Integrated Assessment of Neurobehavioral and Cardiotoxic Effects of Pyrrolidine-Containing Cathinones in Zebrafish: Structural Determinants of Safety Margins

The rapid emergence of New Psychoactive Substances (NPS), particularly pyrrolidinophenone derivatives, poses a significant challenge for public health and forensic toxicology. While their neuropharmacological profiles as dopamine transporter inhibitors are well-documented, their cardiac toxicity remains poorly understood. This study employs a high-resolution New Approach Methodology (NAM) using zebrafish embryos to integrate neurobehavioral and cardiotoxic endpoints for comparative hazard prioritization. We evaluated nine pyrrolidine-containing cathinones, including α-PVP, MDPV, α-PiHP, MDPiHP, α-D2PV, 3-Cl-, 4-Cl-, and 3,4-Cl-α-PVP, as well as 4-F-3-Me-α-PVP, on locomotor activity and cardiac rhythmicity in zebrafish embryos using high-speed video microscopy and dynamic pixel analysis.. Across the series, compounds induced concentration-dependent negative chronotropy and, in most cases, locomotor suppression. Crucially, we identified a functional dissociation between atrial rate control and atrioventricular (AV) conduction. The 3,4-dichloro substitution (3,4-Cl-α-PVP) was the most potent inducer of negative chronotropy (EC50 = 52.6 μM), whereas 4-Cl-α-PVP exhibited a distinct pro-arrhythmic liability, increasing the incidence of 2:1 AV block. Time-course locomotor profiling indicated that α-PVP and chlorinated analogs were among the most potent behavioral modifiers. Using a Functional Safety Index (AV block EC50 / locomotor EC50-like), we show that most compounds exhibit wide separations between neurobehavioral inhibition and severe conduction impairment, while specific substitutions—particularly para-chlorination—are associated with comparatively reduced safety margins. Overall, these data demonstrate that subtle structural changes within the pyrrolidinophenone scaffold can shape distinct arrhythmic phenotypes and functional safety profiles, supporting zebrafish-based integrated screening as a rapid platform for prioritizing emerging synthetic cathinones with disproportionate cardiac risk.