Investigating dose-dependent nephrotoxicity and tolerable daily intake of melamine (MEL) and cyanuric acid (CYA) co-exposure via a sequential three-stage experimental framework.

Co-exposure to melamine (MEL) and cyanuric acid (CYA) exhibits synergistic toxicity, yet existing toxicological studies have several limitations, including suboptimal doses, short exposure durations, and lack of synergistic effect data in current TDIs and environmental guidelines. To address these gaps, this study conducted a three-stage experiment in male Sprague-Dawley rats to explore nephrotoxicity and dose-response relationships under co-exposure conditions. In Stage 1 (CYA at 10× MEL), the toxicity threshold for MEL was assessed, with trace crystal formation and mild tubular injury first observed at 2 mg/kg bw/day. In Stage 2 (MEL fixed at 4 mg/kg bw/day and CYA ranging from 0.5 to 10× MEL), the toxicity threshold for CYA was evaluated, revealing that a 1:1 ratio of MEL to CYA exhibited the highest nephrotoxic potential. In Stage 3, with exposure duration extended to 12 weeks, the no-observed-adverse-effect levels (NOAELs) were established as 0.5 mg/kg bw/day (based on tubular injury) and 2 mg/kg bw/day (based on crystal deposition). Applying an uncertainty factor of 200, the derived TDIs for combined exposure were calculated as 2.5 μg/kg bw/day (tubular injury-based) and 10 μg/kg bw/day (crystal deposition-based). These findings represent a critical advancement toward updating the current risk assessment framework for MEL and CYA co-exposure.