Data generated from in vivo testing has been a foundational requirement in drug development for decades. Not least among its usefulness is its critical role in assessing the safety and toxicity of new medicines before they advance to human clinical trials. Regulatory agencies worldwide have long required in vivo studies to support safety protocols and reduce the risk of adverse reactions in human participants.
These studies often involve repeat-dose toxicity assessments, pharmacokinetic (PK) profiling, and safety pharmacology, typically conducted under Good Laboratory Practice (GLP) standards. Drug developers and sponsors also rely on in vivo data to investigate disease mechanisms and inform personalized medicine.
However, the scientific and regulatory communities are starting to question the utility of in vivo testing. In the United States, more than 90% of drugs that appear safe and effective during in vivo testing do not receive regulatory approval for human use.1 In response to this reality, there is a growing global movement to embrace the principles of the 3Rs: replacement, reduction, and refinement.
The FDA Modernization Act 2.0, enacted in December 2022, and the agency’s recently announced plan to phase out in vivo studies when developing monoclonal antibodies (mAbs) and other drugs are two strong signals of where the industry is heading.2,3 New Approach Methodologies (NAMs) — i.e., organ-on-a-chip systems, advanced in vitro assays, and computational modeling — are poised to take over drug safety assessments, with the long-term goal of making animal studies the exception rather than the norm.
But one element of the discussion that is sometimes overlooked is data integrity from in vivo testing. Specifically, is data garnered from stressed species valid and/or reliable? Is it reproducible? These and other important questions need to be asked.
How Animal Welfare Directly Impacts Data Integrity
Animal welfare is indeed an ethical consideration, but it also speaks directly to scientific validity in toxicology. When laboratory animals experience stress — from housing conditions, handling practices, or broader husbandry issues — their physiology changes in ways that can inadvertently mask or mimic the effects of the drugs being tested. This cuts to the core of data reliability and interpretability.
Cortisol, the primary stress hormone, rises predictably in animals exposed to acute or chronic stress. These elevations can significantly affect hemodynamics (blood pressure and cardiac output), immune function, metabolism, and vital study markers, making it difficult to determine whether observed changes are due to a drug or to stress itself. Stress response is indeed a challenging metric to understand. Routine handling or restraint can raise cortisol levels, but repeated exposure to poor conditions can also blunt or elevate this hormone. Both scenarios undermine the usefulness of the data generated for safety assessments.
Most importantly, prolonged or unpredictable cortisol exposure can suppress immune cell production, disrupt cytokine signals, and cause immune organs like the thymus and spleen to shrink. These effects compromise immune-related study endpoints and weaken responses to infections and vaccines.4 Chronic high cortisol not only increases infection risk but also disrupts the body’s regulatory balance, leading to inflammation, muscle loss, metabolic issues, and faster physiological decline.
These broad, stress-related disruptions mean that animals suffering long-term welfare deficits no longer represent normal physiological baselines. As a result, the data generated under these conditions exhibits excess variability and diminished reliability, casting doubt on the reproducibility and translational value of the research itself.
The bottom line is that long-term animal welfare deficits create scenarios that no longer represent the normal physiological baseline. That makes animal welfare inseparable from data integrity. When stress-induced physiological changes jeopardize reproducibility, it undermines the foundational premise of preclinical safety studies: to predict human outcomes reliably.
Enzyme Modulation and Its Ripple Effects
Stress also directly affects a drug’s absorption, distribution, metabolism and elimination (ADME) characteristics. Altered feeding patterns and baseline metabolic changes in stressed animals impacts how drugs are processed and frequently produce inconsistent or unreliable PK profiles.
At the molecular level, the effects of stress are equally profound. Activation of the hypothalamic-pituitary-adrenal (HPA) axis raises circulating glucocorticoids like corticosterone and cortisol. These hormones have a widespread effect on liver enzymes. Specifically, stress impacts the cytochrome P450 (CYP) family’s activity. This network of enzymes is almost entirely responsible for metabolizing drugs in both animal models and humans.
Studies have demonstrated that stress can suppress or alter the function of key CYP isoforms, including CYP3A, CYP2C, CYP2D, and CYP1A.5 Scientists have documented raised corticosterone levels and significant reductions in hepatic enzyme activities, including aminopyrine N-demethylase and aniline hydroxylase. These PK deviations introduce unpredictable and systematic biases into the parameters at the heart of drug safety evaluation.
Study Variability & Reproducibility
Around the world, standards for housing, enrichment and handling laboratory animals are far from uniform, creating significant disparities between countries and even among institutions in the same region. The lack of standardization produces marked differences in baseline physiology and stress levels, introducing a layer of variability that can undermine confidence in collaborative projects or pooled research.
Animals living in human-made confines may also exhibit a host of behavioral and biological changes. For example, bare or stressful environments can lead to stereotypic behaviors, including repetitive pacing or bar-biting. These behaviors are often accompanied by irregular activity cycles and fluctuating hormone levels, including chronic or dysregulated cortisol secretion. Such physiological stress responses can alter immune function, metabolism, and even gene expression, further increasing variability in key toxicological and pharmacological endpoints. This variability makes it more challenging to attribute observed behavior to a specific intervention versus differences in environmental conditions.
Even minor environmental differences — i.e., cage size, floor type or ambient noise — have been shown to affect experimental outcomes. These factors, often overlooked in protocol design, can alter study results, further undermining reproducibility.6,7 Reducing chronic stress and supporting positive welfare states lowers allostatic load in animals and leads to more robust, interpretable study outcomes.
Enhancing Welfare
When drug development programs embrace best practices in animal welfare, the benefits are measurable and meaningful. Facilities that prioritize appropriate group housing, environmental enrichment and stable human-animal interactions consistently observe reductions in stress markers. This translates into steadier behavioral patterns and physiological baselines that are less prone to fluctuations andinconsistency.
Thoughtful enrichment — e.g., nesting materials, opportunities for exercise, and social housing — promotes psychological well-being and allows animals to engage in natural behaviors. These improvements are visible in daily observations, and reflected in metabolic, immunological and reproductive data. When animal welfare is prioritized, key physiological endpoints stabilize, study variability decreases and research teams can potentially achieve statistical significance with fewer subjects.8 This aligns directly with the 3R’s goals of reduction and refinement.
Equally important is the expertise of research staff. Technicians trained in gentle, species-sensitive handling techniques can alleviate acute stress during any procedure. A calm, positive human presence also generates less physiological volatility — e.g., changes in food intake, body weight or immune response — that might otherwise obscure true experimental data.
A Final Word on Animal Welfare
The evidence for a direct link between animal welfare and data integrity is clear. Poor welfare can distort toxicology data, increase variability, and compromise the reproducibility of scientific studies. But a strong commitment, including better housing, enrichment and staff competency, can deliver data that is more reliable, consistent, and predictive of human outcomes.
The drug development industry has another strong incentive to uphold high welfare standards. Lapses in oversight and inconsistent environmental conditions invite a host of consequences that can lead to business risk. A culture of continuous refinement is starting to take hold across the industry and throughout regulatory agencies, but a meeting of the minds is not enough. It will take a global commitment and a collaborative approach to generate better data and safer drugs.
References
- U.S. Food and Drug Administration. (2025, April). Roadmap to reducing animal testing in preclinical safety studies.
- Zushin, P., et. al. (2023, Nov). FDA Modernization Act 2.0: transitioning beyond animal models with human cells, organoids, and AI/ML-based approaches. The Journal of Clinical Investigation. 133(21), 175824.
- U.S. Food and Drug Administration. (2025, April 8). FDA announces plan to phase out animal testing requirement for monoclonal antibodies and other drugs.
- Alotiby, A. (2024 Oct). Immunology of Stress: A Review Article. Journal of Clinical Medicine. 13(21), 6394.
- Konstandi, M., & Johnson, E. O. (2023). Age-related modifications in CYP-dependent drug metabolism: role of stress. Frontiers in Endocrinology. 14, Article 1143835.
- Gaskill, B., & Pritchett-Corning, K. (2015, Sept). Effect of Cage Space on Behavior and Reproduction in Crl:CD(SD) and BN/Crl Laboratory Rats. J Am Assoc Lab Anim Sci. 54(5),497-506.
- Earley, B., et. al. (2015, Oct 31). Effect of floor type on the performance, physiological and behavioural responses of finishing beef steers. Acta Vet Scand. 57(73).
- Weed, J., & Raber, J. (2005). Balancing Animal Research with Animal Well-being: Establishment of Goals and Harmonization of Approaches. ILAR Journal. 46(2) 118-128.