Better ways to do research: An overview of methods and technologies that can replace animals in biomedical research and testing. 2 Humane Research Australia
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Better ways to do research:
An overview of methods and technologies that can replace animals in biomedical research and testing
Monika Merkes PhD (public health) President, Humane Research Australia Honorary Associate, La Trobe University, Australia
May 2019 4 Humane Research Australia
Foreword
Why I wrote this overview “But isn’t animal research a necessary evil?” This is a common response when I mention that I am the president of Humane Research Australia. For several years now, I have wished for a publication I could recommend to people who are genuinely interested in alternatives1 to animal research2, but I have found scientific research articles hard to locate and difficult to read. Summaries of alternative methods are available, but they are usually limited to a few pages. This overview in plain English is intended to fill this gap. My sources are mostly scientific publications in peer-reviewed journals and government reports, and I provide references so that the interested reader may find out more from the original article or report3. I have summarised what I found in the scientific literature and use many quotations because I think researchers who have done the work can often explain it best. The focus here is on animal research conducted mainly for human medicine or other human “benefit”, which is often questionable. Animals are also used for studies in veterinary medicine. This overview of alternatives to animal research and testing, as well as the use of live animals in education and training, was written in late 2018 and early 2019. In this rapidly growing area of research, existing methods and technologies are being constantly improved and new ones added. It is also a very complex area that is difficult to access for a layperson. I have done my best to give a sense of the available information at the present and hope that readers may find it informative and useful.
1 The term “alternative” is used as relating 2 I use the terms animal research and animal 3 While some articles are behind paywalls, to methods that depart from or challenge experimentation interchangeably with animal authors are usually willing to provide a copy traditional norms. It is not used as offering referring to non-human animals. of their publication when contacted via email. another, equally valid possibility or choice. A contact email address is usually included in the article abstract, which is available for free. Better ways to do research 5
Acknowledgements I would like to thank the following friends and colleagues for critical and helpful comments on drafts of this document: Scott Anderson, Rob Buttrose, Dr Eleonora Gullone, Dr John van Holsteyn, Helen Marston, Reema Rattan and Cheryl Veitch. In the spirit of peer review, three experts on novel non-animal methods in research and testing have generously provided feedback on the manuscript: I am deeply grateful to Dr Andrew Worth, European Commission, European Union Reference Laboratory for Alternatives to Animal Testing; Dr Malcolm Wilkinson, Kirkstall Ltd; and Vy Tran, Center for Alternatives to Animal Testing, Johns Hopkins University, for their thoughtful suggestions. For design and layout I thank Adam Foran and his team at U-bahn. Finally, for company during the long hours I spent researching and writing this overview, I thank Sheba who patiently lay under my desk, perhaps dreaming of chasing balls in the sunshine. 6 Humane Research Australia
Contents
Foreword 4 In-silico tools for the evaluation of toxicity 42 Why I wrote this overview 4 RASAR 42 Acknowledgements 5 OECD QSAR Toolbox 42 REACHacross 42 Contents 6 Toxtree 43 Toxmatch 43 Summary 7 Other in-silico approaches for the Introduction 13 evaluation of toxicity 44 AOPs 44 What types of research use animals? 13 IATA 44 How many animals are used? 16 Computer modelling of health and disease 45 What animal welfare measures are meant to protect animals? 17 Studies with human Why do we need alternatives to animal research? 18 volunteers 48 Post-mortem studies 48 In-vitro methods 20 Population-based studies 49 3D tissues and microfluidic devices 20 Microdosing 50 Organoids 20 Organs-on-chips 22 Simulators 51 Biobanking 27 Efforts by governments and the ‘Omics’ technologies 28 scientific community to replace Stem cell technologies 29 animal experimentation 55 3D and 4D bioprinting 32 Policies and collaborations 55 Robotic testing 35 United States 55 European Union 56 In-silico methods 37 International 57 In-silico prediction methods for the evaluation of toxicity 39 Validation 58 SARs and QSARs 39 Why it matters 59 Read-across 40 PBPK models 41 Glossary 62 Expert systems 41 References 65 Better ways to do research 7
Summary
Better ways to do research: An overview of methods and technologies that can replace animals in biomedical research and testing.
Each year, millions of non-human animals4 worldwide are harmed by animal experimentation. It has been estimated that more than 115 million animals are used per year to supply the biomedical industry5. The countries that use the most animals include China, the US, Japan and Australia. Broadly, the types of research that use animals consist of a) fundamental research (also called basic research), b) applied (or human disease) research, and c) testing (or regulatory testing). There are alternatives to using animals. New – and not so new – methods and technologies that can replace live animals in research, testing, education and training include: 1. In-vitro methods (performed with microorganisms, tissues, whole cells or parts of cells in test tubes, Petri dishes etc.) 2. In-silico (computer-based) methods 3. Studies with human volunteers 4. Simulators
1. In-vitro (test tube) methods
3D tissues and microfluidic devices: Organoids, organs-on-chips Organoids are a miniature and simplified version of a (human) organ. Organoids are grown in-vitro in three dimensions. They allow researchers to study disease and treatments in the laboratory. Mini organs can also be grown on microchips. Researchers have used microchip manufacturing methods to engineer microfluidic6 culture devices that can mimic the structures and functions of living human organs.
Organoids, organs-on-chips – Useful for/ can replace animals in: Chemicals/ Regenerative Personalised/ Organ/ Large scale & Education & Provides drug testing medicine precision disease fast studies/ training access to for toxicity & & other medicine studies tests human effectiveness treatments tissues ✓ ✓ ✓ ✓ ✓
4 hereinafter referred to as animals 5 Akhtar, 2015 6 At the micro scale, often flowing through channels. 8 Humane Research Australia
Biobanking To study human cells and tissues, researchers need a readily available supply of these human biological samples. They are stored in so-called biobanks or tissue banks. Biobanks use tissue that is left over from clinical procedures such as surgery, from dead bodies, or they collect tissue specifically for research. They also store organoids.
Biobanking – Useful for/ can replace animals in: Chemicals/ Regenerative Personalised/ Organ/ Large scale & Education & Provides drug testing medicine precision disease fast studies/ training access to for toxicity & & other medicine studies tests human effectiveness treatments tissues ✓
Omics technologies The term “omics technologies” refers to areas of study in biology whose names end in “omics”, such as genomics (the study of the genome of an organism). The science of “omics” reflects diverse technologies with a focus on studies of life processes, such as comprehensive studies of genes, proteins and metabolites of an organism.
Omics technologies – Useful for/ can replace animals in: Chemicals/ Regenerative Personalised/ Organ/ Large scale & Education & Provides drug testing medicine & other precision disease fast studies/ training access to for toxicity & treatments medicine studies tests human tissues effectiveness ✓ ✓ ✓
Stem cell technologies Stem cells are unspecialised or undifferentiated cells with the ability to self-renew, and to differentiate to produce specialised cell types in the body.7
Stem cells – Useful for/ can replace animals in: Chemicals/ Regenerative Personalised/ Organ/ Large scale & Education & Provides drug testing medicine & other precision disease fast studies/ training access to for toxicity & treatments medicine studies tests human tissues effectiveness ✓ ✓ ✓ ✓
7 Stem Cells Australia, 2018 Better ways to do research 9
3D and 4D bioprinting Bioprinting involves the precise layering of cells, biologic scaffolds, and growth factors with the goal of creating bioidentical tissue for a variety of uses.8 4D bioprinting aims to create dynamic 3D patterned biological structures that can transform their shapes or behaviour under various stimuli. For example, 4D bioprinted materials are capable of changing their shape over time.9
3D and 4D bioprinting – Useful for/ can replace animals in: Chemicals/ Regenerative Personalised/ Organ/ Large scale & Education & Provides drug testing medicine & other precision disease fast studies/ training access to for toxicity & treatments medicine studies tests human tissues effectiveness ✓ ✓ ✓ ✓
Robotic testing Researchers, in particular those in the pharmaceutical industry, have developed automated methods to test biological activities of thousands of chemicals that used to be tested in animals. This is called high-throughput testing or robotic testing.
Robotic testing – Useful for/ can replace animals in: Chemicals/ Regenerative Personalised/ Organ/ Large scale & Education & Provides drug testing medicine & other precision disease fast studies/ training access to for toxicity & treatments medicine studies tests human tissues effectiveness ✓ ✓ ✓
8 Bishop, et al., 2017 9 Li, Zhang, Akpek, Shin, & Khademhosseini, 2017 10 Humane Research Australia
2. In-silico (computer-based) methods
Prediction methods and tools A range of in-silico prediction methods and tools for the evaluation of toxicity have been developed, such as structure-activity relationships (SARs) and quantitative structure-activity relationships (QSARs), read-across, physiologically-based pharmacokinetic (PBPK) models, expert systems, read-across structure activity relationships (RASAR), OECD QSAR Toolbox, REACHacross, Toxtree, and Toxmatch.
Prediction methods and tools – Useful for/ can replace animals in: Chemicals/ Regenerative Personalised/ Organ/ Large scale & Education & Provides drug testing medicine & other precision disease fast studies/ training access to for toxicity & treatments medicine studies tests human tissues effectiveness ✓ ✓ ✓
Other in-silico approaches Adverse Outcome Pathways (AOPs) provide the biological explanation for a single toxic event.10 Integrated Approaches for Testing and Assessment (IATA) are approaches for making decisions about the toxicity of substances that are based on multiple information sources.
AOPs and IATA – Useful for/ can replace animals in: Chemicals/ Regenerative Personalised/ Organ/ Large scale & Education & Provides drug testing medicine & other precision disease fast studies/ training access to for toxicity & treatments medicine studies tests human tissues effectiveness ✓ ✓ ✓
Computer modelling A computer-based model or simulation is a computer program that is designed to simulate a physical or biological system or situation. Computer models can link many processes together, something which is not possible to achieve with animal models.
Computer modelling – Useful for/ can replace animals in: Chemicals/ Regenerative Personalised/ Organ/ Large scale & Education & Provides drug testing medicine & other precision disease fast studies/ training access to for toxicity & treatments medicine studies tests human tissues effectiveness ✓ ✓ ✓ ✓
10 Taylor, 2019 Better ways to do research 11
3. Studies with human volunteers
Post-mortem studies Donated tissues after the death of a person can be studied to gain insight into cell-level changes in human illnesses, and cadavers can be used in training surgical skills. For example, post-mortem brain studies are useful to gain more knowledge about psychiatric illnesses, in particular in combination with the approaches of genomics and proteomics (the study of the structural and functional aspects of total proteins of an organism or system using high-throughput technologies).11
Post-mortem studies – Useful for/ can replace animals in: Chemicals/ Regenerative Personalised/ Organ/ Large scale & Education & Provides drug testing medicine & other precision disease fast studies/ training access to for toxicity & treatments medicine studies tests human tissues effectiveness ✓ ✓ ✓
Population-based studies Epidemiology is the study of diseases and other health-related states in groups (populations) of people, in particular how, when and where they occur. Epidemiologists want to discover what factors are associated with diseases (risk factors), and what factors may protect people against disease (protective factors).
Population-based studies – Useful for/ can replace animals in: Chemicals/ Regenerative Personalised/ Organ/ Large scale & Education & Provides drug testing medicine & other precision disease fast studies/ training access to for toxicity & treatments medicine studies tests human tissues effectiveness ✓ ✓
Microdosing Microdosing involves the administration of very low doses of a substance (sub-therapeutic). When testing a new compound or drug, microdosing can provide useful information to help decide whether the new compound or drug should be developed further, and whether it may be safe to progress to further human testing.
Microdosing – Useful for/ can replace animals in: Chemicals/ Regenerative Personalised/ Organ/ Large scale & Education & Provides drug testing medicine & other precision disease fast studies/ training access to for toxicity & treatments medicine studies tests human tissues effectiveness ✓
11 Yadav, Tanveer, Malviya, & Yadav, 2017 12 Humane Research Australia
4. Simulators Simulators are either virtual reality (VR)-based or physical model (PM)-based. Apart from replacing live animals in education and training, VR simulators have great potential for training people in remote locations, for example, training students and surgeons in developing countries.
Simulators – Useful for/ can replace animals in: Chemicals/ Regenerative Personalised/ Organ/ Large scale & Education & Provides drug testing medicine & other precision disease fast studies/ training access to for toxicity & treatments medicine studies tests human tissues effectiveness ✓ ✓ ✓
Collaborative efforts Why it matters to replace animal For a long time, the use of animals Animal researchers argue that the in research, testing, training and new methods can’t replace all areas of experimentation education has been considered a current animal research. Considering Governments, the scientific community, necessary evil. More and more, people the ethics of using animals for the industry and other stakeholders, question the ethics of this approach. purported benefit of humans and in particular in the EU and the US, At the same time, the animal research the many shortcomings of animal have started to make efforts to pool community increasingly recognises the research, this is a compelling argument knowledge and resources to replace problems with animal research: it is for speeding up the development of animal experimentation with more costly, lengthy and not very effective. human-relevant research and testing humane, more human-relevant, and Also, it may have held back the without animals. often cheaper and faster methods. This discovery of treatments and cures for We need urgent change. From an includes, for example, the development humans because they did not work well animal rights perspective, it was never of policies and tools, working together in animals. okay to inflict pain and suffering on to build large databases, developing The main alternatives to the use of animals for the real or perceived benefit plans for the further development animals in the laboratory are new in- of humans. For proponents of animal of particular technologies (such as silico and in-vitro approaches. Studies welfare, the use of animals is justified organs-on-chips), and collaborating with human volunteers and simulators as long as harm is minimised. With on the validation of new methods also play an important role. Some of awareness of the many shortcomings and technologies. these methods are used in combination of animal research and testing and for greater effectiveness. So far, increasing availability of better ways, most progress in the development of animal research is no longer justified. alternatives has been made in the area With greater investment in innovative of toxicology. The new methods and and promising non-animal methods, technologies are not yet perfect, and firm policy initiatives and robust some of the current methods that are collaborations of all interested parties, deemed to be alternatives might still better treatments and cures for human use animal parts. diseases can be developed. This will also end the suffering of millions of animals. Better ways to do research 13
Introduction
This is an overview of methods and technologies that can replace live animals in research, testing, education and training. It is divided into four sections:
• In-vitro methods (performed with microorganisms, tissues, whole cells or parts of cells in test tubes and Petri dishes) • In-silico (computer-based) methods • Studies with human volunteers • Simulators This is followed by a section on efforts by governments and the scientific community to replace animal experimentation, as well as concluding reflections on why the availability and further development of alternatives matter. A glossary is also included. But first of all, I provide a few general observations and facts about animal research.
What types of research use animals?
The types of research that use animals education and training. Individual Available statistics differ in how they are usually called fundamental or basic scientists decide what is worth describe and count procedures that research, applied or human-disease studying and whether or not they will use animals. They don’t usually explain research and testing (regulatory use animals. the purposes of animal research by testing). Basic research is curiosity these three categories. The descriptions The situation is different for the third driven and, unlike applied research, it provided by different countries also type of research, regulatory testing. is not necessarily designed to answer vary, as does the use of animals by Government regulators require that specific questions or solve practical category. But generally, basic research new consumer products, medicines, problems. It is exploratory and aims accounts for roughly half of all animals and industrial and agricultural to increase and advance scientific used in research. The three figures chemicals are tested to identify knowledge. Applied research aims (below) are examples of statistical potential dangers to human and animal to solve specific practical problems, reporting on the use of animals for health, as well as to the environment. such as using animals as a model scientific purposes. These are the most For some product types (drugs and to seek a cure for a human disease recent statistics available from the vaccines, biologicals12), animal testing or condition. Animals are also used European Union (EU), the United States includes testing for efficacy as well as in education and training – from (US) and Australia. safety (toxicity). Current laws in many preschool to postgraduate level and countries make it difficult to avoid in professional development. There using animals for regulatory testing, is no legal requirement for animal although with the development of new experimentation in basic and applied methods and technologies this has research, nor for it to be part of started to change.
12 Biologicals, or biologics, are drugs that are made from living organisms or contain components of living organisms. 14 Humane Research Australia
Purposes of experiments, European Union 2011 2% Education & training 2% Diagnosis of disease 7% Other 9% Toxicological & other safety evaluation 46% Biological studies 3% of a fundamental Production and quality nature control of products for veterinary medicine 11% Production & quality control 19% for products for human Research & medicine and dentistry development - human, veterinary, dentist Source: European Commission13
Purposes of experiments, US 2016
12% Others 9% 65% Toxicity testing Research & Development, fundamental research 14% Quality control of products and devices, production
Source: Meigs et al.14
13 European Commission, 2013, p. 6 14 Meigs, Smirnova, Rovida, Leist, & Hartung, 2018 Better ways to do research 15
Purposes of experiments, Australia 2016 0.27% Stock Maintenance 0.30% Regulatory product testing 11% 2% Stock Breeding Achievement of educational objective 48% Understanding human or animal 28% biology Environmental study
1% Production of biological products
Source: Humane Research Australia15 4% Mice and rats are the most commonly Improvement of animal management or production used animals in research. In 2011, rodents and rabbits represented 80% of all animals used in research in the EU. The second most-used group were 0.02% cold-blooded animals, such as reptiles, Diagnostic procedures amphibians and fish (12.4%) while birds accounted for 5.9%.16 However, in recent years the use of zebrafish has soared, partly due to their lower cost 6% compared to mammals. In the UK, they Maintenance and improvement of human are now the second most-used animals or animal health and welfare after mice.17
15 Humane Research Australia, 2016 16 European Commission, 2013 17 Home Office, 2015 16 Humane Research Australia
How many animals are used?
We do not know how many animals are used worldwide for research, testing and teaching purposes. Not all countries release statistics about animal use. In Australia, for instance, only four states provide statistics while in the US, the numbers of mice, rats, fish and birds used are not known because these animals are not counted. From published data and estimates we can ascertain that: • In Australia, around 9 million animals Around Just under were used in 201618 • In the EU’s 28 member states, just 9 million 11.5 million under 11.5 million animals were used in 201119 Animals used in Animals used in the • In the US, an estimated 26 million Australia (2016) 28 EU countries (2011) animals were used in 2010, with 96% of these being mice, rats, fish and birds20 • In Canada, in 2016, 4.3 million animals were used in research, teaching, and testing. The majority of animals (57.3%) were used in basic research21 • In South Korea, 3 million animals were used in 201722 • An estimate of worldwide use suggested a figure of 115.3 million animals used for research in 200523 An estimated The number of animals From what statistics are available, it appears that, overall, the number of 115.3 million used in the laboratory animals used in research has been Animals used has been constant stable in recent years24 although in worldwide (2005) over recent years some countries, it has increased. In particular, the number of genetically- altered (transgenic) mice has risen, especially in basic research.25
18 Humane Research Australia, 2016 21 Meigs, et al., 2018 24 Meigs, et al., 2018 19 European Commission, 2013 22 Meigs, et al., 2018 25 Daneshian, Busquet, Hartung, & Leist, 2015; 20 The Hastings Center, no date 23 Taylor, Gordon, Langley, & Higgins, 2008 Timoshanko, Marston, & Lidbury, 2017 Better ways to do research 17
What animal welfare measures are meant to protect animals?
In animal experimentation, researchers Animal experimentation regulations are permitted to use procedures vary around the world. Many countries that would be illegal outside of require an institutional or project the laboratory, such as artificially license before research using animals producing spinal cord injury in can be carried out. There is also usually rabbits.26 However, there are laws, a requirement for a group made up The 3Rs – regulations and codes of practice of representatives of veterinarians, that direct researchers to limit harm scientists, animal welfare organisations Replacement, to the animals they use. The most and the general public to oversee the widely accepted guidance on limiting ethical conduct of individual projects. Reduction, harms to animals in biomedical In Australia, these are called Animal research comprises the principle of Ethics Committees (AEC) and in the EU, Refinement the 3Rs: Replacement, Reduction and all states have a national committee – are meant to Refinement. The 3Rs were proposed by for the protection of animals used for protect animals William Russell and Rex Burch in the scientific purposes. In Australia, the in research testing. late 1950s and are today embedded members of AECs are usually appointed in many laws, regulations and by the institution that carries out the codes governing animal use.27 It was animal experiments. The public does summarised in a 2015 article as: not have easy access to their minutes and reports. It is generally not possible “In essence, they allow animals for a member of the public then, to to be used in scientific research assess whether the use of animals only when they cannot be replaced in a particular project could have with non-animal alternatives, been avoided because alternatives when the number of animals has were available. In other words, the been reduced as much as possible transparency of the process that given the research goals, and determines decisions on the use of The public does when procedures and housing animals is questionable.29 have been refined to minimize not have enough welfare impacts”.28 information to judge whether or how the 3Rs protect animals.
26 Leonard, Thornton, & Vink, 2014 28 Franco, 2013, p. 2 27 Franco, 2013 29 Merkes & Buttrose, 2019 18 Humane Research Australia
Why do we need alternatives to animal research?
The use of animals in research – and the On the other hand, supporters of “Cardiovascular toxicity claims the moral status of animals more generally the animal welfare position find it highest incidence and severity of – has been and remains surrounded morally acceptable to use animals in adverse drug reactions in late-stage by ethical controversy. Answers to the research as long as their wellbeing clinical development. For example, question “do we have the moral right is considered. The 3Rs represent an Vioxx (Rofecoxib), originally to use animals for our purposes?” have animal welfare position, particularly designed to treat pain related to been approached from many angles. in the requirements for refinement osteoarthritis and approved by the Broadly, there are two positions: animal and reduction. Food and Drug Administration (FDA) welfare and animal rights. Animal in 1999, was linked to over 27,000 Recent surveys indicate a growing welfare is concerned with minimising cardiovascular-related deaths concern with the suffering of animals suffering, while the animal rights and myocardial infarctions (MI). used in research, and the public is position considers the use of animals as It was withdrawn from the market increasingly questioning whether our resources to be morally wrong. in 2004, although later relicensed animals ought to be considered as for more specific indications, with Animal liberationists, for instance, call means to an end or as sentient beings implementation of regulatory for equal consideration of interests. with inherent value. In 2018, for and transparency safeguards. In They question the right of humans to instance, the Pew Research Center preliminary clinical investigations, assume that our interests must always reported that 52% of Americans the drug showed effectiveness in prevail. For example, Peter Singer noted are opposed to using animals in its target treatment and adverse 30 years ago: scientific research.31 events were not significant. It was “And the basic right that animals Ethics aside, there are other reasons not until four years of long-term should have is the right to equal why the use of animals in research clinical studies that it became consideration. This sounds like and testing for human purposes evident that the risk of heart attack a difficult idea, but essentially it should be replaced with better ways to and stroke was actually two-fold means that if an animal feels pain, do research. higher with Vioxx compared to the pain matters as much as it does the control group. Some other In-vivo32 and traditional in-vitro when a human feels pain - if that compounds, such as Micturin methods (also called test tube pains hurt just as much. Pain is (Terodiline, for urinary incontince), experiments) are not good at predicting pain, whatever the species of being Fen-phen (Fenfluramine/ therapeutic outcomes and possible that experiences it”.30 phentermine, anti-obesity side effects during clinical trials with treatment), Seldane (Terfenadine, humans. As many as 95% of drugs allergy medication), Zelnorm that appear safe and effective in (Tegaserod, for irritable bowel animals fail in humans.33 Drugs for syndrome), Meridia (Sibutramine, Alzheimer’s disease, for instance, have appetite suppressant), and a 99.6% failure rate34 while those for Darvon/ Darvocet (Propoxyphene, cancer35 and heart disease also have a analgesic drug), have all had a particularly high failure rate. A group similar record in terms of adverse of Canadian researchers made the cardiovascular effects”.36 following observation about the safety and effectiveness of specific drugs:
30 Singer, 1989, p. 1 33 e. g., Cummings, Morstorf, & Zhong, 2014; 34 Cummings, et al., 2014 31 Strauss, 2018 Marshall, Austin, Casey, Fitzpatrick, & Willett, 35 Hutchinson & Kirk, 2011 32 In vivo means “in the living”. In vivo methods 2018; Pound & Ritskes-Hoitinga, 2018; Seok, 36 Savoji, et al., 2018, p. 1 are methods using a living organism/animal. et al., 2013; Thomas, et al., 2016; Triunfol, Rehen, Simian, & Seidle, 2018; van der Worp, et al., 2010 Better ways to do research 19
How can these adverse drug reactions Opportunity cost, the loss of other be explained, given that the drugs options when one option is chosen, had to undergo safety (toxicity) presents an additional problem. Given testing? Thomas Hartung, Director of that animal research and testing has the Center for Alternatives to Animal a high failure rate, we don’t know to Testing (CAAT) at Johns Hopkins what extent new drugs that would University in the US offered the be beneficial to humans have been following explanation: overlooked, because they were harmful to animals. “Toxicology and effective risk Animals are not assessment depend on scientific In addition to producing misleading good models for and technological information and results, animal tests are also costly and should constantly adapt to these lengthy.39 The cost of drug development predicting human advances in this information. has been increasing and the number clinical outcomes However, toxicology still largely of new drugs approved every year relies on traditional assessment has been decreasing over the last two because of methods that were established decades.40 The drug development differences in decades ago and that have process takes around 10 years and has changed little despite scientific been estimated to cost up to US $2.5 physiology, and technological progress. billion.41 Economic considerations are metabolism and Consequently, safety assessments in favour of more human-relevant, are often based on tests of unknown cheaper and faster methods. other differences relevance and reliability and whose “Unlike crude, archaic animal between human predictive validity has never been tests, non-animal methods usually assessed objectively“.37 and non-human take less time to complete, cost The extensive use of animals in basic only a fraction of what the animal animals. research raises another issue. Basic experiments that they replace cost, research does not aim to result in and are not plagued with species “For decades, laboratory biologists practical outcomes and it often doesn’t. differences that make extrapolation have regarded animal models 42 For example, a group of researchers difficult or impossible”. as a necessary evil. While some examined articles in six highly cited activists decry their use on moral basic science journals over a five-year grounds, even the most practical- period. They found that fewer than minded researchers acknowledge 10% of highly promising basic science fundamental problems with them. discoveries enter routine clinical use Animals are expensive, provide 38 within 20 years. From a utilitarian only imperfect replicas of human perspective, which is often used by biology, and introduce numerous animal researchers, it can be argued variables into experiments that can that the many animal lives lost in basic be difficult or impossible to control. research do not justify the benefits. These flaws aren’t purely academic. Pharmaceutical researchers have struggled for years with late-stage development failures, in which drugs that look promising in multiple animal systems turn out to be useless or even toxic in humans. Nonhuman models have simply been the least bad tool for detailed studies on human biology”.43
37 Hartung, 2009a, p. 93 39 Meigs, et al., 2018 42 Ranganatha & J, 2012, p. 32 38 Contopoulos-Ioannidis, Ntzani, & Ioannidis, 40 Zhang, Korolj, Lai, & Radisic, 2018 43 Dove, 2018 2003 41 Ahadian, et al., 2018 20 Humane Research Australia
In-vitro methods
In-vitro methods (the Latin phrase means “in the glass”) are also commonly known as test-tube methods although this kind of research is traditionally done in flasks or Petri dishes as well as test tubes. In-vitro tests and experiments are largely performed outside living organisms and involve tissues, microorganisms, cells or other small parts of biological material. This section provides an overview of recently developed in- vitro methods that can replace experiments or tests with live animals. They include 3D tissues and microfluidic devices, such as organoids (mini versions of organs) and organs-on- chips, biobanking, omics technologies, stem-cell technologies, 3D and 4D bioprinting, and robotic testing.
3D tissues and microfluidic devices
For almost a century, scientists have Organoids grown cells, such as bacteria or human tissue, in Petri dishes. A Petri dish is Lab-grown mini organs were first Organoids have many applications. 44 a shallow glass or plastic dish with developed in 2013. They are miniature They can be used for: versions of human organs and can be a lid that is used by scientists for • Regenerative medicine – organoids grown from many different organs. microbiological studies. It contains grown from healthy tissue could be Healthy or tumour cells are taken via a culture that feeds the cells so they placed back into a patient to help a small biopsy from a person’s organ, can grow. The cells rest at the bottom repair damaged tissue. bathed in a culture that stimulates of the dish and spread out as they • Toxicity testing – toxicologists can them to grow over a few weeks or multiply in a two-dimensional (2D) use organoids to test the effects of months and to organise themselves direction. Since this is not how organs chemicals on the liver and other into mini versions of human organs. grow in a living body, scientists have human organs. developed ways in which cells can be For example, the process of growing • Drug testing – drugs can be tested grown in a three-dimensional (3D) way. a gut organoid has been described in on organoids to help predict their By studying these 3D tissues, such as three steps in the following way: effects in patients. mini organs (also called organoids) and • Microbiome studies – scientists can 1. “Take a tissue sample. A very small organs-on-chips grown from human study how normal human intestinal biopsy is taken from the epithelium, cells, the research directly translates to bacteria interact with gut organoids. the tissue lining the gut. human health and saves animals from • Modelling infections – organoids 2. Incubate. The tissue is bathed in a being experimented on. It is also often can be infected with viruses mix of growth factors designed to cheaper and faster. or bacteria to study how these let gut stem cells replicate. affect cells. 3. Organoids, a millimeter or less in
diameter, emerge in up to 3 weeks
and can be frozen for later use”.45
44 Grens, 2018 45 Sinha, 2017, p. 4
Better ways to do research 21
Brain organoid
• Personalised medicine – organoids grown from individual patients can help predict their response to new or existing drugs. • Cancer studies – scientists can study how cancer develops by introducing mutations in organoids grown from healthy tissues.46
These mini organs are not capable of reproducing all biological responses like a real human organ, but they allow researchers to study a variety of physiological responses to specific manipulations and treatments. They allow for “surrogate” trials before conducting clinical trials, “providing a vital, physiologically relevant bridge between pre-clinical investigations and clinical outcomes and bringing the possibility of personalised medicine closer”.47 Source: National Institutes of Health, US48
Inner ear organoid
Source: National Institutes of Health, US49
46 Sinha, 2017 48 NIH Image Gallery, 2019 47 Archibald, Tsaioun, Kenna, & Pound, 2018, 49 NIH Image Gallery, 2018 p. 2 22 Humane Research Australia
Organs-on-chips Lung-on-a-chip Mini organs can also be grown on microchips. Researchers have used microchip manufacturing methods to engineer microfluidic50 culture devices that can mimic the structures and functions of living human organs. These organs-on-chips are made of a clear and flexible polymer (molecules of a simple compound joined together) and contain hollow microfluidic channels lined with living human cells. Microfluidic channels contain tiny amounts of liquid ranging from submicron (smaller than one millionth of a metre) to a few millimetres. They are equipped with mechanical forces that can mimic the physical environment of organs, such as This lung-on-a-chip serves as an accurate model of human lungs to test for breathing motions (lung-on-a-chip) drug safety and efficacy. and peristalsis-like movements (intestine-on-a-chip). When nutrients, Source: National Center for Advancing Translational Sciences, US51 air, blood or drugs are added, the cells replicate some of the key functions of Human-body-on-a-chip the organ. These organs-on-chips, in which cells can grow and fluids can flow, are usually the size of a computer memory stick.
Multiple tissue chips can be connected in a system to simulate a human- body-on-a-chip.
Source: National Center for Advancing Translational Sciences, US 52
50 At the micro scale, often flowing through 51 National Center for Advancing Translational channels. Sciences, no year-d 52 National Center for Advancing Translational Sciences, 2018d Better ways to do research 23
The term organ-on-a chip was thought endocrine systems. The capabilities “Organs-on-chips are bio- up by Donald Ingber, the Founding and interactions in multiple organ-on- engineered devices that mimic Director of the Wyss Institute for a-chip systems are only in the early key aspects of the physiology Biologically Inspired Engineering at stages of development. However, and function of human organs, Harvard University.53 Together with his assuming future collaboration between replicating some of the complexity multidisciplinary team he developed organ-on-chip innovators, users, of the human body environment on in 2010 a lung-on-a-chip.54 Professor regulators and funders, the participants a microscopic scale. They are able to Ingber is often credited with having of this workshop anticipated real mimic blood, air and nutrient flow, developed the first organ-on-a-chip, patient benefits by replacing poorly as well as mechanical forces such as but similar work had been undertaken predictive animal models with these peristalsis and can be continuously some years earlier by a team from more physiologically relevant human- monitored to obtain a profile over Seoul National University and based models. time. Organs-on-chips enable the Cornell University.55 study of basic biological processes, Organ-on-chip technology is the modelling of diseases and Organs-on-chips use different types of still mostly used in-house by the investigation of the effects of drugs. human cells: primary cells (cells directly companies and laboratories that They can potentially identify safety taken from an organ or a tissue), developed it. However, researchers in and efficacy issues earlier and more immortalised cell lines (cells that have academic institutes and biochemical, reliably in the drug development been modified by chemicals or a virus pharmaceutical, cosmetics and process, enabling the design and in order to survive and stay active chemical companies have started to use selection of drug candidates that indefinitely), or stem cells.56 the technology, and it can also be used are more likely to succeed in human by hospitals for personalised medicine. Organ-on-a-chip platforms that have clinical trials”.60 Researchers have also developed already been developed include liver, disease models, such as chips with Organ-on-chip technology is skin, vascular structures (such as tumour cells to study cancer. still quite new and there is little arteries, veins, capillaries), cardiac standardisation: “Each team is muscle, skeletal muscle, lung, bone and “Chip-based in vitro organ models developing its own approach, with bone marrow, brain, eye, gut, spleen are ranked 6th among the top ten its own unique technology. The and kidney. There are also multi-organ- emerging technologies by the World players are mainly start-up companies on-a-chip platforms that mimic the Economic Forum in 2016, thus commercializing prototypes developed interplay between different organs.57 highlighting the potential of organ- in the local universities”.61 It is a fast on-a-chips to improve lives and A recent workshop attended by experts developing industry. transform the health care system. from industry, academia and the In other words, organ-on-a-chip Medicines and Healthcare products technology is expected to speed up Regulatory Agency (MHRA) held in pharmaceutical drug development Liverpool UK identified the advantages efforts, improve translation of organ-on-chip technologies: of basic research to clinically “These dynamic and responsive relevant patient scenarios and biological test platforms have the provide personalized intervention potential to revolutionise drug strategies”.59 target identification and validation Organs-on-chips can replace many studies without the need for animal different species of animals currently Organs-on-chips models. This will improve compound used for research, such as mice, rats, efficacy, safety and targeted have great rabbits, guinea pigs and non-human drug delivery”.58 primates, which are all now used for potential for The experts also identified the drug testing and vaccine development. personalised technical, funding and regulatory This technology has great potential challenges still to be overcome. Some for pharmaceutical research and medicine, of the technical challenges include personalised medicine. Apart from something not physically-relevant cell interactions, saving the lives of millions of animals, possible with scaling ratios between organs, it is also cheaper and faster than and incorporation of immune or animal experiments. animal testing.
53 Zhang, et al., 2018 56 Ahadian, et al., 2018 59 Rothbauer, Rosser, Zirath, & Ertl, 2019, p. 84 54 Huh, et al., 2010 57 Ahadian, et al., 2018 60 Archibald, et al., 2018, p. 2 55 T. H. Park & Shuler, 2003 58 Haddrick, et al., 2018, p. 4 61 Wilkinson, 2019, p. 648 24 Humane Research Australia
Mini guts for Brain-on-a-chip on drugs personalised treatment A brain-on-a-chip has been dosed with the street drug crystal meth (crystal of people with cystic methamphetamine, also called “ice”). Crystal meth is a stimulant that speeds up fibrosis the messages moving between the brain and the rest of the body. Researchers from the US, Sweden and Israel connected three chips with different types of Cystic fibrosis is a genetic condition brain cells to model the blood-brain barrier and then added crystal meth to that primarily affects the lungs and observe how the drug affects the brain. They were able to observe previously digestive system. There is currently unknown interactions between blood vessels and neurons in the brain. no cure for the condition, which is caused by mutations in the CFTR “The human brain, with its 100 billion neurons that control every gene that affect the function of the thought, word, and action, is the most complex and delicate organ in the CFTR protein. There are drugs for body. Because it needs extra protection from toxins and other harmful the treatment of cystic fibrosis, but substances, the blood vessels that supply the brain with oxygen and different drugs work for different nutrients are highly selective about which molecules can cross from the mutations of the CFTR gene. Not all blood into the brain and vice versa. These blood vessels and their unique drugs work for all patients. network of supporting pericyte and astrocyte cells comprise the blood- brain barrier (BBB). When the BBB is disrupted, as happens with exposure Researchers in the Netherlands to drugs such as methamphetamine (‘meth’), the brain’s sensitive neurons have shown how these drugs could become susceptible to harmful damage”.64 be tested for individual patients. They took rectal biopsies from Many different species have been used in brain research. While rats and mice are patients, grew them into mini guts, more likely to be used to study simple cognitive functions, non-human primates and tested the available drugs for have been subjected to invasive brain research because scientists believe their effectiveness. They showed that brain processes are more similar to those of humans. With the development the drug responses observed in the of brains-on-chips, many complex processes in the brain can now be studied mini guts could be used to predict without inflicting pain and suffering on animals. The new methods are also faster 65 which cystic fibrosis patient would and cheaper. respond to which drug. This test can help to quickly identify the best drug therapy even when patients carry very rare CFTR mutations.62 Cystic fibrosis research has been carried out on different species of animals, for example, mice, pigs and ferrets.63 But these “animal models” can’t predict which drug therapy will work for an individual patient with cystic fibrosis.
Blood-brain barrier on a chip
The blue dye shows where the brain cells would go, and the red dye shows the route for blood circulation.
Source: National Center for Advancing Translational Sciences, US66
62 Dekkers, et al., 2016 64 Brownell, 2018 66 National Center for Advancing Translational 63 Lavelle, White, Browne, McElvaney, & 65 Maoz, et al., 2018 Sciences, no year-b Reeves, 2016 Better ways to do research 25
Placenta-on-a-chip Skin-on-a-chip Scientists at a university in Vienna Microchips can test pharmaceuticals and cosmetics on human skin. A team have created a placenta model in Singapore has developed a credit-card sized device that can overcome on a microchip made up of two a limitation of traditional skin culture systems, which use cell cultures on a areas: one represents the mother, collagen matrix that easily shrinks. Instead, the team developed a new method the other represents the foetus. A that prevents skin contraction, using a fibrin69-based matrix. Their microfluidic gelatin-based material was used to chip has several chambers. This allowed the researchers to grow skin in the provide a structure to which human device and conduct tests without having to transfer the skin. umbilical-vein endothelial cells67 and The chip is made of thermoplastics70 and can be mass produced. This skin-on- cells from the placenta were added. a-chip is suitable for high-throughput screening. The researchers wrote that it Many studies show that conditions has “enormous potential to revolutionize many pharmaceutical, toxicological, of the mother, such as diabetes and cosmetic applications, including safety and efficacy, which currently rely on and high blood pressure, can have animal testing”.71 an effect on the unborn child. Cosmetics have been tested on many species of animals, but perhaps best known Compared with other research is the Draize test. In 1944, John Henry Draize (1900-1992) and his colleagues at methods, this can now be studied the US Food and Drug Administration (FDA) developed Draize rabbit irritation in greater detail on the placenta- tests for identifying and evaluating toxic reactions when test materials are on-a-chip. The researchers who in contact with the skin, penis, and eyes. The tests were originally used for developed this chip intend to use evaluating the safety of cosmetics and then extended to include insecticides, it to study how nutrients such as sunscreens and antiseptics. glucose are transferred from the mother to the foetus, specifically in Draize tests are painful and many animals are killed after the tests. These tests situations that involve a health risk have been criticised because of large variations in test results and because of for the foetus.68 differences in the skin and eyes of humans and rabbits.72 This placenta on a microchip models the human situation much better than animal experiments. It can also be used to study conditions in individual patients, something that is not possible with animal models.
67 Cells from the umbilical cord. 70 Thermoplastics are types of plastic materials 71 Sriram, et al., 2018, p. 338 68 Mandt, et al., 2018 which become soft when they are heated 72 Lee, Hwang, & Lim, 2017 69 Fibrin is a protein. and hard when they cool down. 26 Humane Research Australia
Spinal cord nerve and blood vessel cells on a tissue chip
Source: National Center for Advancing Translational Sciences, US73
Heart-on-a-chip (as compared to a dime) Kidney-on-a-chip
Source: National Center for Advancing Translational Sciences, US75
Source: National Center for Advancing Translational Sciences, US74
73 National Center for Advancing Translational 74 National Center for Advancing Translational 75 National Center for Advancing Translational Sciences, 2018f Sciences, 2018c Sciences, no year-c Better ways to do research 27
Biobanking
To study human cells and tissues, for lab use. Another obstacle is the researchers need a readily available long-standing cultural reliance on supply of these human biological animal models, which can make samples. They are stored in so-called researchers resistant to change, biobanks, which are simply tissue often because of concerns about banks. Biobanks use tissue left over historical data compatibility and from clinical procedures such as losing ground in a competitive surgery and from dead bodies, or they environment while new approaches collect tissue specifically for research. are embedded in lab practice”.77 Biobanks also store organoids. However, these obstacles can be “Organoid biobanking overcome by improving infrastructure and collaboration. The collection of is a promising and tissue also raises ethical issues, such exciting new field with as donor consent. Unless the tissue considerable potential samples are used for personalised medicine, they are de-identified for scientific research, for biobanking. But complete de- precision medicine, identification may not always be and regenerative possible and a de-identified sample medicine”.76 can’t be used to benefit the donor.
While biobanking has great potential, there are also obstacles: “A number of key factors limit the wide adoption of non-animal human tissue models in cancer research, including deficiencies in the infrastructure and the technical tools required to collect, transport, store and maintain human tissue
76 Bredenoord, Clevers, & Knoblich, 2017 77 Jackson & Thomas, 2017 28 Humane Research Australia
‘Omics’ technologies
The term “omics technologies” refers “Traditional toxicology evaluates to areas of study in biology whose end points such as death, disease Metabolomics approach names end in “omics”, such as or observable changes in the genomics. There are too many omics organism or cells of the organism, to identify gum disease areas of study to list here. The most while ‘omics’ measurements are in its early stages common omics include: made across multiple levels of Periodontitis is an advanced gum biological organisation and provide • Genomics – the field of science disease that involves inflammation information that may be used to focusing on the structure, function, of the gums and the supporting understand cellular processes evolution, mapping, and editing of structure of the teeth. If untreated, as an integrated system rather genomes.78 it leads to the loss of teeth. The than as a collection of disparate • Metabolomics – the study of the condition is very common and often measurements”.83 set of metabolites79 in an organism, diagnosed late when substantial cell, or tissue. It holds great promise damage has already occurred. It is Using several of these omics for precision medicine. Small preventable when diagnosed early. approaches together can help scientists numbers of metabolites have learn about toxicity pathways in human A group of researchers in France been used for decades to diagnose cells and study the underlying cause tested saliva from people with metabolic diseases, such as the of disease. and without periodontitis, using development of blood glucose nuclear magnetic resonance test strips in the 1950s to test for “The growing use of ‘omics’ (NMR) spectroscopy together with diabetes. With metabolomics, much technologies (e.g. transcriptomics, multivariate statistical analysis to larger numbers of metabolites than proteomics and metabonomics) identify the metabolic signature of are presently covered in standard in combination with in vitro test active periodontitis.85 They looked clinical laboratory techniques systems allows a comprehensive for a range of metabolites and can be examined.80 This allows analysis of the impact of a found that a combination of lactate, for personalised diagnosis and chemical at the molecular level GABA, and butyrate predicted the treatment of individual patients. and can indicate potential toxicity presence of periodontitis. The • Proteomics – the large-scale study of pathways that may lead to adverse study showed that this simple and proteins, particularly their structures health effects”.84 non-invasive method could be used and functions. for early diagnosis and follow-up of • Transcriptomics – the study of periodontitis. messenger RNA (ribonucleic acid) molecules produced in an individual Rats, mice, hamsters, rabbits, ferrets, or population of a particular sheep, pigs, cats, dogs and non- cell type.81 human primates have been used for modelling human periodontal Recently, a number of beliefs about our diseases and treatments. Of understanding of human biology have these animal species, non-human been called into question following primates and dogs are considered studies using omics approaches. to be closest to humans in their Such beliefs include the idea that anatomy and the way in which the genome is static throughout an dental disease develops.86 This organism’s lifetime, that it is identical new method developed by the in all cell types, and that all of the researchers in France is more necessary information for cellular accurate than animal models. function is contained within the gene sequence.82
78 A genome is the genetic material of an 81 Our genetic material is encoded in DNA. RNA 83 McBride, 2017, pp. 69-70 organism. is similar to DNA, but has another function: 84 European Commission, 2018b 79 Metabolites are small molecules necessary it communicates genetic information to the 85 Rzeznik, et al., 2017 for metabolism. rest of the cell. 86 Struillou, Boutigny, Soueidan, & Layrolle, 80 Clish, 2015 82 McBride, 2017 2010 Better ways to do research 29
Stem cell technologies
When cells are used to test drugs or multipotent. Embryonic stem cells, other substances, it is important that derived from a small group of cells Parkinson’s disease they are reliable representatives of in the early embryo (5-7 days), and cells or cellular systems in the human iPS cells are undifferentiated and identified as a possible body. Many traditional in-vitro methods are considered pluripotent as they autoimmune disease that have been used for decades, such can become every type of cell in A group of researchers at a German as growing cells in a two-dimensional the body. university has gained new insights way on a Petri dish, do not meet these What are induced pluripotent stem into the development of Parkinson’s criteria as they are using immortalised cells (iPS)? disease by using a stem cell cell lines or isolated primary cells. approach. The researchers had Recently, scientists discovered that Immortalised cells87 can be grown observed an unusually high number a mature fully specialised cell, for indefinitely and they are easily cloned of T cells93 in the midbrain and example a human skin cell, in the and cost effective, but they are the blood of Parkinson’s patients. right conditions could be induced genetically altered and may react in These T cells – a type of T cell to mimic the characteristics of an different ways to cells that have not similar to those found in people embryonic stem cell. These are been altered. Isolated primary cells with autoimmune diseases – attack known as induced pluripotent stem are cells from human or animal tissue. and kill nerve cells that produce cells (iPS cells)”.89 They are of varying quality and may dopamine in the midbrain. not react in a consistent fashion.88 Stem Induced pluripotent stem cells were With this observation in mind, the cells are a promising alternative source first developed in 2006 through genetic researchers took small skin samples of human cells for toxicity testing, reprogramming of adult cells.90 The from healthy people and people studying and treating disease. Nobel Prize in Medicine in 2012 was with the disease, and differentiated awarded for the discoveries that cells Stem Cells Australia, an initiative these cells into stem cells that can be reprogrammed to become that links Australia’s leading experts function like midbrain nerve cells. pluripotent stem cells, and that cells in bioengineering, nanotechnology, These were then brought into from individual patients could be stem cell biology, advanced molecular contact with fresh T cells from the harvested and reprogrammed to analysis and clinical research, people who had donated the skin become any tissue type found in the explained these technologies in the samples. The researchers found human body.91 following way: that the stem cells from people with The use of stem cells has great potential Parkinson’s disease killed a large “What type of stem cells are there? for studying and treating diseases.92 number of their nerve cells, but Stem cells can be divided into two In theory, there is no limit for the healthy people’s cells did not react broad groups: tissue specific stem type of diseases that could be treated in this way.94 cells (also known as adult stem with stem cell methods. However, cells) and pluripotent stem cells new methods have to be tested first (including embryonic stem cells to ensure they are safe and effective. and iPS cells). Tissue specific stem Some stem cell therapies are already cells are derived from, or resident being used in cancer treatments and in, adult tissues, and can usually bone marrow transplantation. Human only give rise to the cells of that stem cells are also now being used to tissue, thus they are considered test drugs.
87 cells that continue to divide indefinitely 90 Brevini, et al., 2016 93 T cells are a type of white blood cells. 88 McBride, 2017 91 Rothbauer, et al., 2019 94 Friedrich-Alexander Universitaet Erlangen- 89 Stem Cells Australia, 2018 92 Triunfol, et al., 2018 Nuernberg, 2018; Sommer, et al., 2018 30 Humane Research Australia
Human stem cells could potentially Neural stem cells in 3D culture models be used as a renewable source of replacement cells and tissues to treat Scientists at the University of North Carolina at Chapel diseases such as macular degeneration, Hill (UNC) determined the anti-tumour effects of neural 99 spinal cord injury, stroke, burns, heart stem cells in 3-D culture models. Stem cell aggregates, or disease, diabetes, osteoarthritis and spheroids (blue in the images below), were placed next to rheumatoid arthritis.95 Stem cell banks brain cancer cell spheroids. Fluorescent images captured over are being established worldwide and seven days showed that the stem cell therapy decreased the the global market for human-induced volume of cancer spheroids. The researchers are working to pluripotent stem cell technology advance the therapy towards first-in-human clinical trials. looks promising.96 The use of embryonic stem cells has Neural stem cells in 3D culture models raised questions about the ethics of their use, as each embryonic stem cell line has been grown from a human embryo created through in-vitro fertilisation (IVF) or through cloning technologies.
EBiSC In Europe, a large public-private partnership project has set up biobanks for induced pluripotent stem (iPS) cells, the European Bank for induced pluripotent Stem Cells (EBiSC).97 EBiSC anticipates that its capacity will be 10,000 cell lines. At the time of writing, EBiSC’s online catalogue included more than 800 different cell lines and an additional 240 were in the process of being tested for quality.
The use of human stem cells can replace many experiments on animals, in particular “humanised mouse 100 models”, that is mice that have been Source: National Center for Advancing Translational Sciences, US engrafted with human cells and tissues to give them human diseases. Although scientists have been able to cure cancer in mice for decades98, these animals are still being used to study human cancers.
95 National Institutes of Health, 2016 98 Cimons, Getlin, & Maugh, 1998 100 National Center for Advancing 96 Archibald, et al., 2018 99 Neural stem cells are cells that generate Translational Sciences, 2018e 97 European Bank for induced pluripotent Stem the neurons and glia of the nervous Cells, 2018 system during embryonic development. Better ways to do research 31
Glomerulus chip with Using induced Human pluripotent stem stem cells to study pluripotent stem cells cells for modelling liver kidney disease to understand the development and disease Researchers from the Wyss Institute development of cancer in Pluripotent stem cells can help for Biologically Inspired Engineering children scientists understand liver disease at Harvard University have much better than traditional Reprogramming of human cells that developed a protocol to grow kidney methods, such as studies using mice. can be easily obtained from biopsies cells from induced pluripotent stem Mice are different from humans or blood samples to induced cells (iPS cells) within a microfluidic in diet, genetics, gene expression pluripotent stem (iPS) cells can help organ-on-a-chip to build a human and physiology. Mouse studies as a prognostic tool and to discover kidney glomerulus chip that mimics require time and are unsuitable familial cancers early. Patient- the structure and function of the for high-throughput methods. In derived iPS cells from a tumour can kidney glomerular capillary wall. contrast, iPS cells can be expanded also help develop specific drugs for The glomerulus chip can be used to indefinitely and differentiated that individual child.102 study human kidney development, into liver-like cells that have many function and disease. It has the “The discovery of iPS cells functional characteristics of human potential to be used in regenerative opens up a wide spectrum of livers. Mutations can be introduced medicine as a cell therapy for people possible future applications through genome engineering to help with kidney diseases.101 including development of new researchers study liver development treatments in regenerative or to model rare liver diseases. medicine, generation of better Studying liver disease, such as and more accurate disease hepatitis A and C, and disease models, and improving drug development using iPS cells can be discovery. Where other tools undertaken with semi-automated, fail, using patient-specific iPS high-throughput methods that cells for modeling diseases make it possible to screen a wide have an incredible potential to range of pathways and functions improve our understanding of simultaneously.104 basic mechanisms operating during healthy and diseased human development and differentiation”.103
101 Musah, Dimitrakakis, Camacho, Church, 102 Navarro, Susanto, Falk, & Wilhelm, 2018 104 Heslop & Duncan, 2018 & Ingber, 2018 103 Navarro, et al., 2018, pp. 7-8 32 Humane Research Australia
3D and 4D bioprinting
Bioprinting, or tissue printing, is one of layer by layer from a 3D model but and shape the biomaterials in a desired the technologies of tissue engineering “3D bioprinting involves the use of form. The scaffold materials can be and regenerative medicine (TERM), cell-laden bioinks and other biologics natural or synthetic. which involves researchers from to construct a living tissue while 3D “Bioprinting is no longer confined different disciplines, such as medicine, printing technologies do not use cells to a process for combining one engineering, biology and chemistry. In or biologics”.107 cell type with one material; recent years, they have achieved much 3D printing technologies were the emphasis today is to use a progress in TERM.105 originally designed for non-biological variety of material types to create Three-dimensional (3D) printing was applications, such as metals, ceramics bespoke scaffolds onto which first conceived in 1986 and has since and thermoplastic polymers. The chemical cues can be tethered and influenced fields such as engineering, process used organic solvents, high multiple cell types can be printed manufacturing and medicine.106 While temperatures or materials that are with precision”.109 there are different technologies of 3D not compatible with living cells and printing, it basically involves processes biological materials. A challenge for where materials or liquids are joined, TERM scientists then, is to keep the layer by layer, under computer control, cells alive during the printing process. to create three-dimensional objects. For 3D bioprinting, the researchers had to find materials and printing processes The terms 3D printing and 3D that are compatible with living cells and bioprinting have different meanings. tissues.108 They also had to find suitable Both processes build a 3D object materials to build scaffolds that contain
3D image of bioprinted skin tissue 3D printed eye tissue
Source: National Center for Advancing Translational Sciences, US110 Source: National Center for Advancing Translational Sciences, US111
105 K. M. Park, Shin, Kim, & Shin, 2018 108 Murphy & Atala, 2014 111 National Center for Advancing Translational 106 Bishop, et al., 2017 109 Mehrban, Teoh, & Birchall, 2016, p. 13 Sciences, no year-a 107 Vijayavenkataraman, Yan, Lu, Wang, & Fuh, 110 National Center for Advancing Translational 2018, p. 2 Sciences, 2018a Better ways to do research 33
Researchers have developed many movement controlled by a computer This technique has been used to different approaches to 3D bioprinting, code/images/CAD files, forming a produce vascular networks with including: 3D structure as the build stage is micrometer precision on biological translated vertically building the gels in vitro”.113 • Laser-based bioprinting – laser object layer by layer.”112 energy is used to pattern bioinks A disadvantage of laser-based laden with cells. “In laser-based writing, which bioprinting is its slow printing speed • Droplet-based bioprinting – cell- consists of a laser beam, a substrate, which makes this method unsuitable laden bioinks are ejected out of the and a focusing system, cells are for printing of large tissues or organs. nozzle in the form of droplets. confined in a laser beam and “In inkjet printing, which consists • Extrusion-based – bioink is pushed deposited in a steady stream on of a reservoir tank, an orifice, and out of the nozzle using pneumatic nonabsorbing surfaces, including a print head, a pressure is created pressure or mechanical force by biological gels. As such, cells in the tank, which pushes the ink means of a piston or screw. This is the can be printed continuously in the orifice and out to the printer most widely used type of bioprinting. and accurately without causing head. As a result, cell droplets Extrusion-based bioprinting has been significant cell death. Laser-based are deposited on a surface, which used to bioprint cells, tissues and writing can pattern cells with high provides the advantage of limiting organ-on-a-chip devices for tissue resolution up to the micrometer contact of cells and materials on engineering, cancer research, drug scale and is advantageous over a surface. Other advantages of testing and transplantation. other bioprinting techniques as it inkjet printing include controllable • Stereolithography bioprinting – can be used with many materials resolution, high printing speed, and where a “layer of photopolymer resin and does not directly bring the relatively low material costs”.114 is cured (or polymerized) by light live cells into contact with the (usually UV) irradiation, the light substrate, improving cell survival. Inkjet printing is the most popular printing method. It is fast, cheap and readily available. The disadvantages include print-head clogging, mechanical stress and unreliability in Organovo bioprinter bioink dispensing.115 “Microextrusion printers use pneumatic or mechanical (piston or screw) dispensing systems to extrude continuous beads of material and/or cells”.116 This method has already been used to produce, for example, aortic valves117 and an ovarian cancer disease model.118 But it has disadvantages, too. They include limited spatial resolution and, as high pressures are used, part of the living material may not survive the printing process.119
Source: National Center for Advancing Translational Sciences, US120
112 Vijayavenkataraman, et al., 2018, p. 11 115 Mehrban, et al., 2016 118 Xu, et al., 2011 113 Ahadian, et al., 2018, p. 19 116 Murphy & Atala, 2014, p. 775 119 Mehrban, et al., 2016 114 Ahadian, et al., 2018, p. 20 117 Duan, Hockaday, Kang, & Butcher, 2013 120 National Center for Advancing Translational Sciences, no year-e 34 Humane Research Australia
A research group from Singapore Different cell types can be used for Current 3D bioprinting technologies described the typical steps involved in bioprinting. Stem cells are particularly have several limitations. Some bioprinting in the following way: appealing “as they are pluripotent techniques don’t allow for the mixing and able to differentiate into other cell of different types of cells or make such “A typical bioprinting process types upon exposure to the correct mixing very difficult, for instance, consists of three major steps namely physical and chemical guidance cues. high temperatures or voltages pre-processing, processing and Within the human body, there are a may compromise the living cells or post-processing. Preprocessing number of viable sources of stem cells, tissues. “Thus, there is always a trade involves imaging of the tissue or such as the bone marrow, periosteum between resolution, compatibility organ using computed tomography and adipose tissue”.122 with cell deposition, cell viability (CT), magnetic resonance imaging as well as mechanical stability and (MRI), and ultrasound imaging Bioprinting has many applications in no one of the existing 3D printing/ techniques and reconstruction biomedical research and the health- bioprinting methods is able to provide of 3D models from the imaging. care sector, such as disease modelling, all advantages”.126 The generated 3D models are drug discovery and testing, high- then converted into STL file throughput screening and regenerative 4D printing, which is a more recent format, which is a commonly medicine. Bioprinting has already development, can overcome some accepted file format by most of the contributed to much progress in skin of the challenges associated with commercially available bioprinters. and cartilage repair.123 3D printing. 4D bioprinting has been The processing step starts with described as 3D bioprinting with the “Advanced biofabrication harvesting primary cells from added dimension of time127, or shape techniques, such as 3D printing patients, culturing and expanding it transformation over time.128 and 3D microfabrication can also ex vivo for the bioprinting process. be applied for the creation of tissue The new printing technique has Though cancer cell lines and other models, and organoid technologies been defined as 3D printing of cell- non-human cells are being used, can explore stem cells for the laden materials in which the printed the ideal condition for fabricating formation of organ- like structures structures would be able to respond transplantable living tissues would to accurately and simultaneously to external stimulus or internal cell be to use the patient’s own cells. capture multiple aspects of forces.129 Simply, 4D bioprinted Suitable bioinks with properties human physiology”.124 materials are capable of changing their mimicking the intended tissue to be shape over time. While 4D printing is printed are selected and the cells are “Bioprinting has a great potential still in its infancy, it has great potential suspended in these bioinks. The cell- to solve this ever-increasing organ for tissue engineering in many areas. laden bioinks are then fabricated shortage crisis. Though bioprinting The current demand for donor into required 3D living tissue/ of fully functional organs has a long organs to replace damaged or lost organ according to the 3D model way to go, considerable progress organs is higher than the availability using a bioprinter. Post-processing has been made to realize the greater of donor organs, for instance, and involves maintaining the bioprinted goal of organ printing. Bioprinted 4D printing could one day provide tissue/organ in a bioprinter for tissues could be used as in vitro individualised organs or part-organs tissue maturation before being testing beds in place of animal to fulfil that demand. Current attempts transplanted into patients or used testing. Given the ethical concerns to genetically engineer animals such as in vitro models for disease surrounding animal testing and the as pigs to become organ donors for modelling, or drug testing”.121 high cost involved, bioprinting is a humans will then be redundant. viable alternate. In pharmaceutical research, bioprinting could be used “The significantly enhanced as in vitro models for testing of drug usability and functionality of the efficacy, toxicity, chemotherapy bioprinted objects due to their or chemo-resistance to reduce the capability to transform with high cost and shorten the time of time will likely find widespread drug discovery”.125 applications in areas including but not limited to tissue engineering, regenerative medicine, bioelectronics, robotics, actuators, and even medical devices”.130
121 Vijayavenkataraman, et al., 2018, p. 3 124 Zhang, et al., 2018, p. 258 127 Ashammakhi, et al., 2018 122 Mehrban, et al., 2016, p. 12 125 Vijayavenkataraman, et al., 2018, p. 2 128 Li, et al., 2017 123 Mehrban, et al., 2016 126 Ionov, 2018, p. 1 129 Ashammakhi, et al., 2018 130 Li, et al., 2017, pp. 4-5 Better ways to do research 35
A 3D bioprinted Robotic testing vascularised liver tissue model for drug testing Researchers, particularly those in “In the United States, Tox21 is the pharmaceutical industry, have a multi-agency collaboration An international collaboration of developed automated methods to to research, develop, validate, researchers has developed and 3D test biological activities of thousands and translate chemical testing bioprinted a vascularised liver tissue of chemicals that used to be tested in methods in order to characterize model that allows them to mimick animals. This is called high-throughput chemical toxicity pathways in [sic] drug diffusion and accurately test or robotic testing. interest of protecting public health. whether a drug is toxic to humans. This initiative became official “The last two decades have seen A vascularised tissue is a tissue through the establishment of a innovations in technology that with vessels, especially blood Memorandum of Understanding have helped to evolve automated, vessels. Drug diffusion refers to the between four major agencies: (1) microprocessor controlled robotic movement of a substance or drug the US Environmental Protection processes called High Throughput from an area of high concentration Agency (EPA)/Office of Research and Screening (HTS). This qualitative to an area of lower concentration. Development, (2) the US National leap in drug discovery paradigm Diffusion is an important process Institutes of Health (NIH), National has been achieved via a synergy in living organisms. It occurs in Institute of Environmental Health of chemistry, biology, engineering liquids and gases, and allows Sciences/National Toxicology and informatics”.133 substances to move in and out of Program, (3) the NIH/National cells. The researchers noted that In the US, high-throughput testing Human Genome Research Institute/ this model could be expanded and is being supported by a government Chemical Genomics Center, used to model other organs for drug initiative called Toxicology in the 21st and (4) the US Food and Drug testing, such as the heart, kidney Century (Tox21). It is a collaboration of Administration (FDA). A central 131 and bone. several government agencies that aims component of this initiative is to “to develop better toxicity assessment employ high throughput screening methods to quickly and efficiently test assays and genome analytical Bioprinting can and has already been whether certain chemical compounds methods to identify mechanisms of used in combination with organs-on- have the potential to disrupt processes chemical induced biological activity, chips. Although it is difficult to recreate in the human body that may lead to prioritize over 10 000 chemicals complex bodily functions, the models 134 negative health effects”. for more extensive toxicological that are created with organ-on-chip and evaluation, and develop bioprinting technologies allow for drug computational predictive models of testing and the study of physiological in vivo biological response. … The functions in-vitro. The combination major objective of Tox21 is to deliver of both technologies can build tissues biological activity profiles that are faster than organ-on-chip technology predictive of in vivo toxicities for alone. It creates high throughput in the the thousands of substances that production of organs-on-a-chip, and it are being studied over the 5-year helps researchers to develop new types collaboration”.135 of organ-on-a-chip.132
131 Massa, et al., 2017 133 Ranganatha & J, 2012, p. 30 135 Valerio, 2014, pp. 1026-1027 132 J. Y. Park, Jang, & Kang, 2018 134 United States Environmental Protection Agency, 2018b 36 Humane Research Australia
The US Environmental Protection Testing of nanomaterials is an area Agency (EPA) contributes to Tox21 The Deepwater Horizon that would greatly benefit from high- through its computational toxicology oil spill in the Gulf of throughput testing. Manufactured research (CompTox), which can nanomaterials (materials with at evaluate chemicals for potential risks Mexico and ToxCast least one dimension <100 nm143) and quickly and at a small cost. These The 2010 BHP oil disaster in the nanoparticles (all three dimensions high-throughput screening assays Gulf of Mexico was the largest <100 nm) are used in many consumer can outperform the predictive ability marine oil spill in the history of the products, but their effects on human of models built with animal toxicity petroleum industry. To break up health are not well known. While some data.136 A key part of EPA’s CompTox is the oil slick, more than 1.5 million high-throughput testing methods of its Toxicity Forecaster (ToxCast). Data gallons of the oil spill dispersant nanomaterials are in development, generated by ToxCast are publicly Corexit 9500 was used. Very little they have not yet been validated.144 available online. They complement the was known about the effects of High-throughput testing is also used European Chemicals Agency (ECHA) the dispersant chemicals on acute with organs-on-chips. This technology database and contain “results from or long-term toxicity in humans or is at present still too expensive for several thousands of in vitro tested marine animals. When the US EPA’s widespread use but work is underway chemicals, measuring hundreds of Office of Research and Development to make its pricing more competitive so endpoints each”.137 ToxCast is a useful was asked to evaluate the potential that it can replace conventional in-vitro resource to inform read-across .138 139 toxicity of oil spill dispersants, the and in-vivo models.145 researchers needed a fast testing “Tox21 has established a method. They decided to use high- library of ~10,000 chemicals throughput in-vitro tools that are for the production phase of the part of the EPA’s ToxCast program.141 program, including the NCATS No animals were used in these Pharmaceutical Collection (NPC), tests. Indeed, animal tests could not which contains drugs used in have provided such quick and low- the clinic. This library has been cost results. screened against 47 cell-based assays in a quantitative high- “ … we were able to detect specific throughput screening (qHTS) format bioactivities in complex chemical generating nearly 70 million data mixtures for time-sensitive points to date”.140 environmental issues and using high throughput screening assays. This is exciting given that one of the challenges of real world chemical toxicity testing is the fact that humans and other organisms are often exposed to complex mixtures, rather than the pure single compounds that are the subject of typical toxicity testing. The in vitro tests used in this study rapidly profiled the complex dispersant formulations without the use of animals, and screened for potential endocrine activity, other endpoints and cytotoxicity. In different circumstances, a similar rapid screening effort could be used to make time-sensitive decisions based on potential hazard and risk”.142
136 Archibald, et al., 2018 140 R. Huang, et al., 2018, p. 2 144 Collins, et al., 2017 137 Maertens, Hubesch, & Rovida, 2016 141 Judson, et al., 2010 145 Probst, Schneider, & Loskill, 2018; van 138 Chesnut, et al., 2018 142 Judson, et al., 2010, p. 5984 de Burgwal, van Dorst, Viëtor, Luttge, & 139 See section on in-silico methods for detail 143 One nanometre (nm) is equal to one Claassen, 2018 on read-across. billionth of a meter. Better ways to do research 37
In-silico methods
Progress in information technology has enabled the development of computer-based (in-silico146) methods for biomedical research and the testing of chemical and biological substances. To date, most progress with in-silico methods has been made in the area of toxicology (the study of the adverse effects of chemical substances on living organisms). Many computational methods have been developed to predict toxicity, and can thus play a role in replacing and/or reducing animal testing. The main prediction methods are described in this section. Computer modelling of health and disease is another area where in-silico methods can replace the use of animals.
“Chemical products are used in making The development of new drugs and “In silico methods for toxicology 95% of all goods”.147 Chemical and pesticides is lengthy and extremely apply modern computing biological substances, such as newly expensive, as the following statements applications and informatic developed drugs, need to be tested to show: technologies as scientific tools determine whether they are harmful to to advance and gain efficiency “Therapeutic development is a humans, animals or plants. But there to improving our understanding costly, complex and time-consuming is limited information about most of toxicity potential, pathways, process. The average length of time substances because testing is very hazards, and risks of chemical and from target discovery to approval expensive. The US Toxic Substance biological substances. Multiple of a new drug is about 14 years. The Control Act inventory includes 83,000 computer technologies and failure rate during this process chemicals, but has data for only 3% of methodologies serve to store, exceeds 95 percent, and the cost per these.148 Safety testing reportedly costs interface, process, or transmit successful drug can be $1 billion US$10 – 20 million per product and information which enable scientific or more”.150 takes several years. It is also difficult to advancements in the toxicological obtain toxicological data for the 1,000 “The probable range confronting sciences through implementation new chemicals created every year.149 developers of new pesticide of predictive models, databases, chemicals appears to be $750,000 detection systems, and simulations to $3.25 million – but the trend is processes”.152 constantly upward. On average it The in-silico prediction methods for costs €2.2 billion and takes 10 years the evaluation of toxicity outlined for a new active substance to be below cover the most common brought to market according to the methods and models but they do European Trade Association”.151 not represent a comprehensive list. It is opportune, then, that computers Several organisations and companies can help researchers in the safety have produced software packages for Computer-based sciences by performing large amounts predicting toxicity or physicochemical of complex calculations at great properties of chemicals. In general, they testing and speed to reveal patterns, trends and contain one or more predictive models. research associations. In silico methods have Rapid progress is being made in this many applications, as a scientist from area and software, models and datasets methods are the US Food and Drug Administration are being constantly updated.153 booming in observed: the toxicology sciences.
146 In silico – performed on a computer or via 148 Maertens & Hartung, 2018 151 Meigs, et al., 2018, p. 289 computer simulation. 149 Chesnut, et al., 2018 152 Valerio, 2014, p. 1026 147 Meigs, et al., 2018, p. 286 150 National Center for Advancing Translational 153 Myatt, et al., 2018 Sciences, 2018b 38 Humane Research Australia
The silico methods for predicting and Computer-based approaches for the study of evaluating the toxicity of chemical and biological substances described in this Parkinson’s disease section include: Parkinson’s disease (PD) is an age-related neurodegenerative disease whose • Structure-activity relationship (SAR) cause or causes are largely unknown and for which a cure has not yet been found. and quantitative structure-activity During the 1950s, it was discovered that the psychiatric drugs reserpine and relationship (QSAR) modelling haloperidol induce Parkinsonian-like symptoms in humans. That discovery led • Read-across to the drugs becoming some of the first used to develop animal models. That • Physiologically-based is, these drugs were given to animals to simulate Parkinson’s disease. Decades pharmacokinetic (PBPK) models and many animal models later, no animal model can fully explain the disease in • Expert systems humans: This is followed by a selection of tools: “More than 60 years have been spent attempting to produce PD-like symptoms in various animal species as a model to study disease • Read-across structure activity pathogenesis and treatment. In addition to rodents and nonhuman relationships (RASAR) modelling primates, Caenorhabditis elegans (roundworms), Drosophila • REACHacross melanogaster (fruit flies), fish (zebrafish and goldfish), and anurans • Toxtree (frogs and toads) have been used. However, each animal model generally • Toxmatch demonstrates only specific subsets of PD characteristics, and no single There are also some approaches that model recapitulates all the known pathological processes associated with are not considered a method or a tool: PD in humans”.155 • Adverse Outcome Pathways New approaches are needed that focus on humans, not on animals. These (AOPs) - a conceptual approach include in-vitro and in-silico approaches. In-vitro approaches consist of, for to representing knowledge about example, methods that use stem cells. In-silico approaches include computer toxicity mechanisms simulations, mathematical algorithms and machine learning to predict • Integrated Approaches to Testing and interactions between molecules and pathways within biological systems.156 Assessment (IATA) Much information about Parkinson’s disease exists already, for example in the Lastly, this section provides an Organization for Economic Cooperation and Development (OECD) iLibrary overview of computer modelling for the on Adverse Outcome Pathways.157 With the help of information technology, study of human organs and the body, existing knowledge can be pooled and shared. The OECD, the US Environmental and how this can be used in clinical Protection Agency, and the European Union Joint Research Centre are already settings154. The use of computers for collaborating on a shared resource “that covers the broad spectrum of biological virtual reality and surgical simulation, pathways that are likely to be involved in human health and ecological risk mainly for purposes of education and assessment, the Adverse Outcome Pathway Knowledge Base”.158 training, will be covered in the section on simulators.
154 Obviously, such modelling can also be used 156 Marshall & Willett, 2018 158 Marshall & Willett, 2018, p. 1956 for the study of non-human animals. 157 Bal-Price, et al., 2018 155 Marshall & Willett, 2018, p. 1952 Better ways to do research 39
In-silico prediction methods for the evaluation of toxicity
SARs and QSARs to make the prediction. Quantitative have pointed out that “many QSARs are parameters enable the development difficult to interpret and cannot be used SARs and QSARs are mathematical of quantitative models. QSARs are to define causal links. They represent models used for predicting biological mathematical models derived from a correlation rather than causation”.167 activities of chemicals. training set of example chemicals. “The Progress in computer-based methods training set includes the chemicals that “A SAR is a qualitative relationships has seen improvements in dealing were found to be positive and negative [sic] that relates a (sub)structure with incomplete data sets and making in a given toxicological study … or to to the presence or absence of a models less complicated: property or activity of interest. induce a continuous response … that 161 The substructure may consist of the model will predict”. “Recent advances in machine learning have resulted in models adjacently bonded atoms, or an The models can be used to predict a that can handle missing data and arrangement of non-bonded atoms qualitative or quantitative endpoint.162 model multiple targets at once that are collectively associated with “It is assumed that chemicals that 159 (multi-label learning, in case of the property or activity”. fit the same QSAR model may work toxicology for example multiple- through the same mechanism”.163 For over a century, scientists have had hazard learning). These models can some knowledge of the relationship QSAR models have been used, for sometimes outperform single-label between the structure of a substance example, for skin sensitisation, eye models by increasing the available and its toxicity. The knowledge of this irritation and other human health data for training and by transferring structure-activity relationship (SAR) hazards. They require a large database concepts applicable to one label to became well known, but was rarely with information on the structure and predictions on another label. Multi- gathered and systematically recorded toxicity of substances. label models have the potential to until the 1980s. With progress in simplify the QSAR space. Rather In 2007, changes in European information technology, this changed: than having a model for every legislation on chemical substances chemical property, a single model “Since the early 1990s, the concept explicitly promoted the use of in-silico can predict many different chemical of applying compilations of models, in particular (Q)SARs164, as an properties. In toxicology, many structural alerts to evaluate and alternative testing method to evaluate hazards are interrelated; thus, they predict toxicity has progressed the safety of regulated substances: rapidly, with the expansion to can inform each others’ predictions. other endpoints and effects. … “The legislation permitting the For example, a skin irritant is likely The development of compilations acceptance of in silico models to also an eye irritant, which means of structural alerts has also been evaluate toxicity is Regulation that information on both labels stimulated by the availability of EC 1907/2006, better known synergizes. So, the prediction of one software that can capture them, and as Registration, Evaluation, hazard (label) informs other labels allow users to apply that knowledge. Authorisation, and Restriction of for the same and similar chemicals Bearing in mind that, until the late Chemical substances (REACH). can improve predictions”.168 REACH specifies conditions for the 1980s, the alerts were available only For all in-silico predictions of toxicity, use of in silico (Q)SAR models, and on paper, e.g. in journal articles or (Q)SAR and other models, standardised the European Chemicals Agency book chapters, they were difficult to protocols need to be developed. (ECHA) located in Helsinki, Finland use and open to misinterpretation, “Such novel in silico toxicology (IST) implements REACH. ECHA offers and may have been applied protocols, when fully developed 160 guidance on how the (Q)SAR in silico differently by different users”. and implemented, will ensure in method can help fulfill or support silico toxicological assessments Quantitative structure-activity information requirements“.165 relationship (QSAR) modelling uses are performed and evaluated in a one or more quantitative parameters So many different QSAR models are consistent, reproducible, and well- derived from a chemical structure to now available, that the proliferation of documented manner across industries a property or activity of interest. The these models has been referred to as and regulatory bodies to support 166 term “quantitative” in QSAR refers to a “zoo of QSAR” , and this can make wider uptake and acceptance of the 169 the nature of the parameter(s) used it difficult to choose the best one for approaches”. the task at hand. Further, researchers
159 European Chemicals Agency, 2008, p. 10 163 Raies & Bajic, 2016, p. 156 166 Luechtefeld, Rowlands, & Hartung, 2018, p. 740 160 Cronin & Yoon, 2018, p. 291 164 SARs and QSARs are collectively referred to 167 Luechtefeld, Rowlands, et al., 2018, p. 739 161 Myatt, et al., 2018, p. 6 as (Q)SARs. 168 Luechtefeld, Rowlands, et al., 2018, p. 741 162 European Chemicals Agency, 2008 165 Valerio, 2014, p. 1027 169 Myatt, et al., 2018, p. 2 40 Humane Research Australia
Read-across Read-across is a method that is Read-across is fast and cost-effective, usually based on chemical structure but it relies on subjective assessments information and uses QSAR of what constitutes a “similar” approaches.170 For example, the OECD substance. When comparing chemical QSAR Toolbox171 can be used to support information for read-across, small read-across. The read-across method changes in the structure of chemicals uses data from a substance for which can result in big differences in the level Read-across toxicity information is available, of toxicity. This can lead to prediction is a fast and to make predictions for a similar errors. Adding other information, such substance about which not much is as data from in-vitro tests, can improve cost-effective known. In other words, a data-rich the accuracy of the prediction.175 Thus, method for substance is used to make toxicity knowledge of biological similarity predictions for a structurally similar but enhances read-across. An international analysing data-poor substance. team of researchers offers the following chemicals. outlook: While read-across is currently based 172 on chemoinformatic approaches, it “The increasing availability substances analysed for the report, is not considered a QSAR: “although of biological data via the data 89% had at least one data endpoint read-across is informed by chemical sharing depositories will augment where an alternative to animal studies structure, it is strictly based on local such support of read-across and was used. similarity of a chemical with similar grouping by big data. The curation chemicals”.173 of such datasets and the respective At present, regulatory agencies in data-sharing by companies, the EU consider read-across the “Read-across uses data on one organizations and individual best method in the areas of skin and or more analogs (the “source”) to 178 researchers needs to be further eye irritation. “Read-across is an make a prediction about a query encouraged and possibly furthered innovative approach that can be compound or compounds (the with some incentives”.176 considered an alternative to animal “target”). Source compounds are testing – and at the moment it is identified that have a structurally In the EU, manufacturers and probably the most effective method of or toxicologically meaningful importers have to register information reducing the use of lab animals”.179 The relationship to the target on chemical substances (that are authors of a review of in-silico methods compound, often underpinned by produced or imported in volumes for the prediction of chemical toxicity an understanding of a plausible over one tonne a year) in a central summarised the practical applications biological mechanism shared database at the European Chemicals of the read-across method and between the source and target Agency (ECHA) in Helsinki. This is provided examples of tools used: compounds. The toxicological part of the REACH regulation system: experimental data from these registration, evaluation, authorisation “Read-across was applied to predict source compounds can then be and restriction of chemicals. ECHA’s carcinogenicity, hepatoxicity, used to “read-across” to the specific 2017 report on the use of alternative aquatic toxicity, reproductive target compound(s). Read-across is methods revealed that the most toxicity, skin sensitization, and an intellectually-derived endpoint- common alternative method on environmental toxicity. Examples of specific method that provides analysing substances was read- tools implementing read-across are justification for why a chemical is across (63%), followed by weight of The OECD QSAR Toolbox, Toxmatch, similar to another chemical (with evidence (combining information ToxTree, AMBIT, AmbitDiscovery, 180 respect to chemical reactivity, from different sources), and QSAR AIM, DSSTox, or ChemIDplus”. toxicokinetics, mechanism/mode of predictions (34%)177. Much of the Read-across prediction is strengthened action, structure, physicochemical information is still based on new or old by the availability of high quality properties, and metabolic profile)”.174 experimental studies using animals, data, as well as agreed principles and but ECHA reported that out of the 6,290 guidance on how to group chemicals.181
170 Zhu, et al., 2016 174 Myatt, et al., 2018, p. 7 180 Raies & Bajic, 2016, p. 152 171 OECD, 2018c 175 Chesnut, et al., 2018; Zhu, et al., 2016 181 Ball, et al., 2016; Patlewicz, et al., 2014 172 Chemoinformatics is focused on extracting, 176 Zhu, et al., 2016, p. 178 processing and extrapolating meaningful 177 European Chemicals Agency, 2017b data from chemical structures. 178 Archibald, et al., 2018 173 Chesnut, et al., 2018, p. 414 179 Maertens, et al., 2016, p. 324 Better ways to do research 41
PBPK models Expert systems Physiologically-based Expert systems are a varied group from the literature or from an pharmacokinetic182 (PBPK) models are of models covering a combinations analysis of data sets generated by “mathematical representations of the of SARs, QSARs and databases.187 scientists. Structural alerts are absorption, distribution, metabolism They derive toxicity predictions and defined as molecular substructures and elimination (ADME) of chemicals estimates from a range of in-silico that can activate the toxicological in humans or other animal species. models. With access to large chemical effect or mechanism. … The purpose They are used for multiple purposes, and toxicological databases, increasing of an in silico expert review is including the interpretation of in computational power, statistical to evaluate the reliability of the vitro toxicity data by in vitro to in vivo algorithms for structure-activity prediction. The outcome of the extrapolation (IVIVE) and the simulation modelling and powerful datamining review provides information to of internal concentrations in the tools, this area of research and testing include in the assessment of the organism of interest”.183 has seen much progress in recent years toxicological effect or mechanism. and has become increasingly relevant As part of this review, the expert “Physiologically-based and important for risk assessment might agree with, or refute, pharmacokinetic (PBPK) models 189 required by government regulators.188 individual in silico predictions”. are computational systems In an overview of in-silico toxicology now commonly used in drug methods, expert systems were development and increasingly in described as follows: regulatory toxicology. They predict the absorption, distribution, “Expert rule-based (or expert/ metabolism and excretion (ADME) structural alerts). This methodology of substances in the body, at uses structural rules or alerts different doses. PBPK models based to make predictions for specific on human rather than animal toxicological effects or mechanisms characteristics avoid the problem of toxicity. These rules are derived of species differences. They can also help predict variations in susceptibility between individuals and at different developmental life stages, which commonly occur but cannot be properly addressed by conventional animal testing. Functional PBPK models have developed alongside the rapid advances made in the use of human in vitro systems and in understanding gene function. It is now possible to predict ADME outcomes in ‘virtual humans’ with increasing confidence”.184 In short, PBPK models are translational tools that can be used to link in- vitro and in-silico toxicity estimates to conditions in a living organism. Ready to use PBPK software tools are available.185 PBPK models are also more generically referred to as PBK (physiologically based kinetic) models.186
182 Pharmacokinetics refers to the movement 183 European Commission EU Science Hub, 187 European Chemicals Agency, 2008 of drugs into, through and out of the 2018a 188 Slikker, et al., 2018 body (drug absorption, distribution, 184 Langley, 2012, p. 24 189 Myatt, et al., 2018, p. 6 metabolism and elimination). 185 Cronin & Yoon, 2018 186 for example, Paini, et al., 2019 42 Humane Research Australia
In-silico tools for the evaluation of toxicity
RASAR OECD QSAR Toolbox The REACH registration requirement predict toxic properties of any chemical The OECD QSAR Toolbox194 is a large, has resulted in the ECHA database, substance more accurately than animal curated database developed by the a large database with information tests. Toxic substances were correctly European Chemicals Agency and the about thousands of chemicals. ECHA predicted in 89% of cases. They OECD. It is freely available online.195 The expected 60,000 registrations in 2018. estimated that by using the RASAR data OECD also provides training in the use This information is publicly available, fusion method, they would have saved of this resource, whose most important but it is not presented in a standardised 2.8 million animals and US $490 million features are described as: way that would make it easy to be read testing costs, and would have received • “Identification of relevant structural by a computer. A group of researchers more reliable data. characteristics and potential has used natural language pattern “It has recently been demonstrated mechanism or mode of action of a matching to make the information that machine-learning software target chemical. machine-readable. They found, for combined with big data can now • Identification of other chemicals example, that many chemicals have be used to create sophisticated that have the same structural been repeatedly tested on animals: read-across-based tools that characteristics and/or mechanism or “Interestingly, many chemicals greatly outperform animal studies mode of action. have been tested more than once, in predicting chemical safety, • Use of existing experimental data to some shockingly often: For example, with an accuracy of 80%–95%, fill the data gap(s)”.196 one of the often challenged animal compared to 50%–70% for the 191 tests is the Draize rabbit eye test, respective animal tests”. REACHacross where for more than 70 years now, In addition to avoiding animal testing test chemicals are administered Building on other in-silico methods, and obtaining superior results, RASAR into rabbit eyes. Two chemicals such as read-across and QSAR, and the can help avoid duplication of testing, were tested more than 90 times, availability of large databases, a group achieves higher throughput, and is 69 chemicals were tested more of scientists added machine-learning faster than conventional testing. But at than 45 times. … Notably, the 9 techniques. “A first implementation of this stage, it can’t be used for complex most frequently done animal tests machine learning-based predictions human health effects, such as cancer. analyzed here consumed 57% of termed REACHacross achieved At the time of writing, RASAR research all animals for toxicological safety unprecedented sensitivities of >80% had just been published, and it is not testing in Europe 2011”.190 with specificities >70% in predicting yet clear whether regulators will accept the six most common acute and After making the information machine- this new testing method. Also, other topical hazards covering about two readable, they combined the ECHA researchers will carefully examine thirds of the chemical universe”.197 database with several other large the claims made about RASAR.192 However, this new tool has not yet databases that are publicly available Thomas Hartung, one of the team that been independently validated. The and developed algorithms that enable developed RASAR, commented that: software tool is, however, available to automatic read-across to model “In the future, a chemist could help companies to meet their REACH chemical properties. They named check RASAR before even requirements. As the REACHAcross™ their innovation read-across structure synthesizing their next chemical website notes, activity relationships—RASAR. to check whether the new structure “it predicts 8 required endpoints With RASAR, the researchers mapped will have problems. Or a product for REACH submissions. Users can the relationships between chemical developer can pick alternatives produce Toxicology Assessment structures and toxic properties, based to toxic substances to use in Reports for 6 key human health on 74 characteristics (such as whether their products. This is a powerful endpoints, including skin a substance will cause eye irritation) to technology, which is only starting sensitization, acute dermal predict the properties of a substance. to show all its potential”.193 irritation, acute eye irritation, acute They found that this method can oral toxicity, acute dermal toxicity, mutagenicity and 2 key ecotoxicity endpoints of acute aquatic toxicity and chronic aquatic toxicity”.198
190 Luechtefeld, Marsh, Rowlands, & Hartung, 193 Hartung, 2018 196 OECD, 2018c 2018, p. 199 194 OECD, 2018c 197 Luechtefeld, Rowlands, et al., 2018, p. 732 191 Archibald, et al., 2018, p. 3 195 Ford, 2016 198 Underwriters Laboratories, no year 192 for example, Alves, et al., 2019 Better ways to do research 43
Toxtree Toxmatch Toxtree is a free in-silico tool “that places chemicals into “Toxmatch is an open-source software application that categories and predicts various kinds of toxic effects by encodes several chemical similarity indices to facilitate the applying decision tree approaches. The software is made grouping of chemicals into categories and read-across. freely available as a service to scientific researchers and The core functionalities include the ability to compare anyone with an interest in the application of computer- datasets based on various structural and descriptor-based based estimation methods in the assessment of chemical similarity indices as well as the means to calculate pair wise toxicity”.199 The Joint Research Centre of the EU Commission similarity between compounds or aggregated similarity of a commissioned the development of the software, and compound to a set. various contributors have since collaborated in its further development. The software is made freely available as a service to scientific researchers and anyone with an interest in the application of Toxtree tool computer-based estimation methods in the assessment of chemical toxicity”.201
Toxmatch tool
Source: European Commission EU Science Hub200
Source: European Commission EU Science Hub202
US EPA’ s GenRA tool helps predict whether a chemical is toxic Current read-across relies on a subjective assessment of information about one drug to make predictions about another drug that is similar. Researchers at the US EPA have developed an automated read-across tool called Generalized Read-Across (GenRA) that aims to encode many expert assessments to make read-across a more systematic and data-based method of making predictions about the toxicity of specific drugs: “In its current form, GenRA lets users find analogues, or chemicals that are similar to their target chemical, based on chemical structural similarity. The user can then select which analogues they want to carry forward into the GenRA prediction by exploring the consistency and concordance of the underlying experimental data for those analogues. Next, the tool predicts toxicity effects of specific repeated dose studies. Then, a plot with these outcomes is generated based on a similarity-weighted activity of the analogue chemicals the user selected. Finally, the user is presented with a data matrix view showing whether a chemical is predicted to be toxic (yes or no) for a chosen set of toxicity endpoints, with a quantitative measure of uncertainty”.203 This read-across resource is freely available on the EPA’s website.
199 European Commission EU Science Hub, 201 European Commission EU Science Hub, 203 United States Environmental Protection 2016 2018b Agency, 2018a 200 European Commission EU Science Hub, 202 European Commission EU Science Hub, 2016 2018b 44 Humane Research Australia
Other in-silico approaches for the evaluation of toxicity
AOPs An adverse Outcome Pathway (AOP) the AOP Wiki, Effectopedia and two “Integrated Approaches to Testing is a conceptual framework for risk other AOP platforms together.210 The and Assessment (IATA) are a flexible assessment. It provides a biological following quote comments on the tool for chemical safety assessment, explanation for a toxic event,204 and has importance of AOP development: based on the integration and been described in the following way: translation of data derived from “The safety evaluation of multiple methods and sources. In “An AOP is a sequence of events environmental and industrial addition to traditional in vitro and that starts by a chemical effect at chemicals is currently propagating in vivo tests, IATA are increasingly the molecular level (termed a the concept of adverse outcome incorporating new approach Molecular Initiating Event) and pathways (AOPs). These methods, such as high-throughput progresses through changes developments have repercussions screening and high-content imaging (termed Key Events) in cells, with drug development and safety methods, along with computational tissues, and organs to produce an assessments of drugs. Under approaches that are used as a means adverse effect in the body. AOPs the umbrella of Organisation of data generation, interpretation act as a bridge between emerging for Economic Co-operation and and integration”.213 methods of safety testing and, Development (OECD) and closely ultimately, what happens in the linked to their chemical safety “Models are no longer applied in body in response to a particular testing guideline program, AOPs isolation to determine chemical substance”.205 are organizing in a crowdsourcing safety; there has been a growing movement the mechanistic global trend towards the AOPs are designed to enable targeted, knowledge on how chemicals development and use of multiple fast, low-cost and tailored assessments. impact on human health and strands of information within They can be continually improved, the environment. This happens, Integrated Approaches to Testing as new information is added to the e.g. in the AOP-Wiki and for and Assessment (IATA) for safety pathways. “The eventual goal is to more quantitative AOPs in the assessment. Nearly all IATA involve create a network, or web, of pathways Effectopedia platform. This is the use of existing data, (Q)SAR that is sufficiently well described that organizing existing knowledge on predictions and/or read-across that the eventual effects of a chemical can shared molecular initiating events are amenable to being integrated be predicted with a limited spectrum of and the subsequent key events into computational workflows”.214 molecular information”.206 along the chain from cellular The OECD, which evaluates to tissue, organ, organism, and international chemical testing population effects”.211 As this approach includes subjective protocols, launched a program on the expert judgement (weight-of-evidence), development and review of AOPs in IATA it is not easy to standardise across 2002. The new AOPs are available on industry sectors and countries. the AOP Wiki207, which allows scientists Integrated Approaches to Testing and However, IATA frameworks for anywhere in the world to share, Assessment (IATA) are approaches skin irritation, skin corrosion, eye develop and discuss their knowledge for making decisions about the irritation, serious eye damage and of AOPs. Taking the comments on toxicity of substances that are based skin sensitisation have already been the AOP Wiki into account, the OECD on multiple information sources, adopted internationally.215 publishes the endorsed AOPs on its such as physicochemical properties, website. These published AOPs can be non-testing methods such as QSAR updated on the Wiki.208 Effectopedia209, models and read-across, and testing an online encyclopaedia of AOPs, is methods such as in-vitro and in-vivo. another example for the collaborative The identification of an AOP is a development and review of AOPs. The key component of this approach.212 OECD’s Adverse Outcome Pathway IATA approaches are mainly used for Knowledge Base (AOP-KB) brings regulatory purposes. IATA work and are used in the following ways:
204 Taylor, 2019 208 OECD, 2018a 212 Clippinger, et al., 2018 205 Ram, 2019, p. 367 209 OECD, 2016 213 Worth & Blaauboer, 2018, p. 301 206 Marshall & Willett, 2018, p. 1956 210 OECD, 2019 214 Cronin, Madden, Yang, & Worth, 2019, p. 40 207 AOP-Wiki team, no year 211 Hartung, 2017b, p. 1 215 Casati, 2018; Zuang, et al., 2017 Better ways to do research 45
Computer modelling of health and disease
Computer models are also used “… complex in silico models have airway management, mechanical to simulate (virtual) organs or the been applied to pathophysiological ventilation and cardiopulmonary human body, and to explore various problems to provide information bypass/ventricular support aspects of diseases. Computer models which cannot be obtained devices”.220 can link many processes together, practically or ethically by traditional Other applications of computer something which is not possible to clinical research methods. These modelling include virtual reality and achieve with animal models. For experiments have led to the surgical simulation. These will be example, atherosclerosis is a common development of significant insights covered in the section on simulators. cardiovascular disease that is caused in subject matters ranging from by a combination of factors and pure physiology to congenital heart can be studied with the help of surgery, obstetric anaesthesia computer modelling: “As a disease that depends on multiple factors operating on Computer modelling in planning and performing different length scales, the surgery in children and young adults with congenital natural framework to apply to atherosclerosis is mathematical heart disease and computational modelling. A Congenital heart disease (CHD) is the most common form of birth defect in the computational model provides US and Canada. Nearly all forms of CHD lead to long-term complications, and the an integrated description of the cardiovascular autonomy of patients varies. Hence, there is great interest and disease and serves as an in silico potential in the application of biomedical engineering (BME)-based modelling experimental system from which and simulations to predict the outcomes of surgical and other interventions. we can learn about the disease and develop therapeutic hypotheses”.216 For example, advanced imaging and 3D printing technology allows the surgeon to print a 3D model of an individual patient’s heart and then perform surgery Computer modelling can integrate on the printed model. Computational fluid dynamics (CFD) involves numerical electrical and mechanical processes analysis to solve problems that involve fluid flows, such as blood flow. into electromechanical models. “Application of advanced CFD analyses on a patient’s preoperative anatomy and These models are useful to study, for on the proposed postoperative solution allows a higher level of confidence that example, implanted cardiac devices the intended approach will yield the desired result”.221 A medical doctor reported 217 such as pacemakers. Modelling of the his observations regarding the opportunities of computer modelling for his work respiratory system is another area of as follows: study.218 Such models can be created for individual patients. Computer “Despite the intensity of the work involved, surgical planning with modelling makes an important computational modelling offers unique insights and tremendous contribution to the discovery of new potential for the care of patients with CHD. Several centres have dedicated knowledge. Below, a team of experts laboratories working in this field, and a few multicentre collaboratives in anaesthesiology219 comment on this have formed. Congenital cardiology is moving into an era of ‘personalized contribution from their perspective: medicine,’ and surgical planning with computational modelling and CFD offers hope that each patient’s interventional procedure can be planned and analyzed preoperatively on the basis of their patient-specific characteristics to ensure optimal long-term outcomes”.222
216 Parton, McGilligan, O’Kane, Baldrick, & 219 A medical speciality that encompasses 220 Colquitt, Colquhoun, & Thiele, 2011, p. 499 Watterson, 2016, p. 562 anaesthesia, intensive care medicine, critical 221 Slesnick, 2017, p. 1160 217 Pluijmert, et al., 2015 emergency medicine, and pain medicine 222 Slesnick, 2017, p. 1168 218 Clark, Kumar, & Burrowes, 2017 46 Humane Research Australia
Computational psychiatry In-silico drug trials can “Translating advances in neuroscience into benefits for patients with be more accurate than mental illness presents enormous challenges because it involves both animal testing the most complex organ, the brain, and its interaction with a similarly A group of computer scientists complex environment. Dealing with such complexities demands powerful at the University of Oxford have techniques. Computational psychiatry combines multiple levels and demonstrated that in-silico drug types of computation with multiple types of data in an effort to improve trials are more accurate than animal understanding, prediction and treatment of mental illness”.223 tests at predicting clinical pro- Computational psychiatry, similar to other computational predictive approaches, arrhythmic cardiotoxicity, where a includes two methods: data-driven analysis uses methods from machine drug causes an irregular heart beat learning, such as statistics, to improve classification of disease, predict treatment or stops the heart. The researchers outcomes or improve treatment selection; and theory-driven approaches tested 62 compounds at multiple “mathematically specify mechanistically interpretable relations between concentrations and found accuracy variables (often including both observable variables and postulated, theoretically rates of 89-96%, compared to 75- meaningful hidden variables)”.224 Unlike data-driven approaches, theory-driven 85% accuracy from animal testing. methods incorporate prior knowledge or hypotheses. Both approaches can be Different species of animals, such combined. Some of the advantages and limitations of computational psychiatry as rats, mice, rabbits, guinea pigs, are noted below: dogs and pigs, are still used in drug development to predict side effects “Data-driven approaches have started to bear some fruit for clinically for the human heart.226 relevant problems, such as improving classification, predicting treatment response and aiding treatment selection. These approaches, however, are limited in their ability to capture the complexities of interacting variables in and across multiple levels. Theory-driven modeling efforts, on the other RepoTrial hand, have yielded key insights at many levels of analysis concerning the processes underlying specific disorders, but for the most part have yet to Developing a new drug takes 10 be applied to clinical problems”.225 to 15 years and is very expensive. Drug repurposing, that is finding a different use for an already approved drug, is faster, costs less, and requires fewer or no animal tests. One reason for this is that potential side effects of the drug are already known. The RepoTrial project uses computer-based algorithms to find out whether already registered drugs may work for other diseases that have some similar characteristics, but may relate to different organs or body parts. The drugs will be tested on virtual patients, and finally on real patients.227
223 Huys, Maia, & Frank, 2016, p. 404 225 Huys, et al., 2016, p. 411 227 RepoTrial, 2018 224 Huys, et al., 2016, p. 404 226 Passini, et al., 2017; Passini, Rodriguez, & Benito, 2018 Better ways to do research 47
Machine learning can The Virtual Physiological predict individual cancer Human project patients’ responses to In 2005, a small group of researchers chemotherapy drugs and officers from the European Commission started the Virtual Researchers at Georgia Institute Physiological Human (VPH) project of Technology in the US have which a few years later resulted in developed algorithms to predict the establishment of the Virtual the response of individual cancer Physiological Human Institute for patients to chemotherapy drugs. Integrative Biomedical Research They used their algorithms to predict (VPH Institute). In 2011, the VPH the responses of 152 individual Institute announced its three targets: cancer patients to chemotherapeutic drugs, based on gene expression “1. Digital patient. The VPH profiles of each individual’s tumour, for the doctor; patient-specific with an accuracy of over 80%.228 The modeling to support medical new tool is available to clinicians at decisions. no cost and can help them choose 2. In silico clinical trials. the chemotherapy drug best suited The VPH for the biomedical for individual patients. industry; collections of patient- “Accurate predictions in cancer specific models to augment biology, as in all areas of science, the preclinical and clinical can be based upon established assessment of new biomedical cause-and-effect relationships products; in silico technologies or upon significant correlations for the reduction, refinement, and detected in large sets of relevant partial replacement of animal data. While we are well on our and human experimentation. way to the day when we may 3. Personal health forecasting. fully understand the molecular The VPH for the patient/ causes of all cancers and treat citizen; subject-specific real- them accordingly, we are not time simulations, based on there yet. One promising interim data collected by wearable and solution is the application of environmental sensors, that prediction algorithms derived provide advice to individuals from ML-detected correlations affected by conditions requiring between the molecular profiles careful self-management or of large numbers of cancers and to people simply at risk of associated responses to a variety developing certain diseases”.230 of therapeutic drugs”.229 The VPH Institute is an international non-profit organisation whose collaborators envisage that VPH models will enable predictive and personalised medicine. Personalised predictions will support and improve clinical decisions and help with personalised prevention.231
228 C. Huang, et al., 2018 230 Viceconti & Hunter, 2016, p. 107 229 C. Huang, et al., 2018, p. 5 231 Viceconti & Hunter, 2016 48 Humane Research Australia
Studies with human volunteers Studies with human volunteers are not new. Examinations of cadavers have been conducted for centuries, though with modern technologies the methods have become more sophisticated. Population-based studies appeared first during the 19th century, and microdosing goes back several decades.
Post-mortem studies
Donated tissues after the death of a “Diagnosing ‘depression’ in “Indeed, one of the major limitations person can be studied to gain insight a monkey is at odds with the to our knowledge of human into cell-level changes in human successful ongoing process of neurological diseases resides illnesses. For example, the brains of clarifying psychiatric diagnoses partly in the limits inherent to stroke victims can be studied after by using DSM criteria. It is not animal models, which mimic some death232 and cadavers can be used in possible to ascertain feelings of aspects of the human neurological training surgical skills.233 Such studies worthlessness and excessive guilt, disorder without reproducing can replace studies on living non- indecisiveness, and thoughts its complexity arising from human primates and other animals. of death from observations of both genetic and environmental monkeys”.235 236 factors. For example, more than Animal models are also used in 50 different animal models behavioural tests, such as the despair Post-mortem brain studies are useful to have been generated to explore test – where rats or mice are forced to gain more knowledge about psychiatric Alzheimer’s disease (AD) and swim until they are exhausted and stop illnesses, in particular in combination more than 20 models are available swimming, then antidepressants are with the approaches of genomics and for the study of schizophrenia administered to see if this prolongs proteomics. For example, post-mortem without clear consensus about the the time they are able to swim – to tissue from the prefrontal cortex of similarities with human disease. study stress, anxiety, depression, and people who lived with schizophrenia The underuse of post-mortem obsessive-compulsive behaviours. have led to new insights about this human brain tissue also impedes Researchers who conduct these tests disease.237 Some advantages of the deeper understanding of the assume that the animals accurately studying post-mortem tissues are pathophysiological processes mimic human psychiatric conditions. reported below: ongoing in the diseased brain”.238 However, the human brain’s architecture and physiology is much Brain tissues available for research more complex than that of rodents or are kept in brain banks where they monkeys:234 are processed (for example, frozen) by standard procedures. Most brain banks keep tissues donated by individuals or their families, but very few have specimens from longitudinal cohort studies239.240
232 Gordon & Langley, 2008 236 Diagnostic and Statistical Manual of Mental 238 Gomez-Nicola & Boche, 2015, p. 1 233 Thomson, Poudrier, Stranix, Motosko, & Disorders (DSM). It defines and classifies 239 Repeated observation of groups of people Hazen, 2018 mental disorders for the purposes of over long periods of time. 234 Menache, 2012 diagnosis, treatment and research. 240 Paraizo Leite & Tenenholz Grinberg, 2015 235 Menache, 2012, p. 5 237 Lewis, 2002 Better ways to do research 49
Population-based studies
Epidemiology is the study of diseases • Case control studies – Epidemiological studies can replace and other health-related states in observational studies comparing animal studies. For example, if groups (populations) of people, in patients who have a disease or researchers want to find out whether particular how, when and where they outcome of interest (cases) with a high carbohydrate or a high protein occur. Epidemiologists want to discover patients who do not have the disease diet is better for weight loss, this can what factors are associated with or outcome (controls). Also, looking be studied in populations that already diseases (risk factors), and what factors back retrospectively to compare how follow these diets. Similarly, forcing may protect people against disease frequently the exposure to a risk mice into small tubes and making (protective factors). Epidemiological factor is present in each group to find them inhale tobacco smoke242 is not studies discover the correlation out about the relationship between necessary as there are still many between certain behaviours (such as the risk factor and the disease. human smokers who could be asked to smoking), demographic factors (such • Cross-sectional studies – participate in an epidemiological study. as age, sex), constitutional factors (such observational studies of a distinct Many nutrition and diet studies use as blood group or immune status) or population at a single point in time mice or rats. The digestive systems circumstances (such as living in an area or over a short period. Exposure to of these animals are very different to with high pollution) and health-related the risk factor(s) and outcome are those of humans. Some researchers conditions. determined at the same time. even acknowledge that the knowledge A population or group can be studied More recently, omics technologies have gained from studies with animals can’t in different ways, for example, by been used in epidemiological research: be applied to humans. For example, questionnaire, by taking measurements a research group at the University of “-Omics technologies have (such as blood pressure), by analysing Copenhagen found a way to stop mice substantially transformed blood specimens, or by examining gaining weight despite being fed a epidemiological research and healthcare records. Epidemiologists diet high in fat. They discovered that advanced the paradigm of use statistical methods to conclude mice who were lacking the enzyme molecular epidemiology, which whether the differences they find are NAMPT would not put on more weight focuses on underlying biology real or due to chance. on a high-fat diet than on a healthy (pathogenesis) rather than on diet. However, decreasing this enzyme Common types of epidemiological empirical observations alone. … in obese humans is not an option, studies include: The -omics technologies that have because NAMPT is essential for healthy been applied in epidemiological • Cohort studies – observational muscle function.243 research, however, have now studies used by epidemiologists expanded beyond genomics looking into the factors that affect to include epigenomics, the health and illness of populations. proteomics, transcriptomics, and Longitudinal studies are a form metabolomics”.241 of cohort studies, which follow groups of people over time. They Biobanks can become an important can be prospective or retrospective. resource for such research. The cohort study design is a good scientific method for measuring the effects of a suspected risk factor.
241 National Academies of Sciences, 2017, p. 4 242 Hsu, et al., 2015 243 Nielsen, et al., 2018 50 Humane Research Australia
Microdosing The concept of microdosing goes “Microdosing is an back to the late 1990s and is intended to investigate basic drug attractive approach properties. In drug development, a for the study of new microdose is a dose of a compound and existing drugs in (chemical elements of more than one element) that is intended to vulnerable populations be subpharmacologic244 when (children, pregnant administered. It is 1/100th of the known women, elderly, or expected active dose or 100 µg245/ adult, whichever is the smaller.246 hepatically and renally impaired), who are The two assumptions behind microdosing are that the best model routinely excluded from for humans are humans, and that the clinical trials due to 247 pharmacokinetics observed after safety concerns”.250 microdosing is an acceptably accurate predictor of therapeutic doses of Microdosing can replace the use 248 a compound. Also referred to as of animals in safety testing of new Phase 0 of clinical trials, microdosing compounds and drugs. Mostly but not is usually employed at the very start always, these animals are rodents. of clinical tests with humans. It can According to international guidelines, provide useful information to help a single dose given to a mouse or rat is decide whether the new compound “1000-fold the human dose, followed or drug should be developed further, by 2 weeks of observation”.251 and whether it may be safe to progress to further human testing. High failure Non-human primates are also used for rates in the clinical phase of drug animal testing in drug development. development are now at 95%: “This In the US, cynomolgus macaques illustrates the limited predictivity of (crab-eating macaques) are the most our predominant tool – the animal commonly used type of monkeys used 252 model”.249 Microdosing could screen out for this purpose. drugs destined to fail earlier, faster and cheaper. Further, unlike animal testing, it can provide vital information about different groups of patients:
244 A subpharmacological dose is a minute, 247 Pharmacokinetics is the study of the time 250 Lappin, Noveck, & Burt, 2013, p. 818 safe dose of a test compound. duration of drug absorption, distribution, 251 Svendsen, et al., 2016 245 A µg is a microgram. metabolism and excretion. 252 Feister, DiPietrantonio, Yuenger, Ireland, & 246 Rowland, 2012 248 Rowland, 2012 Rao, 2018 249 Hartung, 2017a, p. 26 Better ways to do research 51
Simulators The availability and use of simulators in medical and veterinary training is increasing. Simulators are also replacing live animals in primary and secondary schools. Training simulators are either virtual reality (VR)- based or physical model (PM)-based. Apart from replacing live animals in education and training, VR simulators have great potential for training people in remote locations, for example, training students and surgeons in developing countries.
Virtual reality-based systems can In AR, the real world is not blocked surgical skills and decision making.259 be grouped in immersive VR, non- from the user’s view. Virtual objects “Ultimately, this would lead to greater immersive VR, augmented reality (AR) are superimposed, and users are able efficiency, improved patient care, and and mixed reality. In immersive VR, the to interact at the same time with the minimization of technical errors that real world is completely blocked from real world and the virtual objects. are inherent to the surgical learning view and users experience that they are The virtual objects are transparent in curve”.260 Nevertheless, virtual reality immersed in that virtual world. Non- the real world in daylight, similar to a systems are not yet able to fully immersive VR blocks the real world, hologram. In mixed reality, the virtual simulate the tactile experience of the but users remain aware that they are images appear solid. In AR and mixed clinical situation with a real patient.261 viewing a virtual environment. The reality, users wear glasses that do not Physical model-based simulators user’s view is commonly blocked by a block out the real world.254 are available from companies that head-mounted display (HDM). This is The beginnings of VR and AR go back specialise in their development and how it works: to the 1960s. The US Air Force and production, or can be “home-made” by “In the immersive and non- NASA were early adopters with flight researchers. A well-known example of immersive VR systems, the user simulators designed to train fighter a commercially produced simulator is wears an HMD that occludes the real pilots introduced during the 1990s255 TraumaMan.262 world, and the user can maneuver and head-mounted displays (HMDs) The American College of Surgeons’ within the virtual environment used for astronauts. By 2010, computer (ACS) Advanced Trauma Life Support through movements of his or her technology was advanced enough so (ATLS) program has become the head and by physically walking that truly immersive VR and AR systems accepted standard of care for the initial around. Motion sensors in the could be developed.256 assessment and treatment of trauma HMD track head movements, while AR is now widely used in education patients. Since 1976, the ATLS program external cameras track the user as from kindergarten to high school has been training health professionals they walk. These movements are and can be used with computers or and is now offered in 86 countries. then translated into motion within mobile devices, without head-mounted Initially, surgical skills were practised the virtual world. Alternatively, the displays.257 on the chests, throats, abdomens and user can maneuver through the limbs of live dogs, pigs, sheep and virtual environment and manipulate VR and AR systems have great potential goats. Using these animals is costly objects using a handheld device for the training of surgical skills and, for and the animal models can’t properly with haptic feedback that give the example, replace the use of rat vessels simulate the clinical conditions of illusion of actually interacting with that are now used in microsurgery human emergency situations. Also, the virtual environment and objects training.258 VR and AR avoid the need public disapproval of animal models within it”.253 for animals or cadavers and provide has increased. So in 2001 the ACS a risk-free environment for practicing
253 Pelargos, et al., 2017, p. 2 256 Pelargos, et al., 2017 259 Vaughan, Dubey, Wainwright, & Middleton, 254 Pelargos, et al., 2017 257 Akçayır & Akçayır, 2017 2016 255 Akçayır & Akçayır, 2017 258 Thomson, et al., 2018 260 Pelargos, et al., 2017, p. 2 261 Thomson, et al., 2018 262 Simulab, 2017 52 Humane Research Australia
approved the use of human cadavers A review of studies comparing animal Similarly, a review of VR-based and the TraumaMan system to replace use in veterinary education and training simulators for spine surgery found that live animals in trauma training. with non-animal methods found that the VR training was more effective than TraumaMan is a surgical simulation student learning outcomes were at traditional approaches. In particular, manikin with lifelike skin, tissue, least as good with humane teaching VR training was very effective in internal organs and bones. It bleeds methods, in some cases even better. teaching surgical technical skills and when cut, and simulates breathing Non-animal methods also had other knowledge.266 Compared to the use using a ventilator. The ACS has since benefits, such as: of live animals, simulators have the approved other simulation systems. advantage that students can repeat “time and cost savings, enhanced Nearly all ATLS programs in the US and practising their surgical skills until they potential for customisation and Canada have now replaced animal use are competent. repeatability of the learning with simulation systems.263 exercise, increased student There are also various online data bases A wide range of simulators is available confidence and satisfaction, and digital representations of human and well suited for education, training increased compliance with animal and animal bodies and body parts. and professional development: use legislation, elimination of For example, BioDigital267 claims to be objections to the use of purpose- “The World’s First Human Visualization “Immersive, highly visual and often killed animals, and integration Platform”, and the SPM Anatomy 3D, virtual reality simulators closely of clinical perspectives and toolbox268 offers maps of the human replicate real-life with incorporation ethics early in the curriculum. brain and functional imaging data. of physical interfaces and haptic The evidence demonstrates that feedback. Virtual reality simulators veterinary educators can best are technically advanced, and serve their students and animals, available in a range of sizes and while minimising financial and shapes. Some incorporate gaming time burdens, by introducing well- industry headsets and cellular designed teaching methods not phones, while others involve reliant on harmful animal use”.265 large-scale simulators resembling carnival rides. The field of virtual This review was published more than reality training in trauma is growing ten years ago, and since then many exponentially. Downloadable apps more humane teaching tools have are currently being developed that, become available. with the addition of a $20 headset, can provide an immersive trauma scenario experience”.264
263 Gala & Crandall, 2018 265 Knight, 2007, p. 91 267 BioDigital, 2018 264 Quick, 2018, p. 448 266 Pfandler, Lazarovici, Stefan, Wucherer, & 268 Institut für Neurowissenschaften Weigl, 2017 und Medizin, 2017 Better ways to do research 53
San Antonio Military Medical Center’s simulation center
Source: Airman Magazine269
A simulator for a range of human functions
Froggipedia Froggipedia271 is a virtual dissection app and Apple’s top iPad app of 2018. It lets users dissect 3D frogs and is a great learning tool for the classroom. No frogs are harmed.
A simulator that can breathe, bleed, vomit and respond physiologically to treatment given such as medications and give feedback to procedures such as defibrillation.
Source: Lavit Yaacov, Sheba Medical Center, Israel270
269 Airman Magazine, no year 270 Yaacov, 2015 271 Designmate (I) Pvt. Ltd., 2019 54 Humane Research Australia
Human-relevant research and testing methods/technologies and their application
Methods/ Chemicals/ Regenerative Personalised/ Organ/ Large scale & Education & Provides technologies drug testing medicine precision disease fast studies/ training access to that can for toxicity & & other medicine studies tests human replace animals effectiveness treatments tissues in biomedical research and testing In-vitro methods Organoids, ✓ ✓ ✓ ✓ ✓ organs-on- chips Biobanking ✓ Omics ✓ ✓ ✓ technologies Stem cell ✓ ✓ ✓ ✓ technologies 3D and 4D ✓ ✓ ✓ ✓ bioprinting Robotic testing ✓ ✓ ✓ In-silico methods Prediction ✓ ✓ ✓ methods (SARs & QSARs, Read- across, PBPK models, Expert systems) and tools AOPs, IATA ✓ ✓ ✓ Computer ✓ ✓ ✓ ✓ modelling Studies with human volunteers Post-mortem ✓ ✓ ✓ studies Population- ✓ ✓ based studies Microdosing ✓ Simulators Virtual reality ✓ ✓ ✓ & physical model-based simulators Better ways to do research 55
Efforts by governments and the scientific community to replace animal experimentation
New alternatives to animal research and testing can only be developed with the support and collaboration of governments, the scientific community and industry. Philanthropic organisations also provide financial support. In the following, examples of major initiatives by governments, the scientific community and other stakeholders are listed.
Policies and collaborations
In particular in the European Union United States and the US, governments have issued policies that aim to replace, reduce and • A strategic roadmap for or policy of any specific agency or refine the use of animals in biomedical establishing new approaches to workgroup, and should not be taken research and testing. Government evaluate the safety of chemicals as a commitment by any federal 272 regulators, researchers in academia and and medical products in the agency”. United States industry and other interested parties • Toxicology in the 21st Century are collaborating to implement these “This strategic roadmap is a resource “Toxicology in the 21st Century policies. Examples of major policies and to guide U.S. federal agencies and (Tox21) is a US federal research collaborations include the following: stakeholders seeking to adopt collaboration that is developing new approaches to safety and risk alternative, non-animal methods to assessment of chemicals and medical quickly and efficiently test thousands products that improve human of chemicals for potential health relevance and replace or reduce the effects. These approaches use use of animals. This document was advances in robotics technology to developed with input from members test chemicals for their potential to of 16 federal agencies, multiple disrupt processes in the human body, interagency workgroups, and input which may lead to negative health from the public. As such, it represents effects”.273 a consensus perspective that does not necessarily reflect opinions
272 Interagency Coordinating Committee on the 273 U.S. Environmental Protection Agency, Validation of Alternative Methods, 2018 2017, p. 1 56 Humane Research Australia
European Union • FDA’s Predictive Toxicology • Directive 2010/63/EU on the development, validation and Roadmap protection of animals used for acceptance of alternative approaches scientific purposes to animal use in regulatory testing. In response to the FDA The overall aim is the replacement, Commissioner’s request for a In 2010, the European Parliament reduction and refinement (3Rs) of roadmap for integrating predictive and the Council of the European animal use in regulatory testing”.278 toxicology methods into safety Union adopted Directive 2010/63/ and risk assessments, the FDA’s EU on the protection of animals • Transition to non-animal research Toxicology Working Group released used for scientific purposes, to in the Netherlands the Predictive Toxicology Roadmap come into effect on 1 January 2013, • The Dutch Government aims to be in 2017.274 replacing the previous 1986 directive. a world leader in alternatives to All use of live animals for scientific “The Predictive Toxicology animal research by 2025. In 2016, and educational purposes must be Roadmap of the FDA, which aims the Netherlands National Committee carried out in compliance with this to integrate new approaches while for the protection of animals used Directive. The “Directive represents reducing animal tests, lists the for scientific purposes presented a an important step towards achieving following human-based methods report to the Minister of Agriculture the final goal of full replacement as especially promising new with timelines for phasing out animal of procedures on live animals for technologies for use in predictive research. As a first step, by 2025 scientific and educational purposes toxicology: microphysiological animal procedures will be phased out as soon as it is scientifically possible systems; alternative test methods in regulatory safety research (such to do so”.276 for reproductive toxicity testing; as testing of chemical substances, computational toxicology; in vitro • Ban on animal testing of cosmetics food ingredients, pesticides, alternatives; and read-across medicines and vaccines). It will take “The EU Cosmetics Regulation methodologies. Although evaluation longer to transition to animal-free prohibits animal testing of finished of these methods is crucial to research in the fields of fundamental products since 2004 and of cosmetic understanding their value in scientific research and applied and ingredients since 2009, reinforced predicting human in vivo outcomes, translational research.279 by a marketing ban of cosmetics human-based approaches can be finished products tested on animals • Innovate UK. A non-animal expected to provide data that are since 2004 and for cosmetics technologies roadmap for the UK more relevant than animal-based containing ingredients tested on methods because they enable “This roadmap, vision and strategy animals since 2013”.277 scientists to investigate how drugs for non-animal technologies in the interact with human cells, tissues, • European Partnership for UK has been drawn up by Innovate and biological processes directly, Alternative Approaches to Animal UK, the National Centre for the removing the need for cross-species Testing Replacement Refinement and extrapolation”.275 Reduction of Animals in Research, “The European Partnership for the Biotechnology and Biological Alternative Approaches to Animal Sciences Research Council, the Testing (EPAA) is an unprecedented Defence, Science and Technology voluntary collaboration between the Laboratory, the Engineering and European Commission, European Physical Sciences Research Council trade associations, and companies and the Medical Research Council, from 7 industry sectors. The partners and has been published on their are committed to pooling knowledge behalf by Innovate UK. It is intended and resources to accelerate the to guide the efforts of all those
274 U.S. Food and Drug Administration, 2017 276 European Parliament and the Council of the 279 Netherlands National Committee for the 275 Baker, et al., 2018, p. 625 European Union, 2010, p. 2 protection of animals used for scientific 277 Dal Negro, et al., 2018, p. 33 purposes, 2016 278 European Commission, 2018a Better ways to do research 57
working in this area. The issues International outlined and the recommendations have come out of extensive • PETA International Science • BioMed21 (biomedical research for st discussions between the six Consortium the 21 century) organisations that are endorsing the “The Science Consortium and “The BioMed21 Collaboration roadmap and with many other key its members promote the (https://www.biomed21.org) stakeholders. The participation and implementation of animal-free grew out of a 2015 review paper endorsement by the six organisations testing approaches through multiple authored by a diverse group of reflects their continuing interest in efforts, including: stakeholders representing civil non-animal technologies, but should society, research funding, academic, –– providing financial support toward not be construed as a commitment to regulatory, corporate and other the development and validation of ensuring its delivery”.280 communities, which recognized the non-animal test methods human relevance and translational • ORCHID (organ-on-chip in –– organising expert working groups limitations of the conventional development) to tackle the development of new paradigm in biomedical research approaches to address regulatory The ORCHID project is a collaboration and drug discovery and the need for requirements when non-animal between seven research institutions change”.285 from six European countries. The methods do not exist or require project’s main goal is to create further optimisation • OECD Integrated Approaches to a roadmap for organ-on-chip –– providing technical support Testing and Assessment (IATA) to companies and researchers technology and to build a network of The OECD provides guidance seeking to replace, reduce, or all relevant stakeholders.281 documents for IATA and facilitates the refine the use of animal tests sharing of case studies that “explore • EU-ToxRisk –– publishing manuscripts, the use of novel methodologies in developing technical analyses, This is a major collaborative six-year Integrated Approaches to Testing presenting at international project. It was launched in 2016 with and Assessment within a regulatory scientific conferences, and hosting a budget of €30 million, funded by context”.286 New approaches are free webinars to ensure that the EU. The project “aims to make needed to assess the many chemicals information regarding the use of progress towards animal-free safety in use today that have never been non-animal methods is accessible assessments and tackles complex tested. Testing these chemicals in to all audiences areas of toxicology, such as repeated- the conventional way would increase 282 –– interacting with national and dose and reproductive toxicity”. the use of animals and be too costly international regulatory bodies and slow. • EuroMix and standards organisations to EuroMix (European Test and Risk ensure that opportunities exist Assessment Strategies for Mixtures) to increase and harmonise the is another collaborative project. The use of validated non-animal test project examines how a mixture of methods. This includes attending multiple chemicals affect human Organisation for Economic health – chemicals that may be part Co-operation and Development of the food we eat, may be inhaled, or (OECD) meetings through the may come into contact with our skin. International Council on Animal The project uses computer-based Protection in OECD Programmes methods and in-vitro assays.283 (ICAPO) to ensure the best possible science and widest possible integration of alternatives to in vivo test methods in OECD guidelines”.284
280 Innovate Uk, 2015, p. 2 283 National Institute for Public Health and the 285 Triunfol, et al., 2018, p. 1230 281 Mastrangeli, et al., 2018 Environment, no year 286 OECD, 2018b 282 European Commission, 2018c, p. 6 284 PETA, 2019 58 Humane Research Australia
Validation Any new drug testing method has to These validation centres collaborate If researchers want a new method to be checked to make sure it is safe and in streamlining and standardising the be accepted by regulators, they have effective. Governments regulate drugs validation process. Together with the to demonstrate that the method is and other substances. They have to be Chinese Food and Drug Administration scientifically satisfactory (valid) for convinced that the new methods are and the Guangdong Center for Disease the purpose sought. This is done as good or better than conventional Control and Prevention, they have through a validation process.289 Often, ones. The process of checking whether formed the International Cooperation the validation process consists of a the claims about new methods are on Alternative Test Methods (ICATM), comparison of the new method with accurate is called validation. It is a “working together to promote animal tests. This is problematic, key step towards the acceptance of a enhanced international cooperation because animal tests have not been test method by the regulators. Several and coordination on the scientific validated, as argued below: countries have validation centres that development, validation and regulatory “While insisting that any non- assess new methods and technologies. use of alternative approaches”.287 animal method must undergo The first validation centre, the EURL ECVAM prepares annual validation before it can even be European Union Reference Laboratory reports with updates on the status of considered for regulatory approval, for alternatives to animal testing (EURL alternative methods and approaches. the pharmaceutical industry and ECVAM) was established in 1991, and It “provides updates on the progress the regulatory authorities have since then similar centres have been made in the development, validation conveniently overlooked the fact set up in the US (1995), Japan (2005), and regulatory acceptance and use of that animal experiments have never South Korea (2010), Brazil (2013) and alternative methods and approaches been validated”.290 Canada (2017). and their dissemination”.288 “Currently, new in vitro methods are validated against animal models; However, this approach will not help to improve the prediction of the effects of a substance in the human organism in case the animal model is biased”.291 “A … key problem in toxicology may be called the ‘validation dilemma.’ This is posed by the fact that the point of comparison for any novel toxicological tool will be a traditional, poorly assessed methodology. The challenge faced in toxicology is how to objectively assess the value of new tools”.292
287 European Commission, 2017 289 Eskes & Whelan, 2016 291 Rovida, et al., 2015, p. 179 288 Zuang, et al., 2018, p. 4 290 Menache, 2006, p. 5 292 Hartung, 2009a, p. 93 Better ways to do research 59
Why it matters
For a long time, the use of animals in research, testing, training and education has been considered a necessary evil. More and more, people question the ethics of this approach. At the same time, the animal research community increasingly recognises the problems with animal research: it is costly, lengthy and not very effective. Also, it may have held back the discovery of treatments and cures for humans because they did not work well in animals.
Long-held beliefs and practices are that “significant progress has been with animal models. They have difficult to change. This is also true made in demonstrating how in-vitro great potential for regenerative and for animal research. Besides, there and computational methods can be personalised medicine. are economic interests at play.293 combined to ‘read-across’ toxicological The new animal-free methods and But several developments indicate properties between similar chemicals technologies are not yet perfect and that change is on the way: With to avoid unnecessary animal testing”.295 need further development. Also, some an increasing number of people So far, most progress in the of the current methods that are deemed opposed to animal experimentation development of alternatives has been to be alternatives might still use animal and in response to public pressure, made in the area of toxicology. New parts or animal data collected in the governments - in particular, in the tests are being validated and approved past. Examples include the tissues or EU and the US – are encouraging by regulators, although this is a slow body parts of animals used for food and supporting the development of process and many challenges still (such as eyes from the slaughterhouse human-relevant, non-animal research lie ahead. But only around 10% of for assessing eye irritation), the methods. Rapid technological progress animals are used for regulatory testing. use of foetal calf serum for in-vitro enables the development of these Governments do not require the use of cell culture296, or the use of “lesser” new methods. animals for the remaining 90% used in animals (such as replacing mammals The main alternatives to the use of basic and applied research, education with zebrafish).297 The databases used animals in the laboratory are new and training. for computer-based methods hold in-silico and in-vitro approaches. data from various sources, and this Apart from toxicity testing, the Studies with human volunteers and includes data obtained from previous methods and technologies described simulators also play an important role. animal experiments. in this overview can replace animals Some of these methods are used in used in basic and applied research, There are also other new technologies, combination for greater effectiveness. education and training. As described such as nanotechnologies and genome Many of the new methods, such as in the previous sections, there are editing, that have the potential to organs-on-chips and organoids, many applications for the new replace animal experimentation, but “integrate with in silico approaches and methods and technologies. They at present they use many animals.298 with systems biology, seen by many can be used to model and study For this reason, they are not covered in as having potential to revolutionise diseases in general or for individual this overview. medicine and drug discovery”.294 patients. The new methods can be For example, the European Union used to study conditions in individual Reference Laboratory for Alternatives to patients, something that is not possible Animal Testing noted in its 2018 report
293 e. g., Bottini & Hartung, 2009 295 Zuang, et al., 2018, p. 1 297 Redmond, 2019 294 Pound & Ritskes-Hoitinga, 2018, p. 5 296 for example, van der Valk & Gstraunthaler, 298 Bailey, 2019 2017 60 Humane Research Australia
Animal researchers argue that the Below, two groups of researchers In the near future, industry rather new methods can’t replace all areas comment on the difficulties of moving than academic institutions might of current animal research. Given the away from animal research: be more interested and invested ethical questions raised and the many in progress towards research and “It is important to recognise that shortcomings of animal research, testing without animals. For biotech researchers can be reluctant isn’t this a compelling argument for and pharmaceutical companies, the to invest time and money in speeding up the development of advantages of new non-animal research implementing a new technique, or human-relevant research and testing methods are compelling – they are to replace an animal model that without animals? Governments, the cheaper and faster than conventional has served as the basis of their scientific community and industry in methods, and more acceptable to research for many years. … There EU countries and the US have taken the customers. Stakeholders in UK drug may be concerns about a lack of lead in collaborating to replace animal discovery companies are already calling historic data comparability, or experimentation. But using animals for more “humanised” preclinical invalidating past results. Setting up in research and testing is still the research methods.304 Likewise, a report a new model can require additional predominant paradigm, the standard by Humane Society International technical expertise or development way of doing things. Many researchers stressed the need for change: of new infrastructure. Referees are regard animal testing still as the “gold familiar with data from the ‘gold “Reflecting the new conceptual standard”.299 standard’ animal models, and may approach in safety testing, “Why is this so?” one might ask. request additional in vivo data to fundamental health research We know that we are not 70 kg rats300, be generated to support in vitro and drug development need to be so why is it so difficult to change findings. These factors can delay based primarily on human disease this paradigm? publication in a highly competitive pathways — not on misplaced research environment and efforts to mirror human illnesses Apart from a reluctance to change, result in a lack of motivation in mice and rats. And as safety which is common to most humans, to change models”.302 testing is moving away from a range of other factors are at play. simply causing recognisable Research funding is competitive, “All believe their research toxic effects in animals without and researchers may not want to is important and there is an understanding the toxic pathways take risks by applying for funding for understandable fear to alter involved, so too should research unconventional methods. For example, methods that have worked well shift its focus from creating in Australia only about 15% of National in the past, or to break away artificial ‘symptoms’ in animals and Health and Medical Research Council from established models linked towards understanding underlying (NHMRC) grant applications are to previous funding and the disease pathways in humans”.305 successful. Unlike in the US and the published literature. Pressure in EU, the Australian government does the biomedical research field to We need urgent change. From an not provide incentives for new animal- publish in high-ranking scientific animal rights perspective, it was never free methods. New methods and journals can also force researchers okay to inflict pain and suffering on technologies also require new skills and to use established models rather animals for the real or perceived benefit new infrastructure. For example, new than develop better or new models. of humans. For proponents of animal in-silico methods do not only require Current peer review systems welfare, the use of animals is justified proficiency in computer informatics, do not encourage change to the as long as harm is minimised. With but scientific expertise in other areas.301 extent needed”.303 awareness of the many shortcomings of animal research and testing and increasing availability of better ways, the justification for animal research vanishes.
299 Vonk, et al., 2015 302 Jackson & Thomas, 2017, p. 941 305 Langley, 2012, p. 31 300 Hartung, 2009b 303 Prescott, Langermans, & Ragan, 2017, p. e2 301 Valerio, 2014 304 Catapult Medicines Discovery and UK BioIndustry Association, 2018 Better ways to do research 61
We, the citizens and consumers of health research, need to advocate for better ways to do research and testing. We must persuade our governments to phase out animal research and testing and fund human-relevant and effective research that is based on human biology and not that of animals – research that has real potential to help humans. The new methods and technologies can do more than replacing animals. The future lies in personalised medicine, regenerative medicine, precision medicine, biobanks and personalised drug screening:306 “This new era is marked by a consideration of validity (e.g. reproducibility), by the use of human biomaterials (3D cell cultures, organoids and induced pluripotent stem cells (iPSC)), by the use of ‘high-content’ methods (e.g. Omics), by the combination of computer-based approaches, such as ‘read-across’ and ‘virtual organs’, and by miniaturisation technology (organ/human on a chip)”.307 With greater investment in innovative and promising non-animal methods, firm policy initiatives and robust collaborations of all interested parties, better treatments and cures for human diseases can be developed. This will also end the suffering of millions of animals.
306 e. g., Gorshkov, et al., 2018; Ianelli, 2018 307 Daneshian, 2018, p. 4 62 Humane Research Australia
Glossary
3Rs Applied research Clinical trials These are the principles of Applied research is research that seeks After pre-clinical research, which replacement, reduction and to answer a question and/or to solve a involves in-vitro and/or animal refinement. The 3Rs were proposed by practical problem. studies, promising new drugs or William Russell and Rex Burch in the other treatments are tested in clinical late 1950s and are today embedded Associative network mapping trials. The goal is to find out whether in many laws, regulations and codes the treatment is both safe and governing animal use. “Network representations have recently effective. Clinical trials involve human made the leap from social sciences, volunteers. There are four (sometimes • Replacement - techniques that where they have been applied for five) phases: replace the use of animals must be decades, to systems biology. Network sought and used where possible mappings can provide a picture of Phase 0 – Usually trials go from • Reduction - each project must use the interaction between molecules, pre-clinical research to Phase I, but no more than the minimum number represent the relative abundance sometimes a Phase 0 will be run. A of animals necessary of those molecules, and provide very small number of healthy people • Refinement- projects should be molecular insights into the organization are given a very small dose of a drug designed to avoid pain and distress in of signaling pathways, protein–protein (a microdose). If the drug acts not animals308 interactions, or metabolism, that as expected, additional pre-clinical would not be possible from studying research will be undertaken. Adverse Outcome Pathway (AOP) individual proteins or genes. An Phase I – Safety study. Tests a new drug important finding in many network “An AOP is an analytical construct for the first time in healthy humans analyses is that associative networks that describes a sequential chain of (unless there was a Phase 0). Includes can elucidate relationships that cannot causally linked events at different levels a small number of volunteers (usually be seen when comparing single or of biological organisation that lead to 20-80 people). Shows what side effects small sets of genes, proteins, or other an adverse health or ecotoxicological the new drug may have. About 70% of components”.311 effect. … AOPs are the central element medications move on to Phase II. of a toxicological knowledge framework Bioprinting Phase II – Safety study. Identifies side being built to support chemical risk effects. Tests how well a drug works assessment based on mechanistic “We use the term 3D bioprinting to on a specific disease, such as cancer. 309 reasoning”. describe the precise layering of cells, Includes a small number of patients biologic scaffolds, and biologic factors (around 100-300 people). About 33% of Animal Ethics Committee (AEC) with the goal of recapitulating a medications move on to Phase III. biologic tissue. Compared to traditional AECs oversee the ethical conduct of Phase III – Measures effectiveness tissue engineering methods, the research using animals. In Australia, and monitors side effects. Compares technologies utilized by 3D bioprinting AECs include representatives of new and standard treatments. Often systems allow for greater precision in veterinarians, scientists, animal welfare requires random assignment of the the spatial relationship between the organisations and the general public. new and standard drugs. Can include individual elements of the desired Their primary responsibility is to hundreds or thousands of patients. tissue”.312 ensure that all care and use of animals Roughly 25% to 30% of medications is conducted in compliance with the move on to Phase IV. Australian Code for the Care and Use of Cardiovascular If the new drugs passes Phase III, Animals for Scientific Purposes.310 This term refers to the heart and blood regulatory review and approval follow. vessels. For example, cardiovascular In other countries these committees The new drug then enters the market. may have a different name. For toxicity refers to damage to the Phase IV – Monitors long-term side example, in the UK they are called heart and blood vessels by harmful effects after the drug is on the market. Animal Welfare and Ethical Review chemicals. Testing for interactions with other Bodies (AWERBs). drugs. Testing on certain populations, such as pregnant women.
308 National Health and Medical Research 310 National Health and Medical Research 312 Bishop, et al., 2017, p. 186 Council, 2013 Council, 2013 309 OECD, 2018a 311 Marshall, et al., 2018 Better ways to do research 63
ECHA Induced pluripotent stem cells Omics technologies (iPS) European Chemicals Agency The term “omics technologies” refers to Stem cells which resemble pluripotent areas of study in biology whose names EU embryonic stem cells. They are derived end in “omics”, such as genomics (the from mature, fully differentiated cells of study of the genome of an organism). European Union the body that have been reprogrammed The science of “omics” reflects diverse through genetic manipulation technologies with a focus on studies of EURL ECVAM and other techniques to restore life processes, such as comprehensive 314 European Union Reference Laboratory developmental potential. studies of genes, proteins and for alternatives to animal testing metabolites of an organism. In-vitro, in-vivo, in-silico Organoid Fundamental research In-vitro, in-vivo and in-silico are Also referred to as basic research. This categories of experimental studies. A miniature and simplified version of a type of research is curiosity driven In-vitro (Latin for “within the glass”) (human) organ. Organoids are grown and not designed to answer specific experiments are also called test tube in-vitro in three dimensions. They questions or solve practical problems. experiments. In-vitro research is allow researchers to study disease and Fundamental/basic research is traditionally done in test tubes, flasks treatments in the laboratory. exploratory and aims to increase and or Petri dishes. More recent in-vitro advance scientific knowledge. studies use, for example, organoids and Organ-on-a-chip organs-on-chips. “Organs-on-chips are microfluidic Haptic feedback In-vivo means “in the living”. In-vivo devices for culturing living methods use living organisms, such as The use of touch to communicate with cells in continuously perfused, living animals. users. For example, the vibration in a micrometersized chambers in order mobile phone. In-silico studies are performed on a to model physiological functions of computer or via computer simulation. tissues and organs. The goal is not to build a whole living organ but rather IATA to synthesize minimal functional units Machine learning Integrated Approach for Testing and that recapitulate tissue- and organ- Assessment Computer programs that learn from level functions”.315 data. Immortalised cells Peristalsis Microfluidic chip “Immortalized cell lines are either Wave-like muscle contractions that tumorous cells that do not stop dividing A set of micro-channels etched or move food through the digestive tract. or cells that have been artificially moulded into a material, such as glass, manipulated to proliferate indefinitely silicon or polymer. The micro-channels Pluripotent stem cells and can, thus, be cultured over several are connected together. The size of the generations”.313 channels is in the range of one to tens Stem cells that can develop into all of micrometers. A micrometer is 1000 types of cells in the body. Incidence times smaller than a millimeter. In disease epidemiology, incidence NHMRC means the number of people newly diagnosed with a condition or disease. National Health and Medical Research In contrast, prevalence is the actual Council (Australia) number of people with the disease, both newly diagnosed and diagnosed OECD in the past. Organisation for Economic Co- operation and Development
313 Carter & Shieh, 2015, p. 298 315 Bhatia & Ingber, 2014, p. 760 314 Stem Cells Australia, 2018 64 Humane Research Australia
Physiologically-based Read-across Systems biology pharmacokinetic (PBPK) models “ ‘Read-across and grouping’, or ‘read- “An approach that seeks to study These are “mathematical across’, is one of the most commonly organisms as complete systems— representations of the absorption, used alternative approaches for data networks of interacting genes, distribution, metabolism and gap filling in registrations submitted biomolecules, and biochemical elimination (ADME) of chemicals in under the REACH Regulation. Read- reactions. It thus attempts to integrate humans or other animal species. across involves the use of relevant all relevant structural and functional They are used for multiple purposes, information from analogous information, rather than focusing on, including the interpretation of in substance(s) (the ‘source’ information) say, just one particular gene or protein vitro toxicity data by in vitro to in vivo to predict properties for the ‘target’ at a time. This involves amassing extrapolation (IVIVE) and the simulation substance(s) under consideration”.319 and organizing data obtained from of internal concentrations in the genomics, proteomics, and other areas organism of interest”.316 Regenerative medicine of bioinformatics and managing and analysing the data to identify patterns, Regenerative medicine aims at Quantitative structure-activity formulate hypotheses, and ultimately developing methods to regrow, repair relationship (QSAR) create computer models that will or replace damaged or diseased cells, enable accurate predictions of cellular “A QSAR is a mathematical model organs or tissues. and organismal responses. Such (often a statistical correlation) relating models could have a radical impact on one or more quantitative parameters Regulatory testing medicine and biology in the future”.322 derived from chemical structure to a quantitative measure of a property Testing that is undertaken to comply Toxicology or activity (e.g. a (eco)toxicological with relevant laws, policies and endpoint). QSARs are quantitative (government) regulations. The branch of science concerned models yielding a continuous or with the study of the adverse effects categorical result”.317 Stem cells of chemical substances on living Unspecialised or undifferentiated cells organisms and the diagnosis and Read-across structure activity with the ability to self-renew, and to treatment of exposures to toxins. relationships (RASAR) differentiate to produce specialised cell 320 US EPA Machine-learning software combined types in the body. with big public data sets that can be US Environmental Protection Agency used to create sophisticated read- Structure-activity relationship across-based tools to predict toxicity of (SAR) US FDA chemical compounds. SARs (structure–activity relationships) US Food and Drugs Administration are mathematical models used for REACH predicting biological activities of Validation The EU regulation on registration, chemicals. According to the OECD, validation is evaluation, authorisation and “A SAR is a qualitative relationships defined as “the process by which the restriction of chemicals (REACH). that relates a (sub)structure to the reliability and relevance of a particular presence or absence of a property or “REACH is a regulation of the European approach, method, process or activity of interest. The substructure Union, adopted to improve the assessment is established for a defined may consist of adjacently bonded protection of human health and purpose”.323 the environment from the risks that atoms, or an arrangement of non- bonded atoms that are collectively can be posed by chemicals, while Validity enhancing the competitiveness of associated with the property or 321 the EU chemicals industry. It also activity”. In research, validity is an indication promotes alternative methods for the how sound the research is, how well hazard assessment of substances in a scientific test or piece of research order to reduce the number of tests on actually measures what it intends to animals”.318 measure, how well it reflects the reality it claims to represent.
316 European Commission EU Science Hub, 319 European Chemicals Agency, 2017a, p. 4 322 Hine & Martin, 2016 2018a 320 Stem Cells Australia, 2018 323 OECD, 2005, p. 68 317 European Chemicals Agency, 2008, p. 10 321 European Chemicals Agency, 2008, p. 10 318 European Chemicals Agency, no year Better ways to do research 65
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