Confident words in the introduction
In his opening speech, Prof. Martin Paul, President of Maastricht University, expressed his conviction, that the future is to phase out animal testing. Christian deSaintes, Policy Officer at the European Commission emphasized that the European Commission is a strong supporter of alternatives to animal testing.
He listed that more than 140 projects had been supported with an annual budget of €11 million, the Innovative Medicines Initiative (IMI) budget was also €48 million per year. The IMI project eTox, for example, was funded with €7 million from the European Commission and €12 million from the pharmaceutical industry. This has resulted in a large database with 1000 drug entries, he added. Funding priorities in the EU's Horizon 2020 funding program included research on liver, lung, kidney, and heart toxicity, carcinogenicity and endocrine disruption. Corona research had also been considered: Numerous organoid developments had contributed to the study of the Sars-Cov-2 coronavirus, such as lung organoids. In addition, the European validation authority EURL ECVAM is investigating Adverse Outcome Pathways of Covid-19 pathogens.

Validation in numerous aspects
As reported by its director, Dr. Maurice Whelan, EURL ECVAM has meanwhile validated more than 100 methods since its establishment. One of its current projects includes the development of a testing strategy for assessing the developmental neurotoxicity of substances. For example, he also reported on the validation of defined approaches in the area of skin sensitization. Some approaches already use OECD-validated in chemico and in vitro test data to determine the risk of skin sensitization. Such data are interpreted according to a specific mathematical procedure. The approaches have the same or even higher significance than the conventional mouse test. Methods that can be used to study thyroid dysfunction, as well as PBK models, have also been validated. But ECVAM has also addressed the need for standardization of organ-on-a-chip technologies.

For the validation, a schematic procedure consisting of modules is used, as Prof. Aldert Piersma from the RIVM National Institute of Public Health and Environment Utrecht reports. He describes a new approach in hazard and risk assessment of substances. It starts from a virtual physiological in silico model of humans; missing data are supplemented by specific in vitro tests. The approach takes into account all existing knowledge of chemistry and toxicology. An intact organism as an animal is not necessary for this approach.

The question of what is necessary to improve the efficiency of the acceptance process of new animal-free methods has been explored by Rebecca Clewell of 21st Century Tox Consulting LLC, North Carolina. Key components of biological pathways (adverse outcome pathways; AOPs) should be identified and incorporated into the test system, rather than comparing results from new methods with animal data during validation.

Carl Westmoreland of Unilever explains a new way of looking at risk assessment with a new exposure science. The approach should be to protect humans, not to predict - that is, to move away from high-dose studies in animals to look for a harmful effect of substances, as it were: If no bioactivity is shown in consumer-relevant concentrations, there will be no harmful effects.

Long overdue: elimination of painful 2-year carcinogenicity tests
Dr. Warren Casey of the National Toxicology Program (NTP) presents the Cancer Genome Atlas with over 20,000 molecularly characterized cancer samples, the Pan-Cancer Atlas with information from over 11,000 tumors analyzed, and, for example, the Apollo Network, which has analyzed DNA, RNA, and protein expressions from 8,000 human tissues. These databases can be used for new approaches in carcinogenicity testing. Current tests on rodents were developed more than 50 years ago, and testing protocols from 1976 (!) are still being used for human protection-despite numerous new medical discoveries.

Chris Corton of the Environmental Protection Agency explains how the torturous 2-year rodent carcinogenicity studies can be stopped: namely, by capturing gene expression biomarkers from the short-term rodent studies, which can identify liver cancer-causing agents early on. Pathway-guided computational approaches can also be used for this purpose. This would end the agonizing long-term animal testing and at least allow a reduction in animal testing. Mechanistic data are key for identifying cancer hazards, says Kate Guyton, Ph.D., of the National Academy of Sciences.

Dr. Gina Hilton of PETA informs about the ReCAAP project (ReThinking Carcinogenicity Assessment for Agrochemicals Project), which involves international regulatory agencies and mainly pesticide manufacturing corporations. The project aims to develop a framework to determine when animal testing for carcinogenicity can be eliminated through a new approach.

Natalie Delrue of the OECD reports on a new integrated testing approach for a specific group of cancer-causing substances (non-genotoxic carcinogens). A database of potential assays is currently being evaluated and a guiding document for the testing strategy is being developed.

News from the Organ-on-a-Chip Front
Dr. Donald Ingber, the founder of the Wyss Institute for Biologically Inspired Engineering at Harvard University, believes his already numerous organ chip developments can be integrated into the drug development process. The future, he said, lies in personalized medicine with organ-on-chips. His numerous physiological disease models reflect disease development more realistically than it would be the case using a cross-section of patients with their influences from other patient characteristics. 15-20 organ-on-a-chip models are now available, and up to 10 organ combinations have also been produced by his company, with cell and tissue lifetimes of up to 4 weeks.

Dr. Uwe Marx, the founder of TissUse, has currently 23 different ready-to-use organ-on-a-chip models in his portfolio. Challenges in the near future will be the automation of tests with the organ and multi-organ-on-a-chip models, but also, for example, to replace so-called humanized animal models with NAMs.

To make the replicated organ-like systems on the chip more realistic and physiological, Prof. Andries van der Meer of the University of Twente focuses on the integration of blood vessels. Among other things, he presented a retina on-chip with integrated blood vessels, which can be used to study aspects of age-related macular degeneration, such as drusen formation. Currently, countless mice are still being used for this purpose.

Advances in immunocompetent organ-on-a-chip models such as the lung chip and also the colon chip, both with integrated immune cells, as a model for pneumonia or inflammatory bowel disease (IBD), which will fill an important gap, were presented by Dr. Lauriane Cabon of Roche, Basel.

Prof. Peter Loskill from the University of Tübingen also presented a new generation of eye-on-a-chip for ophthalmological research. His retinal organoids already contain all relevant cell types such as photoreceptors, nerve tissue, ganglion cells, bipolar cells, pigment epithelium and Müller cells. The research team has also developed a choroid on the chip.

Despite all this progress, Adrian Roth, Roche, Basel, and Lorna Ewart, Emulate Bio Boston, highlighted that the acceptance of these systems still leaves much to be desired. Organ-on-a-chip technology could reduce costs by 26% - but there was a lack of standardization, for example of the cells used. The systems are becoming more and more complex, but reproducibility is also decreasing. Concepts and strategies are needed to bring these systems into use. The need on the part of the pharmaceutical industry is definitely there.

Ocean of data no longer manageable without AI
For risk and security assessment, work is also being done to be able to use artificial intelligence (AI). AI are flexible programs that can learn and adapt their solutions to problems. From vast amounts of data sets, AI can extract patterns that are useful for prediction. On the example of drug-induced liver injury (DILI), it was shown that a new so-called deep-learning model was able to correctly predict around 70% of drug-induced liver injuries.

Prof. Thomas Hartung of the Centre for Alternatives to Animal Testing (CAAT) talks about computer-assisted toxicology, for which e.g. predictive models, data analysis tools, PBPK, and QSAR models, among others, can be used. The huge amounts of data generated worldwide are a challenging task that can only be tackled with the help of large powerful computer programs. Alone PubMed publishes approximately 2.5 million articles per year - and this information cannot be analyzed without computer assistance. A cheminformatics tool kit from Underwriter Laboratories (UL), a testing laboratory that certifies compliance with U.S. safety regulations and awards safety test marks, is already recognized in Australia. Scientists in the EU are also working with artificial intelligence: the Ontox project, which is funded by the European Commission with around €17 million, started in May 2021. During the five-year project period, a so-called "generic strategy" for the development of animal-free methods (NAMs) will be elaborated, which will then be applied to any kind of chemical toxicity effects of pharmaceuticals, cosmetics, food, or biocides in case of repeated administration. The individual NAMs will be based on state-of-the-art artificial intelligence. Available biological/mechanistic, toxicological/epidemiological, physicochemical, and kinetic information will form the data basis. If data gaps are identified by AI, they will be filled with targeted in vitro and in silico testing.

Organoid developments and infectious diseases
All in all, organoid developments for the investigation and treatment of infectious diseases were an important topic. Indeed, in many cases, there is no animal model and if there is, the data are difficult to transfer to humans. Dutch scientists have used human organoid culture on microphysiological systems to study human viral infections. To do this, they have, for example, replicated the gut-brain axis with immune cells on-chip or studied the entry mechanisms of human-specific enteroviruses, such as Ikrame Aknouch, who works with gut organoids as part of the EU OrganoVIR project in Amsterdam, or Josse Depla, who researches brain organoids.

Toxicity testing: still a lack of confidence in the new methods
Similar discussions to those held at the EU congress "Putting Science into Standards" in April this year were held in the session NAMs in the field of agrochemicals. Prof. Alan Boobis of Imperial College in London reports that agrochemical manufacturers were not submitting regulatory applications using NAMs because regulators did not trust these results.

Richard Currie of Syngenta sees the greatest challenge in the global harmonization of methods. Irritants that can be inhaled can certainly be characterized using in vitro and in silico methods. The U.S. EPA has already accepted the methods for one substance, he said. An advantage for the manufacturer is also that the (in)safety factor, which has to be taken into account due to species differences through animal testing (thus the product concentration may be higher), is then omitted.

Animal-free antibody production
Prof. Stefan Dübel of the Technical University of Braunschweig sees science and industry on the verge of a radical change in antibody production. He says the scientific shortcomings of animal-derived antibodies should no longer be accepted. In Europe, for example, the ECVAM Scientific Advisory Committee has confirmed the scientific validity of animal-free replacement methods.

In Europe, antibodies are still produced in animals, but in many cases, they cannot be reproduced, reported Dr. Joao Barroso of EURL ECVAM. In a 2019 workshop, EURL ECVAM's Scientific Advisory Committee (ESAC) had come to the conclusion that animal-free antibodies can replace those produced in animals for most applications. This culminated in the ECVAM recommendation to no longer produce antibodies in animals for research, diagnostics, and therapy. The sequences are defined, the antibodies are reproducible, and can be obtained in unlimited quantities. Manufacturers should develop a rapid phase-out strategy from the production of antibodies in animals. However, this should not be understood as a ban.

Call for a European Institute of Health
Many biomedical researchers do not know about NAMs and use animals; they do not have access to such methods, although other researchers often conduct research on the same topic using animal-free methods. Therefore, some participants called for the establishment of a European Institute of Health, where all such information would converge. In addition, mandatory guidelines for the preparation of abstracts with a generally valid anthology of corresponding mesh terms would be necessary, because, with the 2.5 million publications published annually, it is hardly possible to find sufficient information.

It is time to use the new methods also in the research of basics and therapies of human diseases, said Dr. Lindsay Marsh of the Humane Society international. Signaling pathways (AOPs) should also be determined about human diseases similar to those in toxicology. The new EU project CIAO is developing such a framework for Covid19 research. Another such program is the BioMed 21 collaboration, which is developing disease case studies according to this AOP concept.

Great importance is given to NAMs for the principles of Next Generation Risk Assessment. This is understood by scientists, industry, and regulators as an exposure-guided, hypothesis-driven approach that integrates in silico, in chemico, and in vitro approaches. In the case of working with chemical similarities (read-across), NAMs have great importance, said Dr. Gladys Ouédrago of L'Oréal, because they can fill existing gaps, hypotheses can be tested, toxicodynamic and -kinetic similarities or differences with the chemically similar substance can be identified by NAMs.

Some scientists also presented the current development status of the testing strategy for developmental neurotoxicity. A draft of an initial guiding document for this testing strategy is expected in the fourth quarter of 2021 or the first quarter of 2022. However, the approach will not end up being developed without animal use: in vitro testing will include rat cortical neurons and the use of zebrafish embryos as a last resort if all information and test results are insufficient.

Numerous other developments have included printing human skin with hair follicles or a brain model on a chip to study neurodegenerative diseases. In addition, there are now commercially available organoids for studying Parkinson's disease, but these have only been used by researchers to supplement their in vivo research. The countless models cannot all be presented here.

Fetal calf serum: an issue that remains unresolved
In the introduction, the extent of the animal welfare problem was again explicitly presented. The demand for fetal calf serum (FCS) is constantly increasing and is now around 2 million calves per year. The price increased from €160/liter in 2015 to €1216. Although mammalian fetuses are explicitly protected by EU legislation, no requirement exists for blood collection by cardiac puncture, for example, for anesthesia/pain relief. Accordingly, the use of FCS as an additive to cell culture media should be specifically considered in approval processes.

Dr. Jan van der Valk of the 3R Center Utrecht pointed out in his presentation that FCS is meanwhile not only used in in vitro studies but also in the production of laboratory meat. Due to the fact that serum is taken from calves at different stages of its development and often enough under dubious standards, it varies greatly in its composition. He highlighted, in particular, a ring study of A549 cells in which this variability (and concomitant poor reproducibility of research results) was demonstrated.

On the website http://fcs-free.org, 1502 media for 276 cell types have now been published. Efforts to replace FCS are ongoing, as highlighted in particular by Dr. Aline Chary from the Luxembourg Institute of Science and Technology using the example of the A549 lung cell line mentioned above, which was successfully converted to commercially available FCS-free media. Important here was a stepwise approach with continuous monitoring of cell viability/function.

Rehoming of laboratory animals
In the context of Culture of Care (CoC), increasing thought is being given to how to give laboratory animals a "second life" after the end of the experiment - provided they are not genetically modified, are healthy, and can be trained and handled well. Dr. Paulin Jirkof from the University of Zurich and Dr. Julika Fitzi from Swiss Animal Protection presented a successful joint initiative here, the placement of former lab rats in private hands. It is important here that the placement is given into external hands since the university cannot do this work additionally. The Club of rat friends serves here as an intermediate station in the placement, which takes the animals in the meantime and checks the new owners with its experience on heart and kidneys. With the new owners, a contract is signed, which regulates e.g. the consent to controls. The Swiss Animal Protection serves as a contact for interested parties. The initiative has been very well received. At the moment, however, the costs are borne by animal protection. The aim is to write this into the permit application from the outset as a refinement measure in projects where rehoming is possible and to book it as a project cost.

Pascalle van Loo, animal welfare officer at Utrecht University, reported on comparable efforts. The Directive 2010/63/EU encourages animal welfare officers in scientific institutions to promote rehoming, as Dr. Susanna Louhimies from the European Commission in Brussels also pointed out in her presentation. However, despite good successes with dogs, cats, rabbits, etc., rats and mice have been neglected so far. In 2019, negotiations with the university management began; criteria for adoptability (age, health status, etc.) had to be worked out, and appropriate external relinquishment sites found. These places will keep the animals even if new owners cannot be found. Here, too, only healthy, neutered, and not genetically modified animals are handed over, and appropriate contracts are concluded and controls agreed upon. The new owner must also know what kind of experiments the animals come from, what stresses they have been exposed to, and how the previous "handling" took place, in order to be able to better assess reactions of the future pet.

One of the main problems is the immune system of the animals. This could not "get to know" any of the normal pathogens under laboratory conditions.  Thus, cancer and infectious diseases are frequent side effects of rehoming. The animals also have to be prepared for their life outside the laboratory. The animal caretakers are highly motivated to train the animals accordingly and are happy to take on the extra work. Many researchers also handle the animals differently, knowing that they have a life after the lab ahead of them, clarified Prof. Andrew Fenton of Dalhousie University in Halifax. However, common to all rehoming projects is that not all researchers want to take (co-)responsibility for their animals beyond the experimental project. They consider this to be a task for animal welfare officers or animal protection organizations. 

In preparation for the new life with its challenges, but also in everyday laboratory life, PRT (positive reinforcement training) is applied, which reduces stress in humans and animals. Methods of PRT have been presented by Dr. Kathryn Bayne of AAALAC International, among others. Clicker training is well established in dogs and pigs but is rarely used in laboratory rodents. Animals respond very well to training and learn quickly. Via target training, the animal is taught to touch a specific object with its nose. In this way, the animals can be moved from one place to another without stress or handling. The success of these training methods depends on many individual factors, such as the animal's attention span/day shape, species and breed line differences, and the animal's trust in humans.

Culture of Care and Corporate Social Responsibility
Training through positive reinforcement such as rehoming is part of the "Culture of Care" (CoC). Stress is not appropriate for animal welfare and is considered a "confounding factor": it reduces reproducibility. This also and above all includes stress-free handling of the animals. Maria Kiersgaard from Novo Nordisk, Copenhagen, spoke about practical implementation. Tail handling", i.e. lifting by the tail, is now being rejected. Cup handling" and "tunnel handling" are used as alternatives. The beginnings were frustrating: it took longer, the inadequately trained staff chased the mice with the tunnel, and nothing changed in the stress level of the animals. It took intensive and personal training with intensive exchange to achieve the desired success. The animals were calmer, the staff happier. Dr. Mark Prescott of NC3Rs added that changing various lab routines, such as cleaning cages, can have positive effects on male mouse aggressiveness.

Another element of CSR/CoC, presented by Dr. Sabine Juliane Bischoff Animal Welfare Officer of the University Hospital Jena, is learning from mistakes. Damage, near damage, unwanted or unexpected negative events that occur in the course of animal experiments are published, analyzed, and discussed on the world's first platform www.CIRS-LAS.de. Establishing a transparent error culture should help to prevent these events in the future, as incidents of any kind are not published anywhere. It also prevents the repeated failure of animal experiments due to the same mistakes over and over again.

CoC/CSR does not end with animals but includes humans. One phenomenon that has received increased attention in recent years is "compassion fatigue," as outlined by Judy Murray of Charles River Laboratories. It particularly affects animal caretakers who are particularly concerned and compassionate about their animals. The all too often inevitable death/suffering of animals creates feelings of grief that cannot be articulated. They no longer take the time to care for themselves adequately, neglect social contacts and over time become increasingly blunt in their dealings with the animals, which can lead not only to psychological but also to animal welfare problems. The program aims to raise awareness of this problem among experiment managers and those carrying out the experiments and to create places where those affected can be heard and receive support. Helpful in the processing is, for example, a possibility of "commemorating" the animals, the choice to kill the animals oneself after the end of the experiment or to leave this to someone else, or rehoming programs. 

Philosophical and ethical questions
Prof. Ingrid van Visseren of Radboud University raised fundamental questions about the change in a complex social system of which animal experimentation is only a small part. How can we make the transition to an animal-free world while keeping the other systems stable and sustainable? The transformation requires technological, economic, and social change. It can only take place on the basis of a change in values and must keep all sectors in mind, as they influence each other directly or indirectly. In ending animal testing, the question must be asked whether a (yet another) technological solution, namely in vitro methods and the like, are sufficient to ensure health or whether there should not be a much greater focus on a healthy environment and prevention. In such transformations, we cannot avoid profound paradigm shifts. The ultimate goal is the end of speciesism, which will entail profound changes also and especially in the field of agriculture.

Dr. Anna Deplazes from ETH Zurich also took up the topic, critically noting that the 3Rs principle is basically a utilitarian one as well. In animal experiments, a harm-benefit analysis is done to assess the proportionality of an experiment. The 3Rs reduce the cost side (the harm to the animal), but do not increase the benefit/meaning of the experiment. In her presentation, she raised many controversial issues as examples: When using and optimizing human materials such as ESC, it is also necessary to think about the possible use of these cells in the development of biological weapons. Minibrains, which are becoming more and more refined, could independently develop some kind of sentience/consciousness. How do I detect this in an organoid that has no way of expressing itself? When cells are taken from a human donor, the question arises as to who owns the cells. May they be further multiplied and used beyond their original purpose - and who decides on this? What kind of consent on the part of the donor is sufficient - and who ensures that the cells are then destroyed? What are "higher animal species" - and why? This goes far beyond a purely scientific debate. The question of what risks we as a society are willing to bear in terms of what benefits is overdue, and not just in the area of 3Rs. But with the increasing capabilities of in vitro methods, they will soon arise.

The next World Congress will be held in Canada in August 2023.