Evaluation of synthetic cannabinoid receptor agonists toxicity in zebrafish (Danio rerio) through self-administration and Fishbook assays

Abstract New psychoactive substances (NPS) represent an emerging public health concern, with synthetic cannabinoid receptor agonists (SCRAs) among the most prevalent classes worldwide. Although widely reported in seizures and toxicological casework, their pharmacology and toxicity remain poorly characterized. In this study, we investigated the acute toxicity and behavioral effects of two emerging SCRAs—ADB-INACA and CHO-4′Me-5′Br-FUBOXPYRA—using zebrafish ( Danio rerio ) as an in vivo model. Fish embryo acute toxicity (FET) and maximum tolerated concentration (MTC) assays were performed to assess lethality and sublethal effects in early developmental stages. Social behavior was evaluated at 21 days post-fertilization (dpf) using the Fishbook assay, and drug-seeking behavior was investigated in adult zebrafish through a self-administration paradigm. Both SCRAs produced low embryonic mortality across the tested range (0.001–10 µM). CHO-4′Me-5′Br-FUBOXPYRA induced sublethal effects including pericardial edema, loss of posture, and reduced heart rate at higher concentrations, whereas ADB-INACA produced mainly impaired escape responses without significant lethality. Neither compound significantly altered social interaction in juvenile zebrafish, and CHO-4′Me-5′Br-FUBOXPYRA failed to elicit reinforcing effects in adult zebrafish, in contrast to the hydrocodone positive control. Taken together, these results indicate that both SCRAs exhibit limited cannabinoid-like effects at the tested concentrations, with only modest cardiotoxic findings for CHO-4′Me-5′Br-FUBOXPYRA. This study highlights the applicability of zebrafish assays, including Fishbook and self-administration paradigms, for investigating the toxicological and behavioral properties of novel NPS. Graphical abstract