Replace, reduce, refine: Why do we still need to experiment on animals?

Essay submitted to the Biological Sciences Essay Prize (2021) by Newnham College - University of Cambridge

"Kathryn Chia" "Apothekathryn" "Biology"

Animal experimentation and observation is deep-rooted in biomedical research. Exceptional anatomical, pathological and physiological similarities between animals and humans have been the cornerstone of biological and medical discoveries for decades.1 However, such study has traditionally also meant pain, suffering and occasional death for animal subjects, resulting in a persistent debate on the ethics surrounding said studies / research. It thus begs the question: given the approach of replacing, reducing and refining, why is there still need to experiment on animals?


It would be naïve to think that there is a straightforward answer to this, with many stakeholders for and against animal experimentation fighting for their views. Organisations such as the “People for the Ethical Treatment of Animals” (PETA), are strongly against animal testing and describe animals as “languishing in pain, suffering from extreme frustration, aching with loneliness and longing to be free.”2. On the other hand, “Speaking of Research” (SoR) champions the importance of animal research,3 supporting science in furthering research to cure the diseases of the world. These two views are often mired in confusing arguments, preventing the public and politicians from getting a clear view of the issues at stake. This was the case during the evaluation of the European Citizen Initiative (ECI) ‘Stop Vivisection’ which was presented to the European Commission. 4


To get a full picture of why the issue of animal experimentation can be so complicated, it is important to first understand the ethical issues involved. The suffering of animals to further science and benefit humans is often debated. It can be difficult to quantifiably justify that the benefits to humanity from animal experimentation outweigh the suffering of animals involved, especially because the harm done to animals from the experimentation is known whereas the harm done on humans and the world by not doing so is unknown.5



"Kathryn Chia" "ApotheKathryn" "Biology"

On the surface, animals look very different but on a physiological and anatomical level, they are a lot more similar than many may think. Most animals have the same organs as humans, such as the heart, lungs and brain, as well as similar organ systems, such as the respiratory, circulatory and nervous system. This results in similar hormonal regulation, dissemination of microorganisms during infectious disease, immune defence and development of brain function.6 Figure 1 displays this, showing similarities in the anatomy of a human (left) and a mouse (right).


Fighting and overcoming disease depends on understanding the complex biological processes that drive them.7 Because of their physiological similarities to humans, animals work as brilliant models to achieve this; through observation and experimentation over their lifespan, or across several generations, under closely controlled conditions. Humans and animals share hundreds of illnesses, such as cancer in mice, atherosclerosis in rabbits and haemophilia in dogs. 8 As such, animal models of human diseases can be created and induced easily. A non-directed, mutation-driven approach uses radiation and chemicals to cause mutations. Alternatively, a directed, disease-driven approach can use techniques such as transgenesis, single-gene knock ins and knock-outs, conditional gene modifications and chromosomal rearrangements. For instance, transgenesis would involve adding foreign genetic information to the nucleus of embryonic cells, by using a retroviral vector to insert the transgene into an organism’s DNA, inhibiting gene expression. This process is often done in mice models, mutating the genes that are responsible for the human condition and recreating human genetic diseases in mice for scientists to begin to look and test for treatments. Animal experimentation is thus integral in furthering our understanding of how biological systems work in health and disease and in the formulation of new medicines, technologies and treatments.


Testing new medicines in animals also serves to protect the safety of people, animals and the environment. Many potential treatments work in vitro, but legally and ethically, it is key that it still be tested in vivo in animals before humans. Current regulatory guidelines state that new medicines should be tested on two different types of animals; of which one should be a non-rodent such as a rabbit, dog or primate.10 Vaccines may be formulated in petri dishes and appear to work, but they simply cannot replicate the conditions that occur inside a living organism as when body systems work together, they create new conditions which do not exist in tissue cultures.11 In the body system, the drug could be destroyed before it reaches its target, converted into something harmful or useless, passed out of the body before it has time to work, or indirectly cause problems such as organ damage.11 Animal testing aims to demonstrate what happens to a compound in a complete living system and identify the beneficial and harmful effects of a compound on a whole organism before the new treatment is tested in small groups of human volunteers. Drug testing, toxicological screenings, effects of medical procedures and surgical experiments are all treatments that need to first be tested in animals.


From the dawn of scientific research, humans have used animals in their research to make biomedical breakthroughs. Of the 111 times the Nobel prize in Physiology and Medicine has been awarded, 101 were directly dependent on animal experimentation of some form, with the latest Nobel (2020), for the discovery of the Hepatitis C virus having mice underpinning their research.1 Throughout history, animal experimentation has also been indispensable during pandemics, facilitating the development of the smallpox vaccine.



"Kathryn Chia" "ApotheKathryn" "Biology"

Similarly, the polio vaccine was developed through years of research using monkeys, rats and mice. These vaccines allowed for the eventual eradication of these diseases that killed millions of humans.12 Figure 2 shows the traditional vaccine development pathway, of which animals are used in pre-clinical trials for testing of vaccines before clinical trials in humans.


The role of animals in today’s Covid-19 vaccine formulation is no different. In February 2020, when the outbreak began, the WHO assembled an international panel to develop animal models for Covid-19 to accelerate the testing of vaccines. Several animal models in the mouse, hamster and ferret were vital in identifying how the spike protein in the virus played a role in infecting the body systems of these animals and were valuable in the evaluation of vaccines and antiviral agents.13 Covid-19 vaccines will be a powerful tool in ending the pandemic, which has brought about more than 120,000 deaths in the UK so far.14 They could not have been formulated as safely and effectively without animal models and preclinical testing with animals.


Despite the obvious scientific breakthroughs that come with the aid of animal testing, it is important to take animal well-being into account with each experiment. Since animal experimentation began, there have been widespread changes to regulations and types of animals used. Latest figures from 2019 show that the number of procedures on animals decreased by 3% from 2018 to 3.4 million, with 2019 having the lowest number of procedures since 2007. Notably, mice, fish, rats and birds accounted for 97% of animal testing, while cats, dogs and primates only 0.2%.15


To avoid unethical research and maintain animal well-being, the 3 ‘R’s, which stand for Replace, Reduce and Refine were created. They are the standard for which every project involving the use of animals is evaluated and scientific grants to use animals are provided. The UK has gone further than almost any other country in this, providing the strictest regulatory frameworks for the protection of animals used in research.10


Wherever possible, animal research is replaced with non-animal alternatives. These non-animal alternatives include absolute replacement, where in vitro models, cell cultures, computer models and new imaging techniques are used instead.16 A computer model can predict various possible biological and toxic effects of a chemical or potential drug candidate without animal dissection, selecting the most promising molecules obtained for in vivo experimentation. This avoids the testing of unwanted chemicals, reducing the total number of experimental animals.17


Alternatively, replacing a higher grade animal such as a primate, with a lower grade animal such as a zebrafish or an Invertebrate -which does not fall under ethical animal research guidelines, would greatly reduce distress of the animal due to their lower mental cognition as well as their increased tolerance to be held in captivity.16 The zebrafish, or Danio Rerio is an attractive option because of its genetic similarity to higher vertebrates despite its small size (up to 4-5cm). Many critical pathways in humans and zebrafish are similar and hence any type of disease that causes changes in the body of humans can theoretically be modelled in zebrafish. For example, zebrafish have already been used to conclude that the loss of dystrophin gradually leads to necrotic muscle fibres that are replaced by inflammatory cells, fibrosis and abnormally-sized muscle fibres, unlocking the biological processes behind muscular dystrophy.18 Invertebrates, like the Drosophila melanogaster also provide a useful alternative to other types of animal testing. The genome of this organism is completely sequenced and annotated, which enables the study of molecular mechanisms underlying human disease.19 The species has been employed in gene mapping via linkage and recombination studies, to large scale mutant screens to identify genes related to specific biological function.9 Other types of organisms such as prokaryotes, protists and fungi (Escherichia coli, Dictyostelium discoideum and Schizosaccharomyces pombe respectively) can also be used as models for molecular and genetic studies.16


By improving experimental techniques and data analysis and sharing information with other hers, the minimum number of animals needed to achieve the results sought are used.5 Additionally, bans on animal tested cosmetic products have been put in place in the UK due to the development of adequate non-animal techniques in this area, further reducing animals needed for research. Almost all cosmetics are now tested for their toxicity and efficacy with cell and tissue cultures, which involves the growth of cells outside the body in a laboratory environment. At present, these techniques can only be used to prove the safety of cosmetics and not other drugs and medications, due to the more superficial nature (i.e., does not interfere with bodily function) of cosmetics.20 If animals absolutely must be used, procedures are refined as much as possible to minimise the suffering of animals. This is done by using less invasive techniques, giving animals better medical care and better living conditions. In turn, refinement also provides a more accurate model to observe the progression of disease. For instance, it was observed that when mice genetically modified to study Huntington’s disease were provided with a better cage environment, the disease progressed more slowly.16


Though the 3Rs and new techniques reduce the number of animals needed, research on relevant animal models will remain a crucial step for the fundamental understanding of disease, testing hypotheses and the safety of treatments for a long time. Humans and other mammals are immensely complex organisms in which organs achieve distinct physiological functions in a highly integrated and regulated fashion, with intricate relationships. It is true that hypotheses and models can emerge from in vitro studies, but they must be tested and validated in a whole organism, otherwise they remain speculative.


Molecules, cells and sometimes organs can be studied using in vitro approaches, but the exploration of physiological functions and systematic reactions between organs to disease and treatment, hormonal regulations and immune response requires a whole organism.6 Current technology and alternatives to animal research cannot yet reproduce the complexity of a living creature.


Banning animal experiments would effectively mean an end to testing new drugs safely and only using humans for all drug safety tests, which would be even more unethical and dangerous.5 It would also be extremely difficult for researchers to find human volunteers who would provide informed consent to the testing of a drug hitherto untested on animals. Potential dangers of the treatment to the living system will not yet have been identified, subjecting the human to unnecessary and avoidable harm.


Additionally, animals used in research are usually purpose-bred with deliberate gene modifications, with occasional experimentation processes leading to extensive gene and physiological damage to test subjects, requiring eventual euthanisation,21 which would be impossible in humans. The alternatives of human-only experimentation or the pausing of biomedical research are wildly unethical. As such, animal research has to continue.


Furthermore, promising non-animal tests like computer programs are limited by what is already known about a process or disease, working only because they draw on decades of animal research.20 They reveal gaps for further study in organisms and work together with animal experimentation to produce results. A cell culture- an alternative to animal tissue, is also extremely different from the functioning of a complex organism. Despite arguments from animal rights organisations,22 studies on animals cannot be fully replaced by in vitro methods.


Animal rights activists often use the differences between animal models and humans to refute their value.22 For instance, 95% of genes between mice and humans are homologous but there are also differences in members of gene families, in gene redundancies and in the fine regulation of gene expression level, which in turn translate into physiological differences.7 However, these small differences do not mean animal models are ineffective. As far as they can, scientists account for these differences in the experimental design. Another argument animal rights activists make is that due to genetic and physiological variations between animals, clinical presentation varies.22 However this variation is also represented in the human population as human disease is polymorphic among human patients and the difference between reactions of difference species actually provide unmatched opportunity to understand differential host response/s. A striking example is provided by a team who reported that some mouse strains are fully resistant to the Ebola virus while others die without specific symptoms and yet others develop fatal haemorrhagic fever. 23 Results obtained from animals may not always be confirmed in further human studies, but this does not mean that animal models are not extremely useful. Without animal models, many biomedical breakthroughs would never have taken place.


To conclude, misinformation about animal experimentation on animals can pose a huge challenge to science, preventing research that can be done for the greater good of society. In fact, more than half of the medicine formulated for humans by animal testing is also used in veterinary medicine today, benefitting farm, domestic and wild animals, helping them to live healthier and longer lives.7 In today’s world, many serious illnesses such as cancer, heart diseases, HIV, Alzheimer’s and Parkinson’s have no cure and they continue to take millions of human lives every year. In order to further science and continue to save human lives, the 3Rs should be implemented effectively and ethically, whilst allowing science to progress. Animal models must also be constantly improved to be more reliable and informative. The question about the scientific validity of animal experimentation for medical purposes is often confused with questions about complex ethical issues and separation of scientific and ethical questions is essential if greater clarity is to be achieved in the debate about animal research.


References

All references retrieved on 20/2/21 1) Nobel prizes: Ari.info. <http://www.animalresearch.info/en/medical-advances/nobelprizes/>

2) PETA. 2021. The Truth about Animal Testing | PETA. (online) <https://www.peta.org/issues/animals-used-for-experimentation/animal-testing-101/>

3) Speaking of Research. 2021. (online) <https://speakingofresearch.com/>

4) The European Citizens ‘Initiative – Stop vivisection. <http://ec.europa.eu>

5) Bbc.co.uk. 2021. BBC – Ethics – Animal ethics: Experimenting on animals (online) <http://www.bbc.co.uk/ethics/animals/using/experiments_1.shtml#:~:text=Animal%20experime nts%20are%20widely%20used,of%20life%20in%20other%20ways>

6) Speaking of Research. 2021. The Animal Model. (online) <https://speakingofresearch.com/facts/the-animal-model/>

7) Barré-Sinoussi, F., & Montagutelli, X. (2015, November 1). Animal models are essential to biological research: Issues and perspectives.

8) Four main reasons why animals are used in medical research.

http://www.animalresearch.info/en/designing-research/why-animals-are-used/

9) Nature news www.nature.com/scitable/topicpage/the-use-of-animal-models-in-studying-855

10) Animal testing and research.https://www.gov.uk/guidance/research-and-testing-using-animals

11) Animal research and human medicine, ABPI

https://www.abpi.org.uk/media/1451/animal_research_schools.pdf

12) Medical Research Council, M. (2020, May 04). Impact of animal research in the Covid-19 response. https://mrc.ukri.org/research/research-involving-animals/impact-of-animal-research- in-the-covid-19-response/

13) Animal models for covid-19. (2020, September 23) https://www.nature.com/articles/s41586- 020-2787-6

14) Official UK Coronavirus Dashboard. https://coronavirus.data.gov.uk/

15) Animal research numbers in 2019.

https://www.understandinganimalresearch.org.uk/news/communications-media/animal-

research-numbers-in-2019/

16) Doke, S. & Dhawale, S. (2013, November 18). Alternatives to animal testing: A review.

https://www.sciencedirect.com/science/article/pii/S1319016413001096

17) Computer-Aided drug design. https://www.sciencedirect.com/topics/pharmacology-toxicology-

and-pharmaceutical-science/computer-aided-drug-design

18) Bassett, D., & Currie, P. (2003, October 15). Zebrafish as a model for muscular dystrophy and congenital myopathy. https://academic.oup.com/hmg/article/12/suppl_2/R265/620445

19) Drosophila melanogaster. https://www.sciencedirect.com/topics/pharmacology-toxicology-and- pharmaceutical-science/drosophila-melanogaster

20) Cosmetics.https://www.understandinganimalresearch.org.uk/openness/cosmetics/#:~:text=Anim al%20testing%20for%20cosmetics,being%20sold%20in%20the%20EU

21) Kabene, S., & Baadel, S. (2019, November 12). Bioethics: A look at animal testing in medicine and cosmetics in the UK. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7166243/

22) Top five reasons to stop animal testing. (2020, November 25) .https://www.peta.org/blog/top- five-reasons-stop-animal-testing

23) Rasmussen AL, Okumura A, Ferris MT, et al. Host genetic diversity enables Ebola haemorrhagic fever pathogenesis and resistance. Science. 2014; 346 (6212): 987-99


Figure 1. The Animal Model, Speaking of Research. https://speakingofresearch.com/facts/the- animal-model/


Figure 2. Traditional Vaccine Development Pathway, BioPharma. https://www.biopharma- excellence.com/news/2020/8/20/how-far-are-we-with-the-accelerated-development-of-sars-cov- 2-vaccines-a-review-and-outlook















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