Human toxicokinetic study of zeranol and zearalenone for a rigorous discrimination between doping and mycotoxins-contaminated food

Code: 241C01PV

Zeranol (α-ZAL), a prohibited anabolic agent by the World Anti-Doping Agency (WADA), can be present in human urine as a metabolite of zearalenone (ZEN), a mycotoxin occurring in several food commodities. Distinguishing between doping intake and ZEN-contaminated food consumption is challenging. To establish evidence-based criteria, a human intervention trial and toxicokinetic (TK) modeling are fundamental. Up to 74.6% of ZEN is excreted in human urine as reductive metabolites and phase II conjugates. Limited evidence suggests that α-ZAL is metabolized primarily, but not exclusively, into β-ZAL and zearalenone (ZAN) in mammals. Monitoring α-ZAL concentrations relative to other ZEN metabolites helps differentiate illicit intake from food contamination, but WADA LT04 lacks transparent criteria for confident α-ZAL doping identification. This project proposes two independent human intervention trials with 10 healthy adults each to study αZAL and ZEN toxicokinetics. After a wash-out period of two days, the volunteers will ingest a bolus containing α-ZAL or ZEN at the TDI (250 ng/kg bw). From the exposure onwards, the volunteers are requested to collect individual urine and capillary blood samples for 48 hours. ZEN, ZAN, α/β-zearalenol (α/β-ZEL), and α/β-ZAL will be quantified, after extraction and hydrolysis of phase II-conjugates, using GC-MS/MS and GC-HRMS methods validated at ultra-trace levels. We aim to determine the human metabolic excretion profiles over time and use it to build two TK models with Hierarchical Bayesian population structure. The resulting population concentration/time profiles will enable calculating confidence intervals for ZEN/α-ZAL, α-ZEL/α-ZAL, and β-ZEL/β-ZAL ratios, refining WADA report guidelines for Adverse Analytical Finding in urine and capillary blood collected via VAMS Mitra tips. The models will provide population TK parameters of α-ZAL and ZEN and the possibility to estimate intake doses, offering additional proof in legal adjudication.