Supplementary file 1_Animal reuse in safety assessment studies: a review of current practices, challenges and 3Rs opportunities.pdf

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.</p>