Sex as a biological variable in nonclinical studies: Bridging scientific rigor, animal welfare, and regulatory expectations

Abstract

Nonclinical studies in animal models have often treated male animals as the default, based on the assumption that estrous-cycle hormonal variation in females increases variability and complicates interpretation. This convention is a structural limitation in study design, particularly in therapeutic areas where sex differences in pharmacokinetics (PK), pharmacodynamics (PD), and toxicity are substantial and clinically relevant. Clinical experience shows higher adverse drug reaction rates in women and recurring examples of sex-biased toxicities, underscoring the translational cost of limited sex consideration early in development. Sex differences in nonclinical PK and toxicity arise from defined mechanisms, including sex-dependent growth hormone secretion patterns that shape hepatic and renal enzyme/transporter expression, as well as sex-specific determinants of gastrointestinal absorption and cardiac electrophysiology. Omics analyses further show that sex effects are embedded in system-wide molecular networks across immune, metabolic, cardiovascular, renal, and neural systems, supporting a shift from descriptive to predictive and stratified animal study designs. Sex bias is also an animal welfare concern: male-only strategies can generate surpluses of unused females in breeding colonies, challenging the 3Rs—particularly Reduction—when evaluated at the project or facility level. Regulatory expectations are converging through International Council for Harmonization (ICH) principles, the National Institutes of Health (NIH) sex-as-a-biological-variable (SABV) policy, and reporting frameworks such as Sex and Gender Equity in Research (SAGER), with increasing emphasis on transparent sex reporting and analysis across nonclinical evidence streams. This review integrates scientific, welfare, and regulatory perspectives to provide a practical framework for sex-aware nonclinical study design, enhancing translational validity and ethical rigor.

Keywords

animal welfare / nonclinical studies / pharmacokinetics / regulatory science / sex as a biological variable

References

[1] Beery AK, Zucker I. Sex bias in neuroscience and biomedical research. Neurosci Biobehav Rev. 2011; 35(3): 565-572.

[2] Rademaker M. Do women have more adverse drug reactions? Am J Clin Dermatol. 2001; 2(6): 349-351.

[3] U.S. Government Accountability Office. Drug Safety: Most Drugs Withdrawn in Recent Years Had Greater Health Risks for Women. U.S. GAO; 2001. Published January 19, 2001. Accessed February 25, 2026. https://www.gao.gov/products/gao-01-286r

[4] Wewetzer H, Wagenknecht T, Bert B, Schönfelder G. The fate of surplus laboratory animals. EMBO Rep. 2023; 24(3):e56551.

[5] Clayton JA, Collins FS. Policy: NIH to balance sex in cell and animal studies. Nature. 2014; 509(7500): 282-283.

[6] Bale TL, Epperson CN. Sex as a biological variable: who, what, when, why, and how. Neuropsychopharmacology. 2017; 42(2): 386-396.

[7] Heidari S, Babor TF, de Castro P, Tort S, Curno M. Sex and gender equity in research: rationale for the SAGER guidelines and recommended use. Res Integr Peer Rev. 2016; 1: 2.

[8] Shapiro BH, Agrawal AK, Pampori NA. Gender differences in drug metabolism regulated by growth hormone. Int J Biochem Cell Biol. 1995; 27(1): 9-20.

[9] Waxman DJ, O'Connor C. Growth hormone regulation of sex-dependent liver gene expression. Mol Endocrinol. 2006; 20(11): 2613-2629.

[10] Zhang Y, Laz EV, Waxman DJ. Dynamic, sex-differential STAT5 and BCL6 binding to sex-biased, growth hormone-regulated genes in adult mouse liver. Mol Cell Biol. 2012; 32(4): 880-896.

[11] Waxman DJ, Holloway MG. Sex differences in the expression of hepatic drug metabolizing enzymes. Mol Pharmacol. 2009; 76(2): 215-228.

[12] Hu S, Leblanc AF, Gibson AA, et al. Identification of OAT1/OAT3 as contributors to cisplatin toxicity. Clin Transl Sci. 2017; 10(5): 412-420.

[13] Ljubojević M, Balen D, Breljak D, et al. Renal expression of organic anion transporter OAT2 in rats and mice is regulated by sex hormones. Am J Physiol Renal Physiol. 2007; 292(1): F361-F372.

[14] Breljak D, Ljubojevic M, Balen D, et al. Renal expression of organic anion transporter Oat5 in rats and mice exhibits the female-dominant sex differences. Histol Histopathol. 2010; 25(11): 1385-1402.

[15] Du K, Williams CD, McGill MR, Jaeschke H. Lower susceptibility of female mice to acetaminophen hepatotoxicity: role of mitochondrial glutathione, oxidant stress and c-Jun N-terminal kinase. Toxicol Appl Pharmacol. 2014; 281(1): 58-66.

[16] Bellampalli SS, Fonar G, Grynyshyn M, et al. Sex-dependent circadian rhythm impact on murine gastrointestinal transit. Neurogastroenterol Motil. 2025; 37(12):e70114.

[17] Soni KG, Halder T, Conner ME, Preidis GA. Sexual dimorphism in upper gastrointestinal motility is dependent on duration of fast, time of day, age, and strain of mice. Neurogastroenterol Motil. 2019; 31(9):e13654.

[18] Madla CM, Qin Y, Gavins FKH, et al. Sex differences in intestinal P-glycoprotein expression in Wistar versus Sprague Dawley rats. Pharmaceutics. 2022; 14(5): 1030.

[19] Zhu Y, Ai X, Oster RA, Bers DM, Pogwizd SM. Sex differences in repolarization and slow delayed rectifier potassium current and their regulation by sympathetic stimulation in rabbits. Pflugers Arch. 2013; 465(6): 805-818.

[20] Lu HR, Remeysen P, Somers K, Saels A, de Clerck F. Female gender is a risk factor for drug-induced long QT and cardiac arrhythmias in an in vivo rabbit model. J Cardiovasc Electrophysiol. 2001; 12(5): 538-545.

[21] Salama G, Bett GCL. Sex differences in the mechanisms underlying long QT syndrome. Am J Physiol Heart Circ Physiol. 2014; 307(5): 640.

[22] Emoto C, Yoda N, Uno Y, et al. Comparison of p450 enzymes between cynomolgus monkeys and humans: p450 identities, protein contents, kinetic parameters, and potential for inhibitory profiles. Curr Drug Metab. 2013; 14(2): 239-252.

[23] Ngo ST, Steyn FJ, McCombe PA. Gender differences in autoimmune disease. Front Neuroendocrinol. 2014; 35(3): 347-369.

[24] Mustapha AF, Goebel AM, Wells BJ. Sex and gender differences in cardiovascular disease: a review of spontaneous coronary artery dissection. US Cardiol Rev. 2023; 17:e15.

[25] Delco A, Portmann A, Mikail N, et al. Impact of sex and gender on heart failure. Cardiovasc Med. 2023; 26(3): 88.

[26] Mauvais-Jarvis F. Sex differences in energy metabolism: natural selection, mechanisms and consequences. Nat Rev Nephrol. 2024; 20(1): 56-69.

[27] MacGregor K, Ellefsen S, Pillon NJ, Hammarström D, Krook A. Sex differences in skeletal muscle metabolism in exercise and type 2 diabetes mellitus. Nat Rev Endocrinol. 2025; 21(3): 166-179.

[28] Nagral A, Bangar M, Menezes S, et al. Gender differences in nonalcoholic fatty liver disease. Euroasian J Hepato-Gastroenterol. 2022; 12(Suppl 1): S19-S25.

[29] Klein SL, Flanagan KL. Sex differences in immune responses. Nat Rev Immunol. 2016; 16(10): 626-638.

[30] Scully EP, Haverfield J, Ursin RL, Tannenbaum C, Klein SL. Considering how biological sex impacts immune responses and COVID-19 outcomes. Nat Rev Immunol. 2020; 20(7): 442-447.

[31] Gal-Oz ST, Maier B, Yoshida H, et al. ImmGen report: sexual dimorphism in the immune system transcriptome. Nat Commun. 2019; 10(1): 4295.

[32] Sharma A, Kapur S, Kancharla P, Yang T. Sex differences in gut microbiota, hypertension, and cardiovascular risk. Eur J Pharmacol. 2025; 987:177183.

[33] Valeri F, Endres K. How biological sex of the host shapes its gut microbiota. Front Neuroendocrinol. 2021; 61:100912.

[34] Harvey BJ, de la Alvarez Rosa D. Sex differences in kidney health and disease. Nephron. 2025; 149(2): 77-103.

[35] Carrero JJ, Hecking M, Chesnaye NC, Jager KJ. Sex and gender disparities in the epidemiology and outcomes of chronic kidney disease. Nat Rev Nephrol. 2018; 14(3): 151-164.

[36] Veser C, Carlier A, Dubois V, Mihăilă SM, Swapnasrita S. Embracing sex-specific differences in engineered kidney models for enhanced biological understanding of kidney function. Biol Sex Differ. 2024; 15(1): 99.

[37] Lopez-Lee C, Torres ERS, Carling G, Gan L. Mechanisms of sex differences in Alzheimer's disease. Neuron. 2024; 112(8): 1208-1221.

[38] Zelco A, Joshi A. Single-cell analysis of sex and gender differences in the human brain during development and disease. Cell Mol Neurobiol. 2025; 45(1):20.

[39] Moores G, Steadman PE, Momen A, Wolff E, Pikula A, Bui E. Sex differences in neurology: a scoping review. BMJ Open. 2023; 13(4):e071200.

[40] Miller LR, Marks C, Becker JB, et al. Considering sex as a biological variable in preclinical research. FASEB J. 2017; 31(1): 29-34.

[41] Reckelhoff JF. Mechanisms of sex and gender differences in hypertension. J Hum Hypertens. 2023; 37(8): 596-601.

[42] Bager J, Manhem K, Andersson T, et al. Hypertension: sex-related differences in drug treatment, prevalence and blood pressure control in primary care. J Hum Hypertens. 2023; 37(8): 662-670.

[43] McDonough AA, Harris AN, Xiong LI, Layton AT. Sex differences in renal transporters: assessment and functional consequences. Nat Rev Nephrol. 2024; 20(1): 21-36.

[44] Mogil JS. Qualitative sex differences in pain processing: emerging evidence of a biased literature. Nat Rev Neurosci. 2020; 21(7): 353-365.

[45] Ma J, Yao Y, Tian Y, Chen K, Liu B. Advances in sex disparities for cancer immunotherapy: unveiling the dilemma of yin and Yang. Biol Sex Differ. 2022; 13(1): 58.

[46] Kammula AV, Schäffer AA, Rajagopal PS, Kurzrock R, Ruppin E. Outcome differences by sex in oncology clinical trials. Nat Commun. 2024; 15(1): 2608.

[47] Özdemir BC. Removing barriers to address sex differences in anticancer drug toxicity. Nat Rev Cancer. 2024; 24(3): 161-162.

[48] Presto P, Mazzitelli M, Junell R, Griffin Z, Neugebauer V. Sex differences in pain along the neuraxis. Neuropharmacology. 2022; 210:109030.

[49] Soldin OP, Mattison DR. Sex differences in pharmacokinetics and pharmacodynamics. Clin Pharmacokinet. 2009; 48(3): 143-157.

[50] Zucker I, Prendergast BJ. Sex differences in pharmacokinetics predict adverse drug reactions in women. Biol Sex Differ. 2020; 11(1): 32.

[51] Virwani PD, Qian G, Hsu MSS, et al. Sex differences in association between gut microbiome and essential hypertension based on ambulatory blood pressure monitoring. Hypertension. 2023; 80(6): 1331-1342.

[52] Francis-Malavé AM, Martínez González S, Pichardo C, et al. Sex differences in pain-related behaviors and clinical progression of disease in mouse models of colonic pain. Pain. 2023; 164(1): 197-215.

[53] Nicotra L, Tuke J, Grace PM, Rolan PE, Hutchinson MR. Sex differences in mechanical allodynia: how can it be preclinically quantified and analyzed? Front Behav Neurosci. 2014; 8:40.

[54] Westenbroek C, Snijders TAB, den Boer JA, Gerrits M, Fokkema DS, Ter Horst GJ. Pair-housing of male and female rats during chronic stress exposure results in gender-specific behavioral responses. Horm Behav. 2005; 47(5): 620-628.

[55] International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH). M3(R2) Nonclinical Safety Studies for the Conduct of Human Clinical Trials and Marketing Authorization for Pharmaceuticals. 2010 Accessed January 23, 2026. https://www.fda.gov/regulatory-information/search-fda-guidance-documents/m3r2-nonclinical-safety-studies-conduct-human-clinical-trials-and-marketing-authorization

[56] International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH). S6(R1) Preclinical Safety Evaluation of Biotechnology-Derived Pharmaceuticals. Accessed Feb 25, 2026. https://www.fda.gov/regulatory-information/search-fda-guidance-documents/s6r1-preclinical-safety-evaluation-biotechnology-derived-pharmaceuticals

[57] International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH). E8(R1) General Considerations for Clinical Studies. Accessed Feb 25, 2026. https://www.fda.gov/regulatory-information/search-fda-guidance-documents/e8r1-general-considerations-clinical-studies

[58] U.S. Food and Drug Administration (FDA). Advancing Alternative Methods at FDA. 2026 Accessed Feb 25, 2026. https://www.fda.gov/science-research/about-science-research-fda/advancing-alternative-methods-fda

[59] Tan R, Hua H, Zhou S, et al. Current landscape of innovative drug development and regulatory support in China. Sig Transduct Target Ther. 2025; 10(1): 220.

[60] Sewell F, Alexander-White C, Brescia S, et al. New approach methodologies (NAMs): identifying and overcoming hurdles to accelerated adoption. Toxicol Res Camb. 2024; 13(2):tfae044.

[61] Miller NA, Reddy MB, Heikkinen AT, Lukacova V, Parrott N. Physiologically based pharmacokinetic modelling for first-in-human predictions: an updated model building strategy illustrated with challenging industry case studies. Clin Pharmacokinet. 2019; 58(6): 727-746.

[62] Ezuruike U, Zhang M, Pansari A, et al. Guide to development of compound files for PBPK modeling in the Simcyp population-based simulator. CPT Pharmacometrics Syst Pharmacol. 2022; 11(7): 805-821.

[63] Affolter T, Llewellyn HP, Bartlett DW, et al. Inhibition of immune checkpoints PD-1, CTLA-4, and IDO1 coordinately induces immune-mediated liver injury in mice. PLoS One. 2019; 14(5):e0217276.

[64] Jamei M, Marciniak S, Edwards D, et al. The simcyp population based simulator: architecture, implementation, and quality assurance. In Silico Pharmacol. 2013; 1: 9.

[65] Ferretti MT, Iulita MF, Cavedo E, et al. Sex differences in Alzheimer disease—the gateway to precision medicine. Nat Rev Neurol. 2018; 14(8): 457-469.

[66] Jones H, Chen Y, Rostami-Hodjegan A. Physiological and pharmacokinetic modelling of the menstrual cycle and its application to drug development. CPT Pharmacometrics Syst Pharmacol. 2022; 11(2): 139-150.

RIGHTS & PERMISSIONS

2026 The Author(s). Animal Models and Experimental Medicine published by John Wiley & Sons Australia, Ltd on behalf of The Chinese Association for Laboratory Animal Sciences.