A Review on Alternative Methods to Experimental Animals in Biological Testing: Recent Advancement and Current Strategies

ABSTRACT

With an increase in the progression of research and development in the medical field, the experimental use of animals for the efficacy and safety testing of pharmaceuticals is on rise. Every year, millions of animals are used for experimental testing during which these suffer from pain and are then eventually sacrificed. Besides bioethical issues, animal experimentation is associated with many disadvantages like high cost, the requirement of skilled manpower, approval, and is time-consuming. Therefore, attempts have been made by researchers to design and develop a number of alternative methods that could bypass animal experiments. These methods not only give accurate results but can also save lives of millions of animals annually. Research techniques, including computer and robotics together with molecular biology techniques, are applied to discover new methods to replace animal testing. Several alternative methods are discussed in this review. Some of these methods can predict the behavior of drugs accurately and are as reliable as in-vivo animal models. Furthermore, these alternative methods offer a variety of advantages over experimental animals. However, there is still a great need to discover and develop new, accurate, and reliable methods to replace experimental animals.

INTRODUCTION

Humans are constantly exposed to dangerous pathogens, which are known to cause many life-threatening diseases. Since ancient times, mankind strived to discover and develop new chemical entities that could be used effectively to diagnose, prevent, and cure communicable and non-communicable diseases (NCDs). However, a successful discovery and development of a drug molecule involve a series of trials of experimental animal studies to ensure their efficacy and safety before use in humans. In the recent past, the utilization of animals increased dramatically due to the explosion of research information/experimentation in various medical fields. The use of experimental animals as a disease or testing model in academics is to educate the students, although, in research, animal studies help in studying the efficacy, safety, and pharmacokinetics of drug candidate(s). Animals like mice, rats, rabbits, fishes, birds, guinea pigs, hamsters, and dogs are being used for the research work. They are used as a tool to understand the effect of new chemical compounds, medicines, and biologics such as vaccines, medical procedures, and surgical instruments. For infectious and noninfectious diseases, drug testing and toxicological screening are performed with the help of animals.[1,2] Many animal models have been developed to evaluate in-vivo drug efficacy and toxicity studies for a variety of diseases, including cancer, diabetes, and acquired immunodeficiency syndrome (AIDS). The death of experimental animals has been seen as a result of experimentation, especially in the evaluation of lethal dose 50 (LD₅₀).

During scientific experimentation, animals experience pain, distress, and death, which has become a debating issue.[3,4] All over the world, millions of experimental animals are being tortured and sacrificed to conduct scientific procedures for educational and research purposes.[5] However, all these are conducted and regulated under well-established ethical committees, including institutional ones, that maintains the research animal ethics to conduct the scientific experiment through animal models. Some social activists and animal lovers are against the use of animals in experiments as they believe that this practice is too cruel towards animals and thus unethical. Various laws, rules, regulations, and guidelines have been framed internationally during recent decades for performing experiments on animals.[6,7] Ethical review committees in institutions approve only minimum acceptable animals for a particular experiment that causes the least possible suffering, which is consistent with the gain of its scientific purposes.

An alternative, replacement, or nonanimal method is a method or test which uses other methods of testing instead of live animals, that is, an alternative method replaces, reduces, or refines an animal experiment. It comprises “testing methods” like ex vivo, in vitro, in vivo, in silico, or in chemico reduced/refined methods, along with the use of expert systems, which comes under non-testing methods.

Many scientific and validated methods favoring the welfare of animals exist that could potentially replace, reduce or refine the use of experimental animals. These methods have been touted as significant alternative sources for conducting different biological research and experiments.[5] Computer-based drug designing, discoveries, docking, and simulation studies make a ray of hope to develop new therapeutics. Thus herein, we have explored the area of alternative ways that replace, reduce or refine the use of experimental animals in different biological experiments and research.[8]

ALTERNATIVE METHODS TO ANIMAL EXPERIMENTS

The alternative methods are constantly discovered to replace the use of animals, generally on the lines of the principle of 3Rs (replacement, reduction, and refinement). Russell and Burch[9], in 1959, first-time reported that the 3Rs are the supervisory system for much ethical practices of animals.[9-11]

Replacement

It refers to the procedure of replacing larger animals with smaller ones. For example, warm-blooded animals may be replaced with various microorganisms, plants, eggs, reptiles, and invertebrates. New analyzing techniques such as cell cultures, in-vitro models, and computer models can also be used as an alternative to the use of animals.[12] In short, this process of replacement will reduce the use of animals in various processes of research.[13]

Reduction

It simply means to diminish the number of animals in an experiment.[9] Because of the development of modular experimentation design and complex statistical techniques, it becomes possible to use a few animals with suitable results. In-vitro embryonic stem cell culture tests are used to reduce the number of live embryos. To prevent the necessity of animal studies, discovered data can be shared (like characteristics of excipients for the test drug), including results collected from each animal and collected results for more than one experiment simultaneously.[11]

Refinement

It aims to reduce the pain and distress of animals in experimental protocols. To diminish the pain, discomfort, and distress throughout the life of animal and experimental procedures, scientists should refine the animal facility.[9] Results can be varied due to the disproportion in hormonal levels of animals under stress and discomfort. There is a need to repeat the experiments, which include a high number of animals.

Besides the 3Rs system, another R has also been introduced, which emphasizes the 3Rs concept in the alternative methods for animals in research. It describes that research on reproductive studies in animals concerning on immunological response could be a future directive to understand the pathophysiology of many disorders in human pregnancy. Much work has been done to explore alternative ways to substitute research animals, including tissue, bio bank-derived tissue, engineering approaches, organ-on-a-chip devices, or computational methods.[13]

ALTERNATIVES TO ANIMAL MODEL: COMMON MODEL ORGANISMS

During the laboratory procedure of higher vertebrate models such as rats, guinea pigs, dogs, monkeys, etc., restrictions have been imposed due to ethical issues. So, the use of other animals has been explored. Recently, cell culture, embryonic stem cells, and nonmammalian species of reptiles, Amphibia, Ave such as loggerhead sea turtle, saltwater crocodile, northern water snake, corn snake, western hognose snake, eastern diamondback rattlesnake, northern leopard frog, northern pike, soft-shell clam, African black-footed penguin, Adelie penguin, Sulfur-crested cockatoo cockatiel, Plymouth rock chicken have been used as different types of cancer models to study metastasis.[14]

Lower vertebrates

For replacing mammals (higher vertebrates), lower vertebrates are the finest alternative because of their genetic similarity. Plus, in the experimental use of lower vertebrates, there are fewer ethical problems. Lower vertebrates have a short life cycle and are studied in large numbers.

Danio rerio (D. rerio): It is also called as zebra fish. D. rerio having length of 2–4 cm is a small freshwater fish. During the initial level of development, it has a transparent body, due to which internal anatomy can be easily viewed. By opting for D. rerio in place of animals, the cost of working space of experimental chemical solutions required for the test, and the manpower concerned are decreased.[15]

Invertebrates

The use of invertebrates has also been reported extensively as an alternative to the experimental use of animals. These have been used for research on many diseases like memory dysfunction, endocrine Parkinson’s disease, cell aging, diabetes, and toxicological testing. They have become the choice of researchers because of their small size, simple anatomy, and brief life cycle.[10]

Drosophila melanogaster: It is also called as a fruit fly. In research, it is one of the most commonly studied invertebrates. As compared to the other mammal-based models, it requires a low cost of maintenance, propagation, and screening. To examine neurodegenerative diseases like Huntington’s disease, Parkinson’s, and Alzheimer’s disease, it is an important tool.[13]

Caenorhabditis elegans (C. elegans): It is a multicellular eukaryotic nematode having approximately a length of 1 mm and has a very short generation time. This organism is used to study many disorders of neurons, such as Parkinson’s disease, Huntington’s disease, various immune disorders, cancer, diabetes, etc.[16]

Microorganisms

Microbes are greatly used as an alternative for animal experimentation. They have been studied for various biological diseases and their effect on life.

Saccharomyces cerevisiae is a brewing yeast, which is considered as the most vital and admired model organism.[17] S. cerevisiae is used to study various neurodegenerative diseases like Huntington’s disease, Parkinson’s, and Alzheimer’s disease.[16-18] Microorganisms like Escherichia coli (bacterium), Schizosaccharomyces pombe (fungus), and Dictyostelium discoideum (Protista) are excellent models for molecular and genetic studies, although cellular differentiation can be studied with the help of Bacillus subtilis.[5]

TYPES OF ALTERNATIVES TECHNIQUES

Researchers are very interested in creating innovative in-vitro disease models as a result of technology developments in tissue engineering and microfabrication technology [Figure 1]. The following are some of the in-vitro and in-vivo alternatives to animals:

Figure 1.

In-vitro and in-vivo alternative techniques to replace animal experiments in drug screening, development, and approval process. The scope of these techniques to replace and reduce experimental animals is widening, suggesting the potential to refine, reduce, and ultimately replace animal testing

In-vitro cell/tissue culture techniques

Cell culture

Cell culture can replace a number of animal experiments. For various research purposes, different types of organ cultures, tissue culture, callus culture, and cell culture are used.[19]

Usually, the immortalized primary cell culture has been reported to analyze the toxicity in cells cultured in vitro.[20,21] Moreover, this area has potentialities in the field of recombinant DNA technology, cell-based bioassays, screening of drugs, and gene therapy as an alternative to animal models.[6]

In-vitro pyrogen test

This is based on the leukocytes’ response upon pyrogen contamination, and release of inflammatory mediators. The pyrogen testing is performed on rabbits, which is replaced with the following tests:

Limulus Amoebocyte Lysate (LAL): LAL is an aqueous extract of blood cells of Limulus polyphemus or horseshoe crab. LAL is used for testing of pharmaceuticals and testing devices for blood or cerebrospinal fluid.[22]

Monocyte Activation Test (MAT): This test uses cryopreserved human whole blood and produces an interleukin-1β response when pyrogens are identified. It is superior to LAL and rabbit pyrogen tests.[2,23]

Neutral red uptake test

This test is used as a substitute to the Draize Rabbit eye test. In this test, neutral red penetrates into cell membrane, which is accumulated in lysosomes intracellularly.[24,25]

Cell transformation assays

Cell transformation assays are faster, expensive, and involve very few animals. It is an alternative to rodent bioassay as well as a transgenic mouse model for carcinogenicity assay.[25,26] Examples include Balb/c3T3 assay and the Syrian hamster embryo (SHE).

Stem cell models

For in-vitro toxicological evaluation of diseases, the stem cell models are used as an alternative to animal in experimentation.[27-29]

The future potentials of mesenchymal stem cell (MSC) in the field of regenerative medicines have been well explored.[30-33] Recently, in the welfare of human safety and animal, the use of stem cells has been increased to screen the toxicity studies of many novel molecules.[5] These methods have not only advantages but also some disadvantages.

Microbiological tests

About 80–90% of all carcinogenic chemicals are detected by the Ames test, a microbiological-based assay. Primarily it is used as a screening system. After accurate validation, it also has a potential as an alternative to animal testing.[26]

Membrane model

Alternative to the Draize test, irritation response can be evaluated by the hen’s egg test (HET), in which chorioallantoic membrane and/or vessels are used to conduct the experiment. This test is also referred to as the HET-chorioallantoic membrane (HET-CAM) test, which is based on hemorrhage, coagulation, and lysis.

Organ model

Cornea collected from a freshly slaughtered animal, cornea of dead rabbit and chicken can be used as an experimental model to evaluate irritational eye toxicity.[34] Primary human corneal cells have been used commercially (Biosolution Co., Seoul, South Korea) to produce MCTT HCE™ for the evaluation of in vitro eye irritation.[35]

Human skin-derived epidermal keratinocytes

These are used commercially to produce the epithelial model EpiOcular™ as an alternative model for the evaluation of skin irritation.[35] The human keratinocyte viability also has been used in neutral red uptake (NRU) assay to evaluate in-vivo animal skin irritation.[36,37]

In silico methods and computer models

By using specially developed computer programs, the required calculations are performed. There are a variety of computer-based simulation software, which are being used for education purposes all over the world, and this replaces the use of animal models for education purposes.[3,38-40]

Also, with the help of such software programs, a new drug can be modified and made for the specific binding site, and later on, in the final stage, animal testing is performed to obtain the results.

In - vivo alternative techniques

Micro dosing

Micro dosing is defined as less than one hundredth of the pharmacological dose up to a maximum of 100 μg. This method helps to screen drugs that will not work in humans, and thereby there is no need for the government animal testing information upon the safety of an experimental drug and the process of metabolism, which will take place in humans before large-scale human trials.

TECHNOLOGICAL ADVANCEMENT IN ALTERNATIVE METHODS

Researchers have been using two-dimensional (2D) culture, animal models, or cadavers for decades to provide insightful data on disease causes, medication testing, and safety evaluations; nevertheless, the reliability, relevance, and repeatability of human translation are debatable. The idea of creating tissue/organ and in vitro models to repair and/or restore injured tissue/organs, as well as predictive and trustworthy systems for drug testing and disease modeling, has, therefore, rapidly grown over the past few decades.[41-43] Researchers have access to much more sophisticated in vitro models, such as organoids, multiphysiological systems, and organs-on-chips. Several of these in vitro models are at par or superior to animal models.

Human-patient simulators

Computerized human patient simulators that bleed, breathe, shake, talk, and even die are used to teach students physiology and pharmacology. These simulators are superior then cutting up animals.[44-48]

Organ-on-a-chip and biological chip

To mimic the structure and function of a human organ and organ system, human cells are grown in a state-of-the-art system on a chip. These chips can be used for drug testing, disease research, and toxicity studies rather than using animals.

Organoids, computer simulations, and other technologies that replicate drug interactions have recently come to the forefront; as a result, it is extremely feasible that the day of animal-free drug testing is closer than we believe.[49]

Organoids

To research cell behavior in a setting resembling the human body, organoids were created as an alternative to 2D cell culture. They are in vitro simplified and miniaturized organ model systems that have attracted a great deal of interest for cell therapy, drug screening, and personalized medicine as well as for modeling tissue development and illness.[50] The pharmaceutical industry has staked a major reduction in animal testing and significant cost savings on the findings of researchers in this field.[51]

Three-dimensional (3D) bioprinting

Through 3D bioprinting, which is increasingly promising for the creation of customized in vitro models of significant scientific and medical value, 3D cell culture offers itself as an alternative to animal models. In place of using animal models, 3D bioprinting can advance research by lowering expenses, accelerating the pace of research development, and increasing the likelihood that novel treatments will be successful.

In contrast to expensive experimental animal testing, 3D-culture systems offer more accurate in-vivo data and a superior translational model.

3D Human derived models

The recent development of 3D human-derived models used for translation into non-animal technologies represents a significant advancement. Researchers should be able to obtain more vitally pertinent human data that can be used for clinical trial design and enhancing successful clinical outcomes by optimizing these human-derived models with-omics and in silico techniques.

Artificial intelligence (AI): Big data and machine learning (ML) in 3Rs

In the past 10 years, computational approaches have advanced significantly and are now capable of identifying patterns in data that humans have never seen. This is particularly true in the field of computational toxicology, where algorithms that predict toxicity by comparing chemical structures do better than reproducible animal tests. AI models can help in saving the life of millions of animals by using computer vision and trustworthy datasets.[52,53]

As illustrated in, big data and data mining sources could completely transform the way animals are used in the future [Figure 2].[52,53]

Figure 2.

Prospective likelihood/ Future of artificial intelligence (AI) surpassing animal testing in drug development and approval process

The scientists have created an ML model entitled “Simple RASAR” built-in logistic regression to forecast dangers from similarities for each chemical as a way to illustrate this. By using cross-validation, simple RASAR (read-across structure activity relationships) and Data Fusion RASAR are able to forecast hazards with an accuracy of between 80 and 95%. The test reproducibility in relation to OECD guidelines, simple RASAR, and data fusion RASAR modes, respectively, are the second and third primary outcomes reported in the reports. The reproducibility accuracy performed admirably in terms of precision and chemical specificity (more than 90% accurate). It is comparable to animal experiments, in other words.

The advent of quantum computing has opened the door to a plethora of studies and findings.[54]

Bioinformatics and sophisticated analytical methods are required for the curation and comprehension of data. Practically, “click and play” technologies cannot be expected to exist in the future.

CONCLUSION

Several alternative methods to animal testing have been successfully applied to a variety of biological tests. These methods not only give accurate results but also save the lives of animals. It is the responsibility of researchers to bring out better models and methodologies for the betterment of humans and animals. It is expected that several newer, novel alternative methods to animal testing will soon be discovered, which will further help in improving our understanding of various diseases, diagnoses, bio-processes, treatments, and mechanisms of new drug molecules.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.