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Shifting the Balance from Animals to New Approach Methods: We’re Working Toward It!

Barbara L. F. Kaplan and William Slikker, Jr.

            Animals and animal models have been used for centuries to provide us with an understanding of basic physiology, disease processes, and treatment options for disease. For instance, while insulin is recognized today as an essential drug for those with Type I diabetes, it took many years of animal studies to understand basic regulation of glucose levels, identify that lack of glucose control leads to a life-threatening disease, and ultimately, purify and use insulin from animals as a treatment (1). In addition to identification of thousands of drugs, treatments, and medical devices discovered in part through use of animals and animal models, many of these same approaches have been used to establish the safe use of chemicals for myriad purposes, including industry, home, cosmetic, and even food use. However, animals and animal models do not always provide all the information needed to treat human diseases, so scientists employ the “3Rs:” replacement, reduction, and refinement of animal use. In addition, scientists are increasing development of alternatives to animal usage called new approach methods (NAMs). As defined in our recent review, “NAMs are any technology, methodology, approach, or assay used to understand effects and mechanisms of drugs or chemicals with specific focus on applying the 3Rs” (2). 

            The primary objective of our review was to provide a balanced view of the current state of use of animal models and NAMs in the biomedical sciences. As indicated above with the insulin example, animals have been invaluable for many scientific discoveries. Certainly, studying animals is still critical for advances in veterinary medicine, but can also provide insights into human medicine. In some cases, this overlaps; osteosarcoma, for instance, is a cancer in the long bones that exhibits similarities in dogs and humans. In fact, the National Cancer Institute (NCI) in the US is studying osteosarcoma that develops spontaneously in pet dogs to increase the understanding of the disease process and develop therapies that might benefit dogs and humans (3). Even as some animals continue to be used for veterinary and human medical discoveries, scientists are always trying to employ the principles of the 3Rs. As one example, longitudinal study design uses repeat measurements or minimally invasive imaging in the same animal (4, 5). Approaches such as these rely on the reuse of the same animals throughout an assessment, resulting in an overall reduction of animal use.  

            Although the above exemplify continued benefit of some animal use, many in the biomedical sciences are working on alternatives to animal use such as NAMs. NAMs include in vitro assays, which are cells that can be maintained and treated in dishes, typically called cultures. These can be traditional 2-dimensional cultures comprised of one or more cell types, or 3-dimensional cultures, sometimes called organoids, comprised of multiple cell types that have the ability to interact (6). These culture systems can be done with almost any cell, including human cells. NAMs also include microphysiological systems such as organ-on-a-chip (7) or 3D tissue printing (8), which attempt to mimic cellular interactions that occur in a tissue. For instance, lung-on-a-chip includes various cell types specifically arranged to mimic respiratory membranes, other lung cells such as alveolar cells, and might also include immune cells since lungs are often exposed to various diseases. Lung-on-a-chip also contains blood or a blood mimic, which can be altered to assess speed and force of blood flow through the lungs and allows one to examine the air-liquid interface that is critical for gas exchange (7).

            Many of the above NAMs either reduce or replace animal use, but computational models and artificial intelligence (also known as in silico models) might be used alone or in combination with already-obtained data as another means to replace animals (9). In silico models include structure-activity relationship assessments to compare how differences in chemicals affect cellular functions, software that predicts how a chemical might act or whether it might be expected to exhibit an adverse effect based on its structure, and physiologically-based models in which mathematically-derived rates of entry or exit from a tissue might provide insights to its biological distribution and effects. Artificial intelligence and deep learning approaches (i.e., teaching computers by attempting to simulate the human brain) are under development to generate animal model study results of new compounds that are based on existing data without testing the new compound in animals (10).

            While we are all working toward increased use of NAMs, we should be cognizant of the limitations of both animal models and NAMs (2). Neither animal models nor NAMs can completely replicate a human disease. NAMs as in vitro cultures are typically limited to one or a few cell types, and NAMs as in vitro cultures, or microphysiological or in silico systems do not replicate the complexity of human systems, whether healthy or diseased, at this time. Regardless, biomedical scientists are continuing to increase NAMs development and application in their work. Data obtained from NAMs can either complement or reveal different information than we obtained from animals. Most of all, it is important to realize that many scientists are making the effort to minimize animal use. Hopefully, NAMs can eventually further reduce and replace animal use in some areas of science. We’re working toward it!

1.         P. Home, The evolution of insulin therapy. Diabetes Res Clin Pract 175, 108816 (2021).

2.         B. L. F. Kaplan et al., Protecting Human and Animal Health: The Road From Animal Models to New Approach Methods. Pharmacol Rev 76,  (2024).

3.         National Cancer Institute, 2019, Helping Dogs—and Humans—with Cancer: NCI’s Comparative Oncology Studies

4.         E. Anklam et al., Emerging technologies and their impact on regulatory science. Exp Biol Med (Maywood) 247, 1-75 (2022).

5.         S. Z. Imam et al., Changes in the metabolome and microRNA levels in biological fluids might represent biomarkers of neurotoxicity: A trimethyltin study. Exp Biol Med (Maywood) 243, 228-236 (2018).

6.         H. Wang et al., 3D cell culture models: Drug pharmacokinetics, safety assessment, and regulatory consideration. Clin Transl Sci 14, 1659-1680 (2021).

7.         Y. Zhang et al., Recapitulating essential pathophysiological characteristics in lung-on-a-chip for disease studies. Front Immunol 14, 1093460 (2023).

8.         Y. Yan et al., 3D bioprinting of human neural tissues with functional connectivity. Cell Stem Cell 31, 260-274 e267 (2024).

9.         J. C. Madden, S. J. Enoch, A. Paini, M. T. D. Cronin, A Review of In Silico Tools as Alternatives to Animal Testing: Principles, Resources and Applications. Altern Lab Anim 48, 146-172 (2020).

10.       X. Chen, R. Roberts, W. Tong, Z. Liu, Tox-GAN: An Artificial Intelligence Approach Alternative to Animal Studies-A Case Study With Toxicogenomics. Toxicol Sci 186, 242-259 (2022).

Dr. Barbara Kaplan is an Associate Professor in the Center for Environmental Health Sciences, Department of Comparative Biomedical Sciences in the College of Veterinary Medicine at Mississippi State University. She has a BS in Environmental Toxicology from University of California at Davis and a Ph.D. in Pharmacology and Toxicology from Michigan State University. She has mentored 2 DVM/PhD students and 2 PhD students. She and her co-author are members of the Scientific Liaison Coalition (SLC), with the goal of linking together over 10 different scientific societies to “…improve and enhance human and environmental health by promoting and strengthening scientific partnerships…”.

Dr. William Slikker, Jr. received his Ph.D. in Pharmacology and Toxicology from the University of California at Davis. He performed his fellowship in the Perinatal Research Program at the National Center for Toxicological Research (NCTR/FDA) and completed a sabbatical at the Freie Universitat, Berlin, Germany. Dr. Slikker recently retired after serving 16 years as the Director of NCTR/FDA. He has had the privilege to mentor a dozen PhD students and over 20 Postdoctoral Fellows.

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