BACKGROUND: Hexahydrocannabinol (HHC) is a new psychoactive substance known for its mind-altering effects and temporary legal status. It is widely used in parts of the Europe and United Kingdom as a legal alternative to ∆9-tetrahydrocannabinol, yet little research has explored its effects and safety. This study examined how HHC is processed in the body, its toxicity, and its impact on behavior in male Wistar rats. METHODS: A 1:1 mixture of (9R)-HHC and (9S)-HHC was administered via intragastric gavage at doses of 1, 5, and 10 mg/kg. Behavioral effects were assessed using the open field test and the prepulse inhibition of acoustic startle response. RESULTS: Two hours after the highest dose (10 mg/kg), peak concentrations of HHC were detected in blood and brain tissue. The Organization for Economic Co-operation and Development 423 toxicity test classified HHC as a Category 4 substance, estimating a lethal dose of 1000 mg/kg. Compared to controls (administered by sunflower oil), 10 mg/kg HHC reduced movement, increased anxiety, and impaired sensory processing. CONCLUSIONS: Overall, HHC crosses the blood-brain barrier, exhibits mild toxicity, and induces behavioral effects similar to tetrahydrocannabinol. Its dose-dependent anxiogenic properties and impact on information processing highlight the importance of the appropriate dosing in any potential therapeutic use.

3rd Faculty of Medicine Charles University Ruská 87 Prague 10 100 00 Czech Republic

Department of Analytical Chemistry University of Chemistry and Technology Prague Technická 5 Prague 6 166 28 Czech Republic

Forensic Laboratory of Biologically Active Substances Department of Chemistry of Natural Compounds University of Chemistry and Technology Prague Technická 5 Prague 6 166 28 Czech Republic

Institute of Pathology 1st Faculty of Medicine Charles University Studničkova 2 Prague 2 128 00 Czech Republic

Institute of Pharmacology 1st Faculty of Medicine Charles University and General University Hospital Prague Albertov 4 Prague 2 128 00 Czech Republic

Psychedelic Research Centre National Institute of Mental Health Topolová 748 Klecany 250 67 Czech Republic

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