Professional Perspectives: Refinement of Animal Toxicology Research Techniques: Ethical and Scientific Advantages of Microsampling
By Emily Zweig | May 21st, 2026
In 2025, the FDA introduced its “Roadmap to Reducing Animal Testing in Preclinical Safety Studies,” which outlines the agency’s actionable plan to transition away from animal testing and toward the development and validation of new approach methods (NAMs). This announcement marked an unprecedented FDA effort to support the shift to human-relevant research as science advances. In 2022, the U.S. Senate supported this shift by passing the FDA Modernization Act 2.0 to amend the Federal Food Drug and Cosmetic Act, which mandates drug developers demonstrate safety before beginning human clinical trials. This testing was referred to as “preclinical tests (including tests on animals).” Over time, this phrasing came to be seen as limiting, particularly as alternatives to animal testing emerged. The 2022 amendment facilitates NAMs research efforts, broadly referring to "nonclinical tests," including NAMs. As animal research is gradually replaced, conventional procedures can be refined to reduce the number of animals used and improve lab animal welfare—one such technique is microsampling.
The refinement of in vivo methods, as highlighted by the roadmap, is an innovative approach supporting the transition to non-animal preclinical safety studies. Microsampling refers to the refinement of bioanalytical techniques that allow the use of smaller blood samples (~5-50 µL) from animals compared with conventional methods. Many preclinical and clinical researchers are implementing microsampling techniques, with the principles of replacement, reduction, and refinement (the 3Rs) motivating widespread adoption. Continued adoption of these methods can benefit both science and animal welfare while reducing the number of animals used in toxicokinetics (TK) testing (determining the level of drug exposure to cause toxic effects in an animal).
Microsampling has multiple scientific advantages. Traditionally, a rodent toxicity study has commonly used a group of “main animals” to observe adverse drug effects and an additional group of “satellite animals” for TK analysis. Researchers have used separate groups of rodents because conventional analytical methods required relatively large blood volumes to analyze samples. Scientists have since developed analytical methods that use microsamples in place of larger samples required by conventional methods. Microsampling can reduce the number of animals used in a study by reducing or eliminating the need for satellite animals.
Toxicokinetic (TK) testing commonly uses serial blood sampling with multiple timepoints. According to best practices, there is a physiological limit to how much blood a rodent can lose over a 24-hour period. Therefore, when higher blood volumes are required for sample analysis, more rodents may be needed for the study. Using microsamples can minimize blood loss, allowing both drug toxicity and TK analyses to be performed in the main group of animals, reducing the need for satellite animals. This is scientifically advantageous, as researchers can directly correlate toxic drug effects with drug exposure in the same rodent. Using a single group of animals for both toxicity and TK data instead of composite groups reduces inter-animal variations. Smaller samples can also be collected at more timepoints, which may produce a more robust TK profile. Despite these benefits, the number of timepoints should be weighed against the consideration of animal welfare. Too many needlesticks in one animal can pose welfare concerns that complicate data collection and are thus a possible limiting factor in study design.
Toxicology researchers can reduce animal stress by implementing microsampling techniques. Compared to collecting larger blood samples, microsamples can be drawn from more accessible blood vessels more quickly and with less restraint of animals. In addition, technicians can perform each venipuncture (needle insertion into a vein to collect blood) using smaller gauge needles, potentially reducing pain and tissue damage. In these ways, refinements to procedures using microsampling can reduce harm to rodents and larger animals alike.
Animal facilities have cited several barriers to the adoption of microsampling. A survey by the National Centre for the Replacement, Refinement and Reduction of Animals in Research (NC3Rs), published in 2014, identified the primary hurdles to wider implementation. These included uncertainty of regulatory approval, the practicality of implementing new techniques, and concerns about whether microsamples can be adequately analyzed. Toxicology and bioanalytical researchers have collaborated over the past decade to respond to these challenges and demonstrate how they can be overcome, but recent surveys indicate that some of these concerns persist.
Despite a 2017 update to international regulatory guidance in favor of microsampling, certain facilities are still hesitant to use the technique in Good Laboratory Practices (GLP) studies due to a perceived lack of regulatory acceptance. However, many companies report using microsampling in various GLP studies with minimal regulatory concern.
Adoption of microsampling and other alternative techniques developed for scientific research can be challenging when conventional methods have long been routine. Despite the availability of microsampling as a more efficient option, initial adoption can be slow because it requires new equipment and training in the techniques. Moreover, study sponsors may hesitate to request microsampling out of preference for traditional methods, giving fewer opportunities for workers to practice and gain confidence with the new skills.
The most common barriers are concerns that microsample volumes are too small for comprehensive sample analysis and a hesitancy to transition away from conventional bioanalysis methods that are already validated. A preclinical study can require analysis of multiple endpoints, which some survey respondents have had difficulty achieving using microsampling. Some facilities are skeptical that proper bioanalysis can be performed using microsamples, particularly for larger molecules such as monoclonal antibodies. Microsampling was initially used primarily to study small molecules because small molecule studies were more common when the methods were first developed. However, many studies have demonstrated the use of microsampling for large molecules, providing reassurance regarding these concerns.
With the recent FDA regulatory updates and expanded opportunities for the scientific advancement of NAMs, these developments are paving the way toward a future of human-relevant research and a shift away from animal testing. The transition will be gradual as alternative methods are validated and broadly adopted, while animal testing is refined and reduced. Microsampling is a refinement that can reduce both the stress experienced by animals and the number of animals used as these techniques continue to advance and become more widely implemented.
Author Bio: Emily Zweig has a B.S. in Biology from the University of Maryland – College Park. She worked as an in vivo technician for 2.5 years at two preclinical CROs. While working in these facilities, she developed an interest in the 3Rs of animal research, particularly in toxicology. Since then, she has learned about NAMs research and has been inspired to find out more.
The views expressed do not necessarily reflect the official policy or position of Johns Hopkins University or Johns Hopkins Bloomberg School of Public Health.
