Reduce, Reuse, Recycle. These are the three Rs familiar to many with reference to best practices associated with environmental stewardship and a smaller carbon footprint. In the United Kingdom (UK), Europe, and increasingly elsewhere around the globe within the scientific community, the three Rs have a different focus.
The National Centre for the Replacement, Refinement, and Reduction of Animals in Research (NC3Rs) is the lead organization in the UK dedicated to the discovery and application of new technologies and approaches to improve animal welfare and minimize the use of research animals. The 3Rs were first described in the UK in the late 1950s, with a call for Replacement, Refinement, and Reduction formalized under European Union Directive 2010/63/EU, which legislates for the protection of animals used for scientific purposes and sets global standards for animal welfare.
The 3Rs are not a directive for the total elimination of animals in research, but similar to Reduce, Reuse, Recycle, they are a call for best practices and enlightened stewardship. Recommended best practices include 1) development and wide-scale implementation of new approach methodologies (NAMs) that replace animal studies, including the application of advanced analytics; 2) critical experimental design and analysis to reduce the number of animals used, and 3) use of appropriate housing, anesthesia and analgesia, and training to minimize animal distress. As stated on the nc3r.org website, evidence suggests that pain and suffering can alter an animal’s behavior, physiology, and immunology. Such changes can lead to variation in experimental results that impairs both the reliability and repeatability of studies. In addition, animal tests often do not provide mechanistic information on how a drug or chemical causes toxic effects. This can delay and even derail drug discovery and development efforts.
|Replacement||Methods which avoid or replace the use of animals||Accelerating the development and use of models and tools, based on the latest science and technologies, to address important scientific questions without the use of animals|
|Reduction||Methods which minimize the number of animals used per experiment||Appropriately designed and analyzed animal experiments that are robust and reproducible, and truly add to the knowledge base|
|Refinement||Methods which minimize animal suffering and improve animal welfare||Advancing research into animal welfare by exploiting the latest in vivo technologies and by improving understanding of the impact of welfare on scientific outcomes|
Table 1. Definition of Replacement, Reduction, and Refinement according to nc3rs.org (accessed 26Feb2020).
3Rs in the US
Over ten years ago, in 2007, the National Research Council (NRC) published a report for the US Environmental Protection Agency (EPA) outlining existing capabilities to help push forward reduced reliance on animal testing and the need for development and validation of innovative approaches based on human cells and cell components to accelerate progress in this area. According to the NRC, development and incorporation of in vitro human-based approaches into safety testing “would lead to much more informed environmental regulations and dramatically reduce the need for animal testing.” Incorporation of in vitro human-based approaches is a more efficient use of resources, as use of animals is expensive, not amenable to high-throughput screening, and often a poor mimic of chemical and drug responses in humans.
Beyond 2007, 16 US federal agencies and interagency workgroups combined with public input to create a roadmap for establishing new approaches to evaluate the safety of chemicals and medical products in the US. The US Department of Health and Human Services’ National Toxicology Program (NTP) strategic roadmap encourages adoption and use of NAMs by federal agencies and regulated industries, and although NTP is distinct from the US Food and Drug Administration (FDA) and does not have regulatory purview over the biopharma industry, drug developers could benefit from adopting aspects of the strategic plan. The roadmap highlights a call for industry partners and federal agencies to “work together on three goals: to connect end users with developers of NAMs, to foster robust practices to establish confidence in NAMs, and to encourage adoption of use of NAMs by scientists working for government agencies or regulated industries.”
The Concept in Practice
While the use of animals and animal disease models for drug development efforts is a separate issue from testing agents for safety, animal testing continues to be the primary approach for de-risking new drugs, and unexpected toxicities still account for some 20-30% of clinical trial failures.
What does this status quo approach mean for those working in either toxicology or biopharma and who leaps first, government regulators or industry scientists? Within both the EPA and the FDA there are personal stories of federal regulators eager to see data from innovative approaches that can replace animal studies for safety pharmacology and toxicity studies, but without a defined policy of acceptance, the risk for incorporating NAMs into regulatory filings remains on the side of scientists.
There are outstanding scientists working diligently on these efforts from the top down. Nicole Kleinstreuer (recipient of the 2019 Society of Toxicology Achievement Award) at the National Toxicology Program Interagency Center for the Evaluation of Alternative Toxicological Methods (NICEATM) led that center’s effort on the EPA’s Endocrine Disruptor Screening Program. This resulted in the EPA replacing the rodent uterotrophic assay with in vitro and in silico methods to screen chemicals for estrogenic activity. At John Hopkins Bloomberg School of Public Health, Thomas Hartung (notably the second most cited German pharmacologist in 2009) directs the Center for Alternatives to Animal Testing (CAAT), providing scientific innovation, funding, and thought leadership to the replace, refine, and reduce effort.
From the bottom up, scientists in academia and government agencies are coalescing around the concept of an Adverse Outcome Pathway (AOP), a conceptual framework that maps a knowledge pathway of links between a molecular initiating event and a final adverse outcome at a biological level of organization relevant to a regulatory decision. The AOP approach can be used to integrate data from diverse drug development platforms for the purpose of toxicity risk assessment. Details of how this applies to data from target-based platforms, phenotypic assays, more advanced complex systems (such as tissues-on-a-chip), and clinical studies is discussed in “Human Cell-Based in vitro Phenotypic Profiling for Drug Safety-Related Attrition.”
Phenotypic assays use whole living cells (or tissues) to evaluate test agents for native functional consequences. Function-related endpoints can include changes in metabolite levels, biomarkers, proliferation, gene or protein expression, protein localization, cell morphology, or death. In contrast to target-based assays, such as biochemical enzymatic or receptor binding assays, phenotypic assays capture the activity of multiple targets and pathway mechanisms, including compensatory responses, within the complexity of a biological system. Phenotypic assays that utilize human cell culture methodologies are highly relevant to clinical outcomes, including adverse events. For example, BioMAP® Toxicity Signature Analysis was created to alert discovery program leaders to the toxicity potential of early pipeline compounds, based on the power of BioMAP Diversity PLUS® phenotypic profiling. BioMAP Toxicity Signature Analysis was built using the comprehensive BioMAP Reference Database, curated for clinically approved and experimental drugs with similarly known toxicities or adverse effects. In an unbiased way, data mining tools identified common biomarkers from BioMAP profile data that were then developed into toxicity signatures. The nine signatures include Acute Toxicity, Immunosuppression, Skin Irritation, Liver Toxicity, Organ Toxicity, Skin Rash, Skin Sensitization, Thrombosis, and Vascular Toxicity.
Eurofins Discovery Phenotypic Services expertise informs client reports for pipeline progression, key opinion leader activities, and investigational new drug applications, to help innovative leaders drive forward in vitro, human, preclinical data. Visit BioMAP Phenotypic Screening and Profiling Services, and follow the link to learn more about Toxicity Signatures for Adverse Outcomes.