Assessment’, became a reality. Specifically, we wanted to provide comprehensive training on state-of-the-art alternative (non-animal) approaches for use in predictive toxicology, and to promote their use within modern chemical risk assessment practice. One hundred and one students and early-career scientists, from 29 countries worldwide, participated in the four-day programme. In the morning sessions, an overview on the whole process of chemical risk assessment was given, while afternoons were dedicated to interactive activities. We had two poster sessions, each starting with ten flash presentations (the selection was made by the scientific committee out of the submitted abstracts). The three Excellent Poster Award winners, however, were nominated by the summer school participants (each registered person had one vote to cast).

World Café-style sessions allowed for discussion in small groups, with the speakers of the morning sessions. Marketplaces offered hands-on demonstrations of various databases and tools for toxicology, as well as information on career opportunities within the EU and beyond. Further activities were visits to the EURL ECVAM laboratory and to the Visitors’ Centre to gain information on the JRC, as well as a JRC Science Quiz. We also prepared an interactive Adverse Outcome Pathway (AOP) game, in which participants had to seek out people possessing the other modules of their assigned AOP and present themselves as one complete AOP on the last day. The splendid weather allowed for breaks outside in the garden, where all could mingle, also joined by JRC staff members who were not directly involved in the Summer School. Other socialising opportunities were an aperitivo and a social dinner. A booklet, including the programme, bio-sketches, contacts, lecture and poster abstracts, together with an USB pen with relevant background material, were distributed to the participants; a certificate of attendance was given to all students that attended the whole course.

This inaugural event was a great success, with participants and organisers happy with having gained new insights, experiences, inspiration and friends. We asked participants for their feedback, which nearly all participants provided. This information was taken very seriously, and provided guidance for the organisation of the following event. We kept what was appreciated and modified the programme according to the feedback, regarding the content and duration. One major complaint concerned the large amount of plastic water bottles used — this prompted us to completely ban plastic for the second Summer School, held two years later.

Building on the lessons learned

The second Summer School, held in 2019, was entitled ‘Non-Animal Approaches in Science — Challenges and Future Directions’. Its aim was to explore the role of the Three Rs in current science and policy, through discussion and debate. Though we already felt more experienced in the organisation, the challenges associated with the preparation and organisation did not appear any fewer than two years before. The application and registration procedures remained unchanged, but we increased the number of participants to 120. This time, students from 34 countries worldwide (including India, Bangladesh, Korea, China, Brazil and Iran) joined us. The programme itself is offered for free, but the travel costs are at the student’s expense, and this can be a limiting factor for participation in some cases. Therefore, a number of supporting organisations generously provided travel grants for 13 students.

Essentially, we kept the programme structure the same — with lectures, interactive sessions, poster sessions and lab visits. In addition, we introduced a debate format, for which students were divided in eight groups (with mixed backgrounds and affiliations). Students could get appropriate arguments by listening to various presentations. The debates took place on the last morning to give sufficient time for getting prepared during the course. The level and content of these debates were amazing. Sli.do was used as interactive tool for polls and questionnaires. A wide social programme — including an aperitivo, dinner, and a fun-quiz evening — was arranged, with many networking opportunities. The feedback received from the first JRC Summer School, to reduce waste such as plastic water bottles and plastic cutlery, led us to rethink the sustainability and recycling strategies when planning the second event. This effort was rewarded with us being awarded 1st Prize in the 1st Corporate Competition on Sustainable Conference and Events (Category 2 External Events in EC-Premises).

More recent events

In June 2020 the first sister Summer School, ¹ on ‘Innovative Approaches in Science’, was held in the United States, and then in 2021, we proposed the third JRC Summer School, ‘The Three R…evolution’. When we started the preparation for this third JRC event, more than a year ago, the pandemic was already starting to change our lives, but we could not imagine that this situation would persist for so long, and that it would have such an impact on our event. So, in good spirits, we prepared for another engaging and interactive training course, to be held at the JRC Ispra site. With growing concerns, we finally had to accept the move to a virtual event space at the end of 2020. Suddenly, we were confronted with a new situation — the whole schedule had to be reconfigured into a suitable virtual format, and we also had to consider the different time zones of participants; finding a way to transform the interactive parts into virtual formats was especially challenging. We ended up with a programme of five half-days (to help adjust to the different time zones) and decided to further increase the number of participants to 144. Going beyond this number would have been at the expense of active collaboration possibilities for all participants. As was the case for the first Summer School, selection of the participants was based on their submitted motivation letter, CV and poster abstract. The poster abstracts for the 2021 event are featured here in the Appendix.

With support from DG SCIC (the Directorate-General for Interpretation) we were able to use an interactive platform; the specific set-up required took a great deal of time and effort. Content-wise, we kept the lectures, World Café-style sessions for discussion in smaller groups, poster sessions with flash presentations, and the opportunity to vote for the best poster by the participants. Also, the debates were maintained, with topics related to the content of the lectures. We added a market fair presenting various organ-on-a-chip models and a career session to present various career options in the field of NAMs. Instead of travel grants, this time the three supporting organisations (PETA, ERA21 and ESTIV) kindly sponsored poster prizes, which were presented in addition to the three existing Excellent Poster Awards, the latter again being nominated by the summer school participants.

Again, we aimed for an environmentally friendly event and had non-tangible items as give-aways, to avoid the purchase and shipment of gadgets and other such merchandise. We invited all participants to plant trees and the JRC Summer School supported the planting of 2,000 trees in Rondônia, Brazil. ² We also offered the students a specific training course in scientific writing, to be held in the week following the Summer School. This professional training focused on the writing of motivation letters and abstracts, taking inspiration from examples provided by the students themselves in their Summer School applications, as well as providing instruction in general communication.

Speakers at the 2021 JRC Summer School

As was the case for the previously-held Summer Schools, the speakers at the 2021 event, came from a variety of disciplines and had a wide range of expertise to share with the participants. The topics covered by each of these speakers in their presentations are outlined below:

Legal obligations and state of play in the European Union

(Susanna Louhimies; European Commission, DG Environment (ENV), Brussels, Belgium)

Since the 1980s, the EU has had legislation in place to protect animals used for scientific purposes. In 2010, the legislation was comprehensively revised making it unique in the world: Directive 2010/63/EU sets the full replacement of all animal use for scientific purposes as its ultimate goal and requires, by law, the use of alternative non-animal methods as soon as these become available. The Directive has three key aims: to harmonise the legislation to obtain a level playing field and promote EU research and competiveness; to set up high animal welfare standards and speed up the uptake of Three Rs (replacement, reduction, refinement); and to improve transparency. Transparency facilitates the progress towards full replacement. It provides important tools to gain a better understanding of where and how animals are used, and the level of distress and suffering scientific procedures have on animals. This information helps determine where efforts are most needed to have the highest impact on reducing animal numbers and the negative impact on their welfare. The EU has taken a quantum leap in pushing the boundaries of transparency by amending the Directive in 2019. The new transparency measures will be of benefit to all those interested in advancing the Three Rs, whether in the EU or elsewhere in the world. Such tools include detailed statistics on animal use, as well as providing the context of these uses through the requirement for non-technical project summaries of authorised projects. These data will be made available through open access EU databases.

Animals used for scientific purposes: The statistics

(Pierre Deceuninck; European Commission, Joint Research Centre (JRC), Ispra, Italy)

In 2020, the first report of statistical information on the use of animals in procedures became available, in accordance with the provisions of Article 57(2) of Directive 2010/63/EU on the protection of animals used for scientific purposes. EURL ECVAM was requested by Directorate-General for Environment to support the preparation of this EC report, based on data provided by Member States in accordance with Article 54(2). For this purpose, EURL ECVAM performed the statistical analysis, providing a comprehensive overview on the use of animals in procedures in the European Union from 2015 to 2017. The first section of this report focuses on the numbers of animals used for the first time, and their origins. These animals can be both conventional animals or those that have been genetically altered (but excludes animals that have been used for the maintenance or creation of new genetically altered animal lines). The second section focuses on the way in which animals are used in scientific procedures, covering both the first and any subsequent reuse, so that a global picture can be drawn of all uses of animals. This section takes into account the nature of the procedures, their legislative context, reuse of animals, their genetic status and the actual severities experienced by the animal having undergone a procedure. The third section focuses on genetically altered animals, providing information on the numbers and types of purpose of genetically altered animals needed to support scientific research in the Union. It reports on the animals used for the creation of new genetically altered animal lines and the maintenance of colonies of existing genetically altered animals.

Predictive toxicology for a more sustainable future

(Andrew Worth; European Commission, Joint Research Centre (JRC), Ispra, Italy)

This presentation discusses the role of predictive toxicology and chemical safety assessment in the context of broad policy challenges faced by the European Union. The state of the European Environment is considered from the perspective of chemical contributions to the burden of disease and ecosystem damage. This sets the scene for highlighting research and innovation opportunities to further develop New Approach Methodologies (NAMs) for assessing the human health and environmental effects of chemicals. Emphasis is placed on the potential contribution of predictive toxicology in supporting one of the six political priorities of the European Commission — ‘The European Green Deal’ and its zero pollution ambition for a toxic-free environment. The Green Deal sets out an ambitious plan to make the EU the world’s first ‘climate-neutral’ continent by 2050. Of particular relevance is the Chemicals Strategy for Sustainability, adopted in October 2020 under the umbrella of the Green Deal. This strategy sets a pathway toward implementing the vision of a toxic-free environment through a series of actions to support innovation for safe and sustainable chemicals, strengthen the protection of human health and the environment, simplify and strengthen the legal framework on chemicals, build a comprehensive knowledge base to support evidence-based policy making, and set the example of sound management of chemicals globally. ³

What can regulatory toxicologists learn from jazz improvisation?

(Annamaria Carusi; Department for Science and Technology Studies, University College London (UCL), London, UK)

The distance between regulatory toxicology and jazz improvisation seems very large indeed. What can the creativity and spontaneity of jazz improvisation have in common with the much more formulaic and standardised nature of regulatory toxicology? This presentation will aim to show that there are useful similarities between them, especially if we want to see change happen in regulatory toxicology, and new methods adopted in it. I claim that regulatory toxicology can learn some tricks from artists such as jazz improvisers — the combination of deep knowledge and understanding of their own instrument, cultivated without ever losing sight of the potentialities of other instruments, and the abilities of other musicians. From this comes the spark of creating something new, that we see in improvisation. Scientific change and innovation happens through collaborations not unlike those that occur in some art forms. But whereas artists spend a huge amount of time training with others, scientists are not trained for collaboration, and they are especially not trained for interdisciplinarity. This talk will explore what such a training for scientists will look like, taking inspiration from the arts. The main topics will be: learning and practicing disciplines, with a view to opening to other disciplines; sharing ways of knowing; and framing common questions and challenges. Various examples from the history of biomedical science and the history of toxicology, and also from the arts, will be used.

Advanced non-animal models in biomedical research: A new collection of JRC models

(Laura Gribaldo; European Commission, Joint Research Centre (JRC), Ispra, Italy)

According to the last report on the use of animals for scientific purposes in the EU, about 70% of animals are used for research and development in the fields of human and veterinary medicine and in biological studies of fundamental nature. Nowadays, a relevant percentage of drug programmes fail to progress, largely due to a lack of efficacy or unexplained toxicity. Although there are several factors underpinning this failure rate, the use of animals to model human biology and disease is coming under increasing scrutiny. For these reasons, EURL ECVAM launched a series of review studies on available and emerging non-animal models in research in seven disease areas: respiratory tract diseases; neurodegenerative disorders; breast cancer; immune-oncology; autoimmunity; cardiovascular diseases; and immunogenicity of advanced medicinal products. These areas were selected because of disease incidence and prevalence, and the amount of animal procedures conducted. The reviews describe both well-established approaches and the ones under development, based on techniques that use cells and tissues (in vitro methods), computer modelling and simulation (in silico) or cells and tissues explanted from an organism (ex vivo methods). Biomedical researchers will be able to use the knowledge base to identify models that might be useful to tackle their specific questions. Educators could use it to provide their students with the latest information on the current state-of-the-art, while funding bodies will be able to consider trends and target promising areas for investment. Furthermore, the knowledge base will be of use to Competent Authorities, to support the process of project evaluation, ensuring that project proposers have properly considered the use of non-animal models in their research proposals.

Biomedical research models in immune oncology

(Lucia Gabriele; Istituto Superiore di Sanità, Rome, Italy)

The expanding field of immuno-oncology relies on the need to depict tumour–immune system interactions driving cancer progression and immunotherapy response. The tumour–immune system interplay is a dynamic and complex phenomenon characterised by patient dependence and variability. The adoption of animal models complementary to advanced 3-D in vitro human models are essential to further our understanding of the fine mechanisms shaping cancer–immune system crosstalk, to finally develop personalised immunotherapies. Tumour-bearing immunocompetent mice allow investigation in a complex biological organism, providing valuable information on mechanisms of action and toxicity of immunotherapies. Nevertheless, these models pose a caveat relating to mouse and human disease differences. In light of this, newer studies have implanted privileged human tumours into immunodeficient mice reconstructed with a ‘pseudo-human’ immune system. However, these models lack the specific organ-dependent tumour microenvironment and thus permit a limited analysis of immune mechanisms. Holistic models of the tumour microenvironment are represented by in vitro human organoids, which are advanced 3-D culture systems that recreate the architecture and physiology of human tumours in remarkable detail — with the potential for including stromal cells and diverse immune cell populations of the parental tumours. Despite some limitations in culture and growth features, these models have shown to represent a valuable tool with which to evaluate immunotherapy treatments. In addition, the emerging technology of tumour-on-a-chip, combining cell biology, microfabrication and microfluidics, recapitulates the dynamic interplay between immune cells and tumour cells under controlled physical and biomechanics conditions on an individual patient basis. These systems permit the testing of immunotherapy efficacy by the real-time imaging tracking of autologous immune cells and tumour elimination. Pros and cons of these models will be discussed.

New Approach Methods (NAMs) research: To be or not to be original, that is the question

(Marco Straccia; FRESCI by SCIENCE&STRATEGY SL, Barcelona, Spain)

The development of NAMs based on human biology is key to improving human health research with mechanistic data, as well as replacing animal models. Within our projects, information on hundreds of human-based models used in biomedical research was retrieved from reviewing the scientific literature. However, only a few of these models were deeply characterised and exploited to provide mechanistic data and thus provide a robust model. In addition, it was clear that when models were used in similar experimental paradigms, previous works published by others in the same field were ignored, as were publications featuring the use of the model in different applications. How can we help inform you? If you have developed, or want to develop, a human-based model, we suggest firstly focusing on the better characterisation and refinement of already available models, instead of looking for further original applications of a poorly characterised model. In this regard, a systematic review is the first step to undertake in order to get a good overview of the current state-of-the-art of your topic. It also helps you choose the right approach to keep expanding your biomedical research field. Being original may give you a high Impact Factor publication — however, the two main goals in your research career are: to answer the relevant biological question behind a certain health problem; and to transfer your solution from the bench into the real world. It is key to start measuring research success through its real impact on society, and not through journal Impact Factor rating. We need better scientists and better solutions to make a better world (see https://ec.europa.eu/jrc/en/eurl/ecvam/knowledge-sharing-3rs/life-science-research).

Organ-on-chip technology and its application in immunological research

(Lorna Ewart; Emulate, Cambridge, UK)

Organ-on-chip technology is an exciting, developing technology that is inspired by biology and uses engineering to create specific microenvironments for cells such that they can live in a ‘home away from home’. Cells within the body experience cues that are biochemical and biophysical in nature and organ-on-chip technology aims to recapitulate these cues through the involvement of microfluidics, mechanical strain (in organs where this is relevant), 3-D shape and chemical gradients. It is widely regarded that by creating a physiologically relevant model, the data generated will translate well into human risk assessment or therapeutic efficacy determination. Many of the organ-chip models that have been built to date focus on recreating healthy organ-function, but more recently attention is turning toward the ability to model aspects of disease. To this end, Emulate is focusing on developing models that enable immune cell recruitment and studying the effect of this recruitment on biological function. During this presentation, I will cover the design principles of the Emulate organ-chip and show data on how these models can be used in immunological research.

Biomedical research, translational failures and indicators to monitor the impact of EU-funded research

(Francesca Pistollato; European Commission, Joint Research Centre (JRC), Ispra, Italy & Janine McCarthy; Physicians Committee for Responsible Medicine (PCRM), Washington DC, USA)

Animal models have been traditionally used in biomedical research to recapitulate human disease features and develop new drugs, as they are generally purported to resemble some of the major hallmarks of human diseases.^4,5 However, these animals do not develop the disease as it occurs in humans, and their use has not paved the way toward the development of drugs effective in human patients.^4–6 Indeed, despite conspicuous research and economical endeavours, the clinical failure rate in drug development still remains very high, with an overall likelihood of approval from Phase I of about 9.6%. On the other hand, the expanding toolbox of non-animal methods — such as induced pluripotent stem cells derived from patients, next-generation sequencing, ‘omics’ and integrated computer modelling — can be used to study human diseases in human-based settings, identify new potential druggable targets and evaluate treatment effects.^7,8 The rise of new technological tools and models in life science, and the increasing need for multidisciplinary approaches, have encouraged many research initiatives and the launch of new EU calls for proposals. Research proposals based on the use of both animal and/or non-animal approaches have been extensively funded at European level. Nowadays, it is becoming pivotal to define and apply indicators suitable to measure the social impact of research funding strategies, monitor contribution to innovation, retrospectively assess public health trends, and readdress funding strategies when needed. ⁸ Here we discuss such issues, describing a list of indicators to measure the impact and innovation of biomedical research.

Adverse Outcome Pathways — Inflammation as a hub in AOP networks

(Brigitte Landesmann; European Commission, Joint Research Centre (JRC), Ispra, Italy)

An AOP depicts how an injury at the molecular level — the molecular initiating event — propagates through the levels of biological organisation up to an adversity at organism or population level, by describing a sequential series of Key Events (KE; change of a biological state) and their causal relationships (KER; key event relationship). Initially, the AOP framework was built to support chemical risk assessment based on mechanistic reasoning. Meanwhile, in addition to chemicals, nanoparticles, low-dose radiation and microbes have also been considered as potential stressors for initiating an AOP. AOPs combine and integrate data from various methods and disciplines and are an ideal platform for interdisciplinary collaboration and exchange. The modular structure of AOPs, consisting of KEs and KERs, allows sharing of common elements among multiple AOPs and subsequently the building of AOP networks from independently described AOPs. AOP networks, defined as sets of AOPs sharing at least one common element, can more realistically describe the complexity of biology by providing information on interactions between AOPs and potentially revealing links between different biological pathways. Some biological processes are common to many pathways and thus represent highly connected central nodes within the global AOP network. Inflammation is an important and central biological process, and three hub KEs were identified that represent salient features common to the inflammatory process across tissues, thus facilitating the interconnection of various AOPs.

Probing the human immune system during aging in health and disease

(David Furman; Buck Institute, Novato, California, USA)

In addition to its function in the protection against infections, the immune system plays a chief role in the development of chronic disease. However, metrics with which to identify those at risk are lacking. We have developed a pipeline to study the human immune system, utilising an unbiased approach to deconstruct a human blood sample in large cohorts and applying deep analytics — including artificial intelligence and advanced machine learning methods. This ‘systems immunology’ approach to aging and chronic disease has led to fundamental discoveries in cardiovascular senescence and the identification of interventions.

Lack in regulation of inflammation as a key factor in fluoroquinolone-induced liver injury: Translation from in vivo to in vitro

(Raymond Pieters; HU University of Applied Sciences, Utrecht, the Netherlands)

Idiosyncratic drug‐induced liver injury (iDILI) is an example of an adverse immunotoxicological outcome with a poorly understood pathogenesis. iDILI is often associated with inflammatory stress signals, both in human patients and animal models. In some of these models, but particularly in the established iDILI-mouse model with the fluoroquinolone trovafloxacin (TVX), macrophages and neutrophils appear crucial. Since the exact role of these and other leukocytes remained to be defined, we set out to study the kinetics of immunological changes that occur in the liver during the development of TVX‐induced iDILI in mice. In vivo studies show that the TNF-induced liver influx of leukocytes, in particular macrophages and neutrophils, was disturbed by TVX. TVX not only reduced the TNF-induced influx but also delayed the recovery to the normal situation, that in case of TNF-only occurred within 4 hours after induction. Unlike the controls, mice receiving TVX + TNF displayed severe hepatotoxicity within these 4 hours, with clear pathology and apoptosis, coagulated hepatic vessels and increased alanine aminotransferase levels and interleukin 6/10 ratios. In line with these in vivo mouse data, we also found that TVX inhibits in vitro migration of human neutrophils. Overall, findings indicate that TVX causes DILI by interfering with regulation of inflammation, rather than by inducing inflammation. This apparently contradictory finding may represent an additional KE, fitting into the already identified KEs representing the process of inflammation. ⁹ This new KE may help to predict certain cases of DILI, by using selected in vitro methods for assessing cellular migration.

Using non-animal models to understand SARS-CoV-2 and Covid-19

(Maria-Joao Amorim; Instituto Gulbenkian de Ciencia, Oeiras, Portugal)

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is the virus responsible for the worldwide pandemic of coronavirus disease 2019 (Covid-19) that has caused the death of over 2 million people (as of 25 January, 2021) in only one year. SARS-CoV-2 emerged as a novel zoonotic viral infection in humans for which the most basic aspects of infection were unknown. Strategies to control and prevent the infection worldwide were implemented and adapted to reflect the knowledge as it was being acquired. Scientists mobilised in unprecedent manner to understand many different aspects of the infection, including its origin, epidemiology, disease, therapies, evolution, prevention and detection. Many of these aspects have relied on animal models, but for this presentation I will focus on how non-animal models were used to further our understanding of SARS-CoV-2 and Covid-19.

CIAO Project — Integration of knowledge based on the AOP concept

(Penny Nymark; Karolinska Institutet, Solna, Sweden and the CIAO consortium, www.ciao-covid.net)

The AOP framework provides a structured approach for systematic organisation of research data and knowledge. The five key principles of the framework allow for broad application and data integration across a range of diverse disciplines related to human health — including toxicology, pharmacology, virology and medical research. The CIAO project, ‘Modelling the pathogenesis of Covid‐19 using the Adverse Outcome Pathway framework’, aims at a holistic assembly of knowledge to deliver a truly transdisciplinary description of the entire Covid‐19 physiopathology, starting with the initial contact with the SARS‐CoV‐2 virus and ending with a variety of adverse outcomes in various organs. In January 2021, more than 50 scientists from numerous organisations around the world met in the 2nd CIAO AOP Design Workshop, to discuss the depiction of the Covid‐19 disease process as a series of key events (KEs) in a network of AOPs. Seventy-four KEs forming thirteen AOPs were identified, covering Covid‐19 manifestations that affect the respiratory, neurological, liver, cardiovascular, kidney and gastrointestinal systems. In addition, modulating factors influencing the course and severity of the disease, as well as possible framework extensions beyond purely biological phenomena, were addressed. This lecture will provide an overview of the concept underlying application of the AOP framework to Covid-19, and the expected outcomes and research support that it provides, as well as a brief glance at the preliminary results obtained in the project.

Multiclonal antibodies as animal-free replacements for polyclonal antibodies

(Stefan Duebel; TU Braunschweig, Institute of Biochemistry, Braunschweig, Germany)

While phage display, the premier animal-free antibody generation method, is well established for the generation of therapeutic antibodies, most antibodies for research and diagnostics are still made by using animals. This presentation reviews the achievements and prospects of recombinant in vitro antibody generation, demonstrating how animal-derived antibodies could be complemented or replaced in a large number of typical current research applications. Examples of further advantages of in vitro antibody generation will be presented, with respect to their predesigned features and sequence-defined quality. This will also include how polyclonal animal-derived antibodies, which are widely used as secondary antibodies in countless assays, can be replaced by recombinant multiclonal antibodies. The successful generation of multiclonal antibodies for use as replacements for typical secondary antibodies used in research, as well as replacements for horse sera used for therapy by passive vaccination, will be presented.

Room on ‘Noah’s Ark’ — High-throughput screening and New Approach Methodologies for ecotoxicology

(Daniel Villeneuve; United States Environmental Protection Agency, US EPA)

In recent decades, significant progress has been made in development of high-throughput screening and other non-animal assays for evaluating chemical safety. However, to date, these efforts have focused almost exclusively on human and mammalian cells, proteins, and human-oriented exposure and toxicokinetic models. Nonetheless, most regulatory and product stewardship programmes are charged with considering not only safety to humans, but also safety to ecosystems and the diversity of non-human wildlife within those ecosystems. While it is neither feasible, nor desirable, to develop NAMs for all the species we aim to protect, consideration of evolutionary diversity, conservation and divergence of genes and pathways, and life history attributes that influence exposure, can inform the design and application of ecologically-focused NAMs. For example, growing databases of protein sequence information were used to identify molecular targets for which taxon-specific assays may be needed, and to identify which assemblage of species may best reflect diversity in intrinsic susceptibility. Likewise, high-throughput transcriptomic assays are being explored as an efficient and effective — while not overly conservative — means to detect chemical impacts on pathways for which there are no orthologs in humans or traditional mammalian test organisms. Together with an established history of using NAMs to fill the substantial ecotoxicological data gaps that exist, current efforts to develop eco-focused NAMs aim to ensure that the non-human residents of our planet are not left behind in 21st century safety assessments. The contents of this presentation abstract neither constitute, nor necessarily reflect, official US EPA policy.

Endocrine Disruptors and the thyroid validation project

(Sharon Munn & Sandra Coecke; European Commission, Joint Research Centre (JRC), Ispra, Italy)

Criteria to identify substances with endocrine disrupting properties have been recently established in the EU under both the plant protection and biocidal products regulations. In order to be able to apply the new criteria, mechanistic methods are needed to investigate the potential of a chemical to interfere with hormone production, action and clearance. The OECD’s conceptual framework for the testing and assessment of endocrine disruptors has focused on methods to detect chemicals that can interfere with the estrogenic, androgenic, thyroid and steroidogenesis (EATS) pathways. However, in vitro methods for the identification of thyroid disruptors are still lacking. To fill this gap, the EU’s Reference Laboratory for alternatives to animal testing (EURL ECVAM) is conducting a multi-laboratory study aimed at validating a number of in vitro methods. Candidate methods cover the different ways in which chemicals may interact with the thyroid hormone axis, including central regulation, synthesis, transport and distribution in the serum, metabolism and excretion as well as cellular uptake and intracellular (de)activation. Characterising and validating these methods are important steps towards their regulatory use and international adoption. The methods and their potential regulatory uptake within the context of the implementation of the EU criteria for ED identification will be discussed.

OECD guidance on in silico methods (QSAR and PBK)

(Andrew Worth & Alicia Paini; European Commission, Joint Research Centre (JRC), Ispra, Italy)

Computational toxicology is a fast developing field of science that integrates information and data from a variety of sources (e.g. biology, chemistry) that allows the development of mathematical and computer-based models to predict the exposure, fate and interactions of a chemical leading to adverse health effects. The uptake of these mathematical models in chemical risk assessment is still minimal. Driven by good modelling practices, the Organisation for Economic Co-operation and Development (OECD) has published several guidance documents to support the use of computational models in chemical risk assessment. The first OECD Guidance Document covers the Quantitative Structure–Activity Relationships (QSAR) domain. ¹⁰ To keep QSAR applications on a solid scientific foundation, principles for QSAR models were established, covering how to develop, validate and use QSARs for regulatory needs.^11,12 The second set of guidance is on the characterisation, validation and reporting of Physiologically-based Kinetic (PBK) models for regulatory purposes. ¹³ In this case, the goal is to increase confidence in the use of PBK models parametrised with data derived solely from in vitro and in silico methods and how these models can be validated. The use of scientifically valid QSAR and PBK models will allow chemical assessment to rely on the use of these approaches for toxicity predictions, rather than in vivo data derived from animal studies. ¹³ This presentation will introduce the principles and criteria captured in these Guidance Documents and also illustrate the application of computational toxicological models in risk assessment.

Chemical assessment and risk management

(Elisabet Berggren & Federica Madia; European Commission, Joint Research Centre (JRC), Ispra, Italy)

Within the EU as well as in all other parts of the world, management measures are in place to protect consumers, workers and the environment from potential adverse effects caused by chemical exposure. Hazard or risk assessment of a chemical and its intended use are the bases for the risk management. In this lecture, we will present how this is currently performed especially within the EU legal framework. In addition, we will explore how the assessment of chemicals could be further improved and made more efficient, based on known underlying biological mechanisms rather than on information on adverse effects in animal studies. Focusing on long-term health effects, we will identify different modes of action contributing to the adverse outcome. These could then build the bases for integrated approaches to testing and assessment and make use of human relevant information.