Medical Faculty of the Johannes Kepler University in Linz.
Credit: Fivetonine, iStockphoto.com

News in the field of developmental neurotoxicity and endocrine disruptors

Developmental neurotoxic chemicals have the potential to disrupt the normal development of the nervous system, which can impair neurodevelopment. That's why the OECD plans to test thousands of chemicals and hundreds of pesticides on development neurotoxicity (DNT). Dr. Martin Paparella, a regulatory toxicologist working for Austria at the OECD, reports on the limitations of regulatory in vivo testing and assessment of DNT. In animal studies, there is high variability in standard DNT data, and transferability from laboratory animals to humans is questionable, he said. A rodent test of this kind requires around 800 rodents per substance. In an experiment with human material, 20 chemicals were tested for DNT. Here damages were found that had not been detected previously in animal experiments. Moreover, there would be functional differences between rodents; the impairment of complex human behavior by a developmental neurotoxic substance could not even be studied in rodents. Instead, integrated testing and assessment approaches (IATAs) for DNT could lead to more reliable comparative toxicity assessments for a wider range of chemicals.

Prof. Dr. Ellen Fritsche from the Leibniz Institute for Environmental Health Research (IUF), Düsseldorf, presented a testing strategy developed together with international scientists from universities and the European Food Safety Authority. Meanwhile, documents are available that describe the methodological approach in detail and provide guidance on how to interpret data in order to use them for regulatory decisions. The performance of the developed test battery could be improved by closing existing gaps, artificial intelligence could also be used.

Dr. Katharina Koch from Prof. Fritsche's group at IUF surprised the audience by extending its neurosphere test system to endocrine disruptor tests. Endocrine-disrupting chemicals interfere with the body's hormonal systems that regulate growth, development, reproduction, energy, and body weight. The scientist succeeded in modifying in vitro culture models with early stages of the developing brain in such a way that potentially hormone-active substances could bind to the appropriate receptors and be determined. This could be used to develop a comparable test strategy with human cell cultures like in the case of developmental neurotoxicity, since cells of the rat, for example, only show half of the hormone-active substances. A human-specific testing strategy is urgently needed: the OECD plans to test countless chemicals for possible hormone activity, which would mean more than 5 million animal tests. (1) The cell assay is currently being pre-validated by PEPPER, a non-profit association and public-private platform based in Paris. The organization is dedicated to such tasks to characterize endocrine disruptors. It was founded by the French Directorate General for Risk Prevention and associations of cosmetic and chemical companies.

Toxicologist Iris Manga, responsible for pesticides at EFSA, reported on two studies (OECD Case Studies 262 and 263) in which the developed testing strategy for developmental neurotoxicity has been used. The objective was to demonstrate the applicability of the in vitro battery (IVB) for developmental neurotoxicity testing (DNT) under the European Pesticide Regulation (EU) 283/2013 and 1107/2009. The usefulness of the test strategy for hazard characterization could be shown. An OECD test guideline on this will be published in 2023.

Unique professorship for the evidence-based transition to animal-free innovations

In her opening address, Prof. Dr. Merel Ritskes-Hoitinga, holder of the unprecedented Chair for the Evidence-Based Transition to Animal-Free Innovations at Utrecht University in the Netherlands, explained the historical legal basis for animal testing. This was based on disasters in the 1930s in the U.S. after people were poisoned by an antibiotic dissolved in anti-freeze, and the 1960s in Europe by the thalidomide scandal. However, the FDA regulations would not have led to increased drug safety. However, animal testing is no longer up to date. In contrast, 3D cell cultures, organoids, biologically printed tissue, computer models, or organ chip technologies are state-of-the-art. In contrast to long animal testing procedures, a shortened approval time for Covid-19 vaccines would have led to an effective fight against the Sars-Cov-2 epidemic with less animal testing by the regulatory authorities waiving the batch testing of messenger RNA vaccines with animals. Further progress must be made along this path, she said.

There is a need for cooperation between science and the regulatory authorities, but above all for transdisciplinary cooperation - also with society - as well as interdisciplinary cooperation among scientists. The pharmaceutical industry actively supports these developments, so they have expressed their desire to continue on this path. The EMA, on the other hand, is attached to animal testing because it is afraid of the risk, even though animal testing involves such risks.

Prof. Dr. Merel Ritskes-Hoitinga, holder of the so far unique chair for the evidence-based transition to animal-free innovations at the University in Utrecht.
Photo: Christiane Hohensee

Future collaboration between pharmaceutical companies and regulatory agencies fosters the promise of developing new vaccines faster and more effectively through new alternatives involving fewer animals. What is required, she said, is funding for method development, followed by education and training on these methods. The knowledge then causes a change in thinking among scientists; they gain new skills with the new methods. However, research must also deal with the poor test results from animal experiments. In the end, a rethinking takes place in the entire scientific community. For this purpose, the Netherlands has developed the partner program TPI (Transition Programme for Innovation without the use of animals). It is designed to accelerate the implementation of animal-free innovations in drug development, food safety, and chemical evaluation. Ten ministries, national institutes and academies, social groups, and industry associations have joined forces to cooperate despite differing backgrounds. For new innovations, for example, that have not yet been approved or validated, other comparative references than animal testing need to be found.

Prof. Ingrid J. Visseren-Hamakers from Radboud University presented a research project into animal-free safety assessments. Funded by the Netherlands Research Foundation, it will run from 2023-2027 and is intended to provide the transition to the animal-free safety assessment of chemicals and pharmaceuticals in the Netherlands, the EU, and the U.S. by developing an understanding of how the transition should be regulated.

The Netherlands: Transition Programme for Innovation without the use of animals

The Dutch TPI (Transition Programme for Innovation without the use of animals) aims to accelerate the transition to non-animal innovation, such as organ-on-a-chip or artificial intelligence. Since 2018, the program's partners from the government, society, academia, and industry have been seeking to develop various methods in the field of chemical safety and drug efficacy and safety. The aim is to create space for model developments and to increase confidence in these methods. The core of the TPI is formed by ten partners who have joined forces to achieve the Netherlands' goal of leading the international transition to animal-free innovation. A mix of research areas, sectors, technologies, and policy dossiers increase the opportunities for animal-free innovations. The Ministry of Agriculture, Nature, and Food Quality is responsible for the partner program. It organizes regular consultations between partners and also facilitates consultations with other ministries working on alternatives to animal use and animal-free innovations.

In 2022, "TPI Young" was founded, a group of motivated young professionals, Ph.D. students and students with different study and professional backgrounds from the Netherlands, but also from other European countries, especially Germany and Belgium. They are united by the ambition to contribute to the acceleration of the transition to non-animal innovations by providing inspiration to other young people and raising their awareness of animal-free options at an early stage. Moreover, they build a network and provide a space for the exchange of knowledge and experiences between people working with and/or interested in animal-free innovations.

They provide professional development support, make their voices heard within existing structures, and search for new collaborations. They host activities at least twice a year, about which interested parties can inform via a TPI's LinkedIn page or the website's registration form. Young TPI welcomes new members from all over Europe and also worldwide.

The Interdisciplinary Centre for 3Rs in Animal Research (ICAR3R for short) has established a 3R Skills Lab "Improve your Skills" for (prospective) researchers. The main goal is to increase awareness of the 3Rs and foster a willingness to reconsider the use of animal models. Knowledge and skills are taught by working at stations.

Policy developments still need improvement

Information was also provided on policy developments in the EU. Dr. Laurence Walder from Eurogroup for Animals, for example, reported on an analysis of the implementation of the EU Animal Experiments Directive by the European member states. There are numerous areas where improvements should be made, especially in transparency. For instance, there would be a gap in measures to ensure that the 3Rs are satisfactorily addressed and sufficient controls are in place. In education and training, there is a lack of transparency on course content, qualification and duration. In many of the Member States, project evaluators have a background in animal experimentation and are rarely experts in non-animal methods. In addition to experts in this field, animal rights activists or patient rights activists should also be involved in the evaluation processes.

Dr. Luisa Bastos, Animal in Science program manager of Eurogroup for Animals criticized that despite the support of a gradual transition to animal-free science by the EU Parliament and European citizens, current EU policy does not fulfill this commitment.

Dr. Samatha Saunders from Cruelty Free International reported that a comparison of the so-called RAT list showed that 10 different animal tests are still being performed, even though an approved alternative is already available. Consistent application of the new method could save 1.5 million animals EU-wide.
Long-term toxicity

Prof. Mathieu Vinken from Vrije University Brussels reported on the European project ONTOX, which started in May 2021 to elaborate a so-called "generic strategy" for the development of animal-free methods (NAMs) in repeated (long-term) testing of substances. Data basis consists of already available biological/mechanistic, toxicological/epidemiological, physicochemical and kinetic information. If data gaps are identified by artificial intelligence, they will be filled with targeted in vitro and in silico tests.

News from 3R Training Platforms

Dr. Susanna Louhimies, a member of the European Commission's Directorate General for Environment, reported that 13 new e-learning modules are currently being developed as part of the ETPLAS - Education and Training Platform for Laboratory Animal Science. While education and training is a Member State's responsibility under the EU Directive on the protection of animals used for scientific purposes, the EU provides the framework. However, participation is voluntary. So far, two of the six English-language modules are relevant to the replacement of animal experiments: "EU-52: Searching for (existing) non-animal alternatives" and "EU-60: Developing in vitro methods and approaches for scientific and regulatory use". Furthermore, materials on how to teach the 3Rs principles to secondary and undergraduate students will be developed and information will be provided on career opportunities in the field of 3Rs.

In silico: new methods on the computer

Dr. Ignacio Tripoldi presented his new research in the field of in silico. Here, he has transformed previously published Adverse Outcome Pathways (AOPs) into testable hypotheses. In the AOP concept, biological processes (cell receptor binding of molecules) lead to adverse effects in the organ, organism or population through several stages. The concept is important in the new perspective on risk assessment and the implementation of new animal-free methods in toxicity testing. For this purpose, the scientist used a subfield of Artificial Intelligence called Natural Language Processing (NLP). NLP is designed to enable computers to understand, interpret and manipulate human language. The goal is to maximize the information potential of AOPs and make it usable for hypothesis testing.
Mieke van Mulders informed about the current development status of the RE-Place database in Belgium. It is based on the old ECVAM database DB-Alm, however the template has been newly constructed. The scientists working on new animal-free methods should enter their information into the database themselves. Currently, more than 200 methods from the fields of basic research, translational/applied and education have been entered. Most of them have been published in peer-reviewed journals. At present, the database is limited to Belgium, but it is planned to make it available throughout Europe at a later stage.

Environmental toxicity

Dr. Stefan Scholz from the Environmental Research Center in Leipzig is a specialist in embryotoxicity tests with fish (FET test). The FET (OECD 236) determines the acute or lethal toxicity of chemicals to embryonic stages of zebrafish. Freshly fertilized eggs are exposed to the test substances for a total of 96 hours and the embryos are then examined. To utilize FET in chemical testing, it must be used in the weight-of-evidence approach (WoE approach). In the WoE approach, interpretive methods are applied to integrate data. The information must be directly plausible or comprehensible and not require proof (evident). To increase the informative value of the FET, the scientist has developed a Bayesian network that can be used to predict a probability of events, such as potential neurotoxicity.
Prof. Dr. Cinta Porte of the Institute of Environmental Assessment and Water Research in Barcelona has studied the environmental effects of plastic additives such as phthalates in fish liver spheroids. A mixture of several such additives generates reactive oxygen species, which can cause damage to the cells.

Human-relevant animal-free methods essential for COVID-19 research

A key feature of the SARS-CoV-2 coronavirus is that it can infect and damage multiple human organs. Currently, there is no animal species known in which the complex symptoms or severe COVID-19 disease can be induced. Therefore, NAMs are very important for the study of coronavirus pathogenesis mechanisms. For example, in the work of Prof. Stefan Hippenstiel from Charité University Medicine Berlin, infected human lung explants and human lung organoids are used, demonstrating that severe lung injury is likely due to immune activation triggered by macrophages rather than direct damage to the alveoli.

Claus-Michael Lehr, Professor of Biopharmacy and Pharmaceutical Technology at Saarland University is working on drug transport to the site of their destination. For this purpose, he and his team had spent the last few years developing the barriers to be overcome, e.g. the lungs, in vitro, and had successfully investigated drug transport in nanoparticles. In the presented work, the team has investigated whether the drugs can end up in the organism, in this case in a bacterium - and kill it afterward. This would be important for patients suffering from cystic fibrosis. This is a congenital metabolic disease in which the salt balance is disturbed and the body's own secretions are viscous, which can lead, for example, to impaired lung function with constant coughing, shortness of breath, and regular infections.  
Doctoral student Jana Wächter from the Institute of Pharmaceutical Technology at Goethe University Frankfurt has also presented important work. Bacteria forming biofilms are a medical problem. The bacteria embedded in this layer of mucus are protected from attack by the immune system, making them less sensitive to antibiotics. The young researcher, who is also one of the 17 winners of the Young Scientist Travel Award, has succeeded in creating a three-dimensional in vitro biofilm wound model on human skin for research and drug development.

Prof. Jens Kurreck of the Technical University of Berlin is specialized in 3D printing of organ-like systems, where the organs do not look organ-like but are functional. An infection model of the lung was presented here. The human fibroblasts, lung cells, and immune cells are embedded in alginate, gelatin, and - Matrigel - the latter of which, unfortunately, is currently derived from the purified secretion of a malignant mouse cancer cell line. The model can also be used for Covid-19 research.

Innovative in vitro and organ-on-chip models

Young scientist Dr. Pauline Zamprogno from the University of Bern is one of the winners of the Young Scientists Travel Award, with which EUSAAT promotes the participation of young scientists at the EUSAAT Congress. She reported on an in vitro pulmonary fibrosis model, IPF (idiopathic pulmonary fibrosis)-on-a-chip. IPF is a chronic, progressive, and severe lung disease. Existing mouse and in vitro models are often unable to predict the effects of drugs in clinical trials. The IPF-on-a-chip model mimics some important features of early and late-stage pulmonary fibrosis, in terms of cellular composition, extracellular matrix and novel biological membrane. The complete biological nature makes the model a promising tool for drug discovery.

Léa Todeschini from Alveolix, a Swiss organs-on-chip company, reported on the Company's own Barrier-on-Chip system, which mimics the dynamics of human organ barriers. The system can be used to model a wide range of biological barriers. Human cells are grown on each side of an ultra-thin porous membrane as mono-, co-cultures or multi-cultures, including immunocompetent cells. Cell differentiation is then stimulated by 3D motion. For example, human primary lung and intestinal cells have been used to generate dynamic lung and intestinal barriers on chips, including an infection model with the pathogen that causes pneumonia. The advanced in vitro models enable efficacy studies and acute toxicology analyses and thus have the potential to identify unsafe drugs early in the drug development phase by reducing or even replacing animal testing. In a single-case study with a pharmaceutical company, toxicological effects comparable to those in animal studies were found.

Tine Haesen from the Bielefeld University and from the Fraunhofer Institute for Toxicology and Experimental Medicine in Hannover, Germany, reported on human precision-cut lung tissue slices as an ideal ex vivo model that can render current in vitro and in vivo experiments obsolete. The sections of lungs or lung tumors can be used to analyze various immune cells and validate targets for cancer therapy. The tissue sections preserve the original microanatomical 3D architecture of the human lung and ensure that all immune cells remain in their complex microenvironment.

Prof. Armin Wolf from the Swiss company InSphero presented a 3D liver microtissue for predicting drug-induced liver injury. These are a major cause of withdrawal of already approved drugs and pose a major threat to human patients. 90% of drug candidates that have not been shown to be liver toxic in animal models fail in clinical trials due to liver damage and lack of efficacy. Animal studies cannot accurately predict drug liver toxicity in humans due to species differences. Insphero developed the 3D InSight™ Liver MPS (microphysiological system), a standardized high-throughput in vitro model based on three human liver cell types found in the liver. The 3D co-culture exhibits the essential structural and functional features of the native liver. Using the so-called liver spheroids, the effect of 63 severely liver-damaging drugs could be predicted very accurately, with a sensitivity of 90.3% and a specificity of 81.3%. Thus, it is a useful tool for in vitro safety assessment in drug development and is also suitable for mechanistic studies and in later stages of drug development to determine complex patterns of damage.

Another in vitro model presented for predicting liver toxicity is the microfluidic model from the German company Dynamic42. The researchers use a microfluidic organ-on-a-chip model based on human blood vessel, liver and immune cells. A blood-like substance with the appropriate drug concentration is passed through the blood vessel-like structure. The liver model was used to reproduce the toxicity of a withdrawn drug, which was not detectable in studies in mice without the addition of inflammatory co-stimuli.

Dr. Brigitta Loretz from Helmholtz Institute for Pharmaceutical Research, Saarland, reports on a complex in vitro model of the inflamed intestinal mucosa for research into new drugs. The model mimics the pathophysiology of chronic intestinal disease such as Crohn's disease and ulcerative colitis. The numerous mouse models have little predictive power for human disease due to differences in organ physiology and disease manifestations, as well as differences in immune response. The presented permeable gut (leaky gut) model has a dysfunctional epithelial barrier and immunocompetent cells in a physiologically relevant microenvironment. It enables studies, on the intestinal barrier as well as on the efficacy and targeted delivery of drugs and carrier molecules.

Sascha Mendjan's research group at Vienna's Institute of Molecular Biotechnology has developed human heart organoids, known as cardioids, that reflect the development and defects of the heart. The most common cause of death in human fetuses is defects in heart development. Previous in vitro models cannot capture the specialized functions of the various heart compartments. With cardioids, the development of the major embryonic cardiac compartments can be recapitulated for the first time with in vivo-like gene expression profiles, morphologies and functions. This will allow researchers to investigate how genetic and environmental factors cause defects in the developing human heart.

Developments in education and training

Nick Jukes of InterNiche presented a documentary film series on animal-free innovations in veterinary education and training. The primary audience for the film series is course instructors, department leaders, as well as students. The film is based on visits to universities around the world. Selected faculties that are at the forefront of progressive change in veterinary teaching are featured. The film includes interviews with lecturers, students and producers. Demonstrations and student labs illustrate the use of methods, from comparative anatomy to abdominal surgery. The range of tools includes virtual laboratories; simulation software for anatomy, physiology, and pharmacology; animal donation programs; synthetic models; simulation models for clinical and surgical skills; and scenery-based simulated clinics for internal medicine. The documentary provides a comprehensive overview and several case studies of humane tools to better meet teaching objectives. It explores the educational, scientific, ethical, economic, and environmental benefits of these innovations. A short clip was shown on Syndaver's canine surgery model, which can be used to train high-risk abdominal procedures.

The goal of the project "Promoting 3Rs Awareness in a Young Audience" presented by Dr. David Pamies (University of Lausanne and Swiss Center for Applied Human Toxicology, Basel) is to stimulate critical thinking in secondary school students and to raise awareness of animal experimentation and the importance of the 3Rs principle. The project provides teaching materials, including a workshop for teachers on how to teach the 3Rs principle. It held events with middle school classes, organized lectures by scientists, and encouraged assessment and discussion.

The Hannover University of Veterinary Medicine and the Philipps University of Marburg are working together on the 3R-SMART project, a 3R training platform, presented by Dr. Melissa Valussi, to support 3R research activities by various stakeholders. At its core is www.3R-smart.de, a platform under development that will provide training tools for students, scientists, technical staff, public agencies, regulators, companies, and ethics committees. Further planned are 3R seminars, a curriculum that can be integrated into laboratory animal science courses and the establishment of a Europe-wide 3R network.

(1) https://www.finanznachrichten.de/nachrichten-2022-03/55633972-huge-increase-in-animal-testing-numbers-predicted-by-cruelty-free-europe-under-plans-for-new-eu-chemicals-regime-008.htm