Please find an introductory article to the concept of the 3Rs in animal testing here
In a world where the role and value of animals in science is becoming increasingly disputed and
ethically critiqued, it is vital that we develop alternative methods to replace these experiments and keep the momentum behind scientific advancements. As introduced in our initial article on the
3Rs (Replacement, Reduction, and Refinement), replacement involves the use of humane methods to either
avoid or replace the use of animal experimentation. The NC3Rs splits replacement into two broad categories; partial replacement and full replacement. This article will explore each of these concepts in more details.
Partial replacement
Partial replacement in research involves using alternative methods or organisms to reduce reliance on traditional experimental animals. It aims to address ethical concerns while meeting scientific objectives. This approach often includes animals less likely to feel pain, such as invertebrates and lower vertebrates. Examples of partial replacement include using
fruit flies (Drosophila melanogaster), nematode worms, social amoebae, and zebrafish (Danio rerio). Additionally, partial replacement can involve using cells or tissues from animals not exposed to harmful procedures. This method balances ethical responsibility with the need for effective research models.
Lower Vertebrates
Zebrafish (
Danio rerio), as lower vertebrates, are valuable alternatives in research due to their genetic similarity to higher vertebrates and fewer ethical concerns. Their transparent embryos allow easy observation of internal structures during early development. This transparency aids studies on organ development, toxicity testing, mutagenesis, and gene expression. Zebrafish are cost-effective, with a short life cycle, high fertility, and low maintenance needs. With a fully sequenced genome, zebrafish provide crucial insights into genetic and molecular research. They are widely used in developmental biology, cancer studies, cardiovascular research, and neurobehavioral disorder models. A study by
Chen et al. utilised zebrafish to investigate social behavior deficits by exposing embryos to valproic acid (VPA), a known teratogen. Such studies are significant as they allow high-throughput screening of neuroactive compounds and facilitate the understanding of genetic and environmental contributions to ASD.
Figure 1: Zebrafish (Danio rerio)
Invertebrates
Invertebrates like fruit flies (
Drosophila melanogaster) and nematodes (
Caenorhabditis elegans) are popular alternatives in research due to their short life cycles, simple structure, and low costs.
Fruit flies (Drosophila melanogaster) are extensively studied and have a well-mapped genome. Around 75% of human disease-related genes have counterparts in fruit flies, making them ideal for genetic research. They are widely used in studies on neurodegenerative diseases, drug development, and behaviour, providing quick and accurate results. Nematodes (
Caenorhabditis elegans) are worms crucial for studying neurological diseases, immune system issues, and cancer. Their transparent bodies and short lifespans allow direct observation of developmental and physiological processes.
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Figure 2: Fruit fly (Drosophila). Figure 3: Nematode (Caenorhabditis)
Cells or tissues from animals or humans
Partial replacement also involves using cells and tissues sourced ethically from animals or humans without causing harm. This method avoids the need for live animal testing while ensuring scientific precision. Human tissues, obtained with informed consent, have been instrumental in studying various diseases. Research on HIV (Human Immunodeficiency Virus) has also greatly benefited from analysing human tissues, particularly blood. Since these diseases primarily affect humans, using human tissue is often more relevant than animal models.
The use of partial replacement strategies reflects a thoughtful
balance between ethical responsibility and scientific advancement. Integrating
lower vertebrates and invertebrates into research practices helps minimise ethical concerns while enabling the pursuit of valuable scientific insights. Ongoing innovations in technology and research methods will further enhance these strategies, paving the way for more ethically sound scientific endeavours.
Full replacement
Full replacement refers to the use of alternative methods that completely bypass the use of animal models. These approaches aim to mimic biological processes and systems without the need for live animal experimentation. As with partial replacement, the push for the development of models to fully replace animal testing has gained significant momentum in recent years, driven by ethical concerns, technological advancements, and the desire for more accurate, human-relevant results.
Computational models
Computational models, or “in silico” modelling, leverage advanced algorithms, machine learning and artificial intelligence to simulate human biology, predict toxicological outcomes, and assess the efficacy of drug compounds. These models can predict how a drug will interact with human biology, from molecular-interactions to organ-specific effects. They rely on vast amounts of biological data and machine learning algorithms to simulate biological systems, offering faster and potentially more accurate results than traditional animal testing. For example,
physiologically based pharmacokinetic (PBPK) models simulate the absorption, distribution, metabolism, and elimination (ADME) of drugs using anatomical and physiological data whilst
quantitative systems pharmacology (QSP) employs mathematical equations representing biological systems to enable simulation of the potential therapeutic effects of new drugs. Established software platform such as
GastroPlus™ and
Simcyp® also utilise computational modelling to predict oral bioavailability and inform formulation strategies. Each of these modelling approaches enables modern pharmaceutical research to predict drug behaviour and safety, without the need for animal research.
One example of how computational models are enhancing drug discovery is in cardiac safety testing in safety pharmacological studies. Until recently, scientists focused primarily on the human ether-à-go-go-related gene (hERG) ion channel to determine whether a drug could potentially cause cardiac arrhythmias (
the hERG channel inhibition assay). If a drug blocked the hERG ion channel, it was considered unsafe. However, this approach oversimplifies the complex physiological mechanisms that contribute to arrhythmias. In reality, multiple ion channels can play a role, and understanding their interactions is challenging. As a result, a drug that fails the hERG test may still be safe and could be prematurely rejected. Dr. Heitmann of the Victor Chang Cardiac Research Institute has led a project to develop a computer model that predicts arrhythmia risk with
approximately 90% accuracy, compared to 75% from animal-based testing. This promising advancement reduces reliance on animal testing and offers a pathway for drugs previously deemed unsafe to progress through clinical trials.
Organ-on-a-chip technology
Organ-on-a-chip (OoC) technology is an innovative approach that mimics the functions of human organs in a controlled, miniaturised environment. These devices are typically composed of microfluidic systems that house living human cells in order to mimic human physiology. The chips are designed to control cell microenvironments and maintain tissue-specific functions, enabling researchers to study the behaviour of organs in response to various drugs, toxins, or diseases. By replicating the mechanical, biochemical, and physiological properties of human tissues, OoC models offer a more accurate representation of human biology compared to traditional animal models. This technology is already being applied in a commercial pharmaceutical setting. For example, Roche have developed an OoC system that models the human gut i.e.
colon-on-a-chip. This technology utilises the patient’s own cells from their colon to replicated the cellular diversity of the human gastrointestinal tract, allowing researchers to test and optimise therapies tailored to the individual patient.
This technology is transforming drug discovery, toxicity testing, and disease modelling by providing a platform for high-throughput screening, personalised medicine, and reducing the reliance on animal testing. Moreover, it holds great potential for advancing our understanding of complex diseases and developing more effective, targeted treatments. Whilst the current focus of these models is on singular organs, it is hoped that with the success and promise of this technology, multiple OoCs could be incorporated together to model a complex biological environment, effectively duplicating a human model.
Alternatives to animal testing will likely become increasingly utilised since the transformative introduction of the
FDA Modernisation Act 2.0, signed into US law in 2022. This legislation permits the use of alternatives to animal based testing, including cell-based assays, sophisticated computational modelling, and organs-on-chips, to obtain FDA exemptions for assessing drug safety and effectiveness during the preclinical stage. This will allow preclinical research to adopt methods that may more accurately simulate human responses, rather than being dependent on outdated, costly and often unreliable animal testing.
Concluding remarks
Collectively, these replacement techniques offer a more ethical, cost-effective and accurate approach to research, hopefully reducing the need for animal testing while advancing scientific progress. However, challenges remain, such as the need for standardisation, regulatory acceptance and continuous refinement of these models. Whilst there is still a need for animal testing within drug development, the continuous development and advancement of these replacement approaches is a significant step forward in reducing the need for animal models and creating more cost-effective and accurate research methods.
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