Comparative in vitro safety assessments of synthetized and plant-derived cannabidiol (CBD), and pharmacokinetics of synthetized CBD: evidence of similar toxicological profiles and accumulation upon repeated oral administration

Comparative toxicology between synthetized and plant-derived cannabidiol (CBD) remains underexplored, as most previous research has focused on a single type of CBD. This leaves uncertainties regarding whether differences in origin, purity, or extraction methods affect the safety profiles or metabolic outcomes of CBD. This study assessed the in vitro safety, in vitro hepatotoxicity, and repeated oral dose pharmacokinetics (PK) of CBD in rats. Two brands of synthetized CBD were evaluated alongside two plant-derived CBD (a high-purity isolate and a lower-purity distillate) in vitro. These findings indicated no meaningful in vitro safety differences among the CBD sources and therefore supported selection of a single synthetized CBD source for an in vivo PK study in male Sprague-Dawley rats dosed orally once daily at 26.5 mg/kg for 21 days. Blood samples were collected at multiple time points on days 1 and 21, and pre-dose samples on selected days to assess baseline accumulation. CBD and its metabolites (7-COOH-CBD, 7-OH-CBD, 6-OH-CBD) were measured in plasma and tissues using liquid chromatography-tandem mass spectrometry. All CBD samples showed comparable safety profiles, with no evidence of genotoxicity or mutagenicity in micronucleus and Ames assays, similar cytotoxicity in HepaRG monolayer cultures, and comparable cytochrome P450 (CYP) inhibition. Over 21 days, repeated dosing increased plasma CBD (AUC INF _D 567 vs. 209 h*kg*ng/mL/mg; Day 21 vs. 1), 7-COOH-CBD (AUC last _D 1006 vs. 581), 6-OH-CBD (AUC last _D 33 vs. 13) and 7-OH-CBD (AUC last _D 43 vs. 31) exposure, and prolonged CBD half-life (41.9 h vs. 5.9 h). The steady-state plasma CBD concentration was achieved by Day 6. Tissue analysis showed marked CBD accumulation in adipose tissue (70,124 ng/g, 1470-fold relative to plasma level) and mesenteric lymph nodes (10,018 ng/g, 201-fold relative to plasma level). Notably, 7-OH-CBD accumulated in adipose tissue (42 ng/g, 21-fold relative to plasma level) and mesenteric lymph nodes (136 ng/g, 73-fold relative to plasma level). Overall, high-purity synthetized and plant-derived CBD showed similar in vitro safety outcomes, while oral administration produced distinct accumulation of CBD and metabolites in plasma and tissue. These findings support flexible CBD source selection for standardized nonclinical research while highlighting the need to consider accumulation dynamics during repeated dosing.