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Kibur Science Spotlight: An Introduction to the 3Rs of Animal Research

Animals from fish and amphibians to rodents and non-human primates have become indispensable as models for human disease, biology and behaviors in biomedical research. They have proven indispensable in developing vaccines, novel medicines and medical devices that have greatly improved human health, longevity and wellbeing.  It is estimated between 11 and 24 million rats and mice are used in the US on an annual basis for biomedical research and approximately 90% of the animals used in research today are mice, rats, birds or fish (University of Pittsburgh 2022). It has been demonstrated that poor animal care can cause physical and emotional stress to the animals and changes in social behaviors that can alter experimental outcomes. Data gained from those experiments may be inaccurate and difficult to replicate (Kiani et al 2022). 

Given their sacrifice to better the human condition, it is essential researchers follow ethical guidelines established by the Animal Welfare Act of 1966, to “ensure that minimum standards of care and treatment be provided for certain animals bred for commercial sale; used in research, teaching, or testing; transported commercially; or exhibited to the public.” (USDA 2023). These guidelines have been codified in procedures established by national funding agencies such as the National Institutes of Health, professional organizations such as the American Veterinary Medical Association and university Institutional Animal Care and Use Committees in the design of their experiments and investigations with animals. 

An important milestone in setting these guidelines was the book The Principles of Human Experimental Technique published in 1959 by William Russell and Rex Burch, proposing new methods and approaches for the treatment of laboratory animals while improving data quality (Tannenbaum and Bennett 2015). They proposed the 3Rs of animal research: Replacement, Reduction and Refinement – to provide a standard framework for use of animals in biomedical research: 

Replacement speaks to methods that directly replace or avoid the use of animals; 

Reduction refers to methods that gain a significant amount of data while minimizing the number of animals consumed.

Refinement addresses methods that minimize or eliminate the animal’s pain or suffering and seek to improve their wellbeing.

A fourth R, Responsibility, has been proposed recently by the biomedical research community to advance responsible animal use in laboratory research. Examples of how the fourth R is put into practice is by investing and collaborating with new alternatives to animal models, connecting with clients to refine the use of animals, developing new models and services that advance quality research and reduce reliance on animal models, and partnering with regulatory agencies to align guidelines with the scientific process.  

This initiative has become public policy as evidenced in the recent FDA Modernization Act 2.0 that allows for alternatives to animal testing(Han 2023). This complements and extends the Federal Food, Drug, and Cosmetics Act of 1938, which mandated animal testing for every new drug development protocol to ensure certain quality and safety standards for drugs and medical devices.  FDA 2.0 recognizes the advancements in science that have begun to offer increasingly viable alternatives to animal testing. In drug development, these alternatives include lab-on-a-chip or organoid technologies that strive to mimic in vitro human biology and disease processes in engineered, artificial systems. The 4R framework and ethical considerations still apply to these in vitro systems since they require cells from a human or animal to function. Despite this progress, animal models of human disease are still preferred given the body of knowledge and the biological tool set that has evolved to exquisitely design and manipulate biological material to craft model systems replicating human biology and disease in an animal. 

The Kibur Medical NanoNail™ is an example of a 4R technology that greatly improves the efficacy and efficiency of oncology drug development by testing the sensitivity of a whole tumor with an intact microenvironment to up to twenty (20) different drugs or drug combinations simultaneously. Deep molecular profiling of the tumor response to each drug or combination reveals mechanisms of efficacy and action, maximizing important data from each study needed by drug developers to make better oncology drugs. The decrease in the number of animals needed for an oncology study is dramatic. In one recent study with our strategic partner Charles River Laboratories to identify effective immunotherapy combinations for treatment of renal cell carcinoma, inclusion of the NanoNail in the study design decreased the number of mice in the study by six-fold compared with conventional study design. Not only was the animal consumption and systemic exposure to toxicity dramatically reduced but the mouse-to-mouse biological variability was also substantially decreased. Combined with the larger number of technical replicates per measurement, the data quality for the study was greatly improved(Charles River Labs 2023).  

The NanoNail also supports Phase 0 microdosing studies whereby similar multi-factorial studies can be performed in tumors in human cancer patients without risk of systemic toxicity.  NanoNails with drugs to be tested are inserted into a patient’s tumor in advance of surgery, drugs on the device at doses 1/100,000th of a systemic dose are released into the adjacent tumor tissue during the indwelling time of typically 3-5 days.  The NanoNails are retrieved post-surgery for processing to reveal the tumor response to all drugs and drug conditions tested. These pre-IND studies will unlikely supplant animal or in vitro studies but are highly synergistic by providing direct, in-human data on the pharmacodynamics of experimental drugs in a whole human tumor with an intact microenvironment. The NanoNail is a prime example of technology supporting the 4Rs in the ethical treatment of animals in biomedical research but also an important step forward to decreasing clinical attrition by direct testing of drug efficacy in humans without exposure to toxicity.



Charles River Laboratories. (n.d.). Animals in research

Charles River Labs 2023. Revolutionizing Oncology Drug Development. 

Congressional Research Services. (2023). The animal welfare act: Background and selected issues. 

Graham M, Prescott MJ. The multifactorial role of the 3Rs in shifting the harm-benefit analysis in animal models of disease. Eur J Pharmacol. 2015 Jul 15;759:19-29.

Han JJ. FDA Modernization Act 2.0 allows for alternatives to animal testing. Artif Organs. 2023 Mar;47(3):449-450.

Kiani A, Pheby D, et al. Ethical considerations regarding animal experimentation. Journal of Preventive Medicine and Hygiene. 2022 Oct. 

National Agricultural Library. (n.d.). Animal Use Alternatives (3Rs). Animal Use Alternatives (3Rs)

National Research Council (US) and Institute of Medicine (US) Committee on the Use of Laboratory Animals in Biomedical and Behavioral Research. Use of Laboratory Animals in Biomedical and Behavioral Research. Washington (DC): National Academies Press (US); 1988. 2, Patterns of Animal Use. 

NC3R The 3Rs. NC3Rs. (n.d.). 

Tannenbaum J, Bennett BT. Russell and Burch’s 3Rs then and now: the need for clarity in definition and purpose. J Am Assoc Lab Anim Sci. 2015

University of Pittsburgh. (2022). Lives Saved, Lives Changed. The Impact of Animal Research

USDA. (2023). Animal Welfare Act and Animal Regulations. 


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