Animal reuse in safety assessment studies: a review of current practices, challenges and 3Rs opportunities

Regulatory nonclinical safety assessment in animals remains an essential part of the drug development process. Historically, naive animals are used within most nonclinical studies to avoid confounding variables such as prior drug exposure. However, opportunities exist to advance the 3Rs (Replacement, Reduction and Refinement) by reusing animals in studies when scientifically and ethically justified, without compromising scientific integrity or animal welfare or exceeding permitted cumulative physiological and psychological burden. Reuse is already standard practice in pharmacokinetic (PK) and safety pharmacology studies, yet its application in toxicology remains limited. This review explores how broader adoption of reuse as a 3Rs strategy can reduce overall animal numbers, while ensuring reuse does not compromise animal welfare or the scientific integrity of the study. Animal reuse can avoid and reduce known stressors prior to study start, such as transportation, quarantine and acclimation to new environments, by allowing animals to remain in a familiar environment for longer periods extending the time for positive reinforcement training and to establish group hierarchy. Appropriate reuse supports the sustainability and ethical use of animal models. Challenges of reuse include managing the cumulative lifetime burden of the animal, ensuring adequate washout and recovery periods, and addressing scientific concerns such as immunogenicity risks associated with developing anti-drug antibodies (ADA) against biologics or pre-existing immunity to gene therapies and the rising frequency of age-related background lesions. We propose a tiered framework to guide reuse decisions: Tier 1 includes studies where reuse is already standard and achievable; Tier 2 covers studies where reuse is generally feasible with appropriate scientific and ethical rationale; and Tier 3 includes studies where reuse is more challenging due to regulatory, scientific or welfare considerations, although enrolling non-naive animals at study start may still be possible without compromising data interpretation. Enabling wider implementation of reuse will require industry-wide collaboration, standardized decision tools, and proactive engagement with contract research organizations. When applied appropriately, reuse can reduce overall animal use while maintaining robust safety assessment. In parallel with the evolving adoption of new approach methodologies, reuse offers an additional practical avenue to support a more sustainable and humane nonclinical testing paradigm.