This study provides a head-to-head comparison of synthetized and plant-derived CBD, assessing whether CBD source and purity influence in vitro genotoxicity and hepatotoxicity, and characterizing accumulation and tissue distribution after repeated oral dosing in rats.

Our finding of no genotoxicity or mutagenicity for CBD agrees with a broad consensus of prior studies. Multiple independent, GLP-compliant investigations–using high-purity CBD from synthetized and plant sources–have uniformly reported negative results in standard genotoxicity assays. For example, Henderson et al. (Henderson et al., 2023b) tested a ≥99% pure CBD isolate in the OECD 471 Ames bacterial reverse-mutation assay (five Salmonella strains and E. coli WP2) at doses up to 5,000 µg/plate and observed no increase in revertant colonies (no mutagenic activity) in any strain, with or without metabolic activation. The same study also evaluated CBD in an OECD 487 in vitro micronucleus test using human TK6 lymphoblastoid cells (up to 30–35 µM) and found no induction of micronuclei above vehicle-control levels. Tallon et al. (Tallon et al., 2025) similarly reported no genotoxic effects of a high-purity plant-derived CBD: in their study, a 97% CBD isolate (in MCT oil) showed no mutagenicity in Ames tests and no clastogenicity in an in vitro micronucleus assay, with no statistically significant increase in micronucleated cells at any dose. These rigorous studies, as well as our study–all following OECD guidelines–provide clear evidence that CBD is non-mutagenic in bacteria and non-genotoxic in human and Chinese hamster cells, reinforcing that CBD lacks intrinsic genotoxic potential in standard test systems. Early safety assessments of CBD-rich hemp extracts are consistent with these findings as well. For instance, Marx et al. (Marx et al., 2018) evaluated a supercritical CO₂ extract of hemp (primary constituent CBD) and found no genotoxicity in the Ames assay or in mammalian chromosomal-aberration tests. Likewise, Dziwenka and colleagues (Dziwenka et al., 2020) reported that a broad-spectrum hemp oil (containing high levels of CBD) was negative for mutagenicity and clastogenicity, with no DNA damage observed in vitro and no micronucleus induction in rodents. More recently, Clewell et al. (Clewell et al., 2023) tested a CBD-rich hemp oil (∼20–30% CBD) and also found no evidence of genotoxicity: the oil did not induce mutations in Ames bacterial assays and did not increase micronuclei in human peripheral blood lymphocytes.

The overall pattern across these diverse preparations–from purified CBD isolates to natural extracts–is that CBD consistently yields negative results in genotoxicity test batteries, irrespective of source or minor impurities. This convergence of evidence suggests that CBD itself does not damage genetic material or cause mutations in standard in vitro systems. Taken together, our results align perfectly with this extensive body of evidence. Across disparate laboratories and test systems, CBD has not shown any genotoxic or mutagenic activity under experimental conditions compliant with international guidelines.

The in vitro hepatotoxicity assessment using HepaRG cells revealed a consistent and steep dose-dependent cytotoxic profile across all four APIs, regardless of their source (synthetized vs. plant-derived) or manufacturing grade (GMP vs. food-grade). After 14 days of repeated exposure, all CBD variants demonstrated similar EC₅₀ values (∼27–31 µM), with no discernible differences in potency or toxicity patterns. This uniformity suggests that the hepatotoxic potential of CBD is intrinsic to the molecule itself and not significantly influenced by its origin or purity. The steepness of the dose-response curves indicates a narrow cytotoxicity window, with a rapid transition from minimal to complete cell death over a small concentration range. This threshold-like behavior was further supported by the temporal dynamics of cellular injury markers: ALT levels spiked within 48 h of exposure to concentrations at or above the EC₅₀, then declined on subsequent days (days 8 and 15), consistent with early-onset cytotoxicity. Such kinetics are characteristic of apoptotic cell death rather than cumulative necrosis. This is in line with previous studies demonstrating CBD-induced apoptosis via cannabinoid receptor activation (Jeong et al., 2019; Greenwood et al., 2024), and the expression of cannabinoid receptors in HepaRG cells (De Nunzio et al., 2023) further supports this mechanism.

Pre-clinical animal research has also provided valuable data for assessing the safety of CBD with varying purities. A majority of the repeated dose oral safety toxicology studies range from 14 to 90 days in duration with additional post-exposure recovery period. Animals were dosed up to a high dose of approximately 150 mg/kg/day (Henderson et al., 2023a; Schwotzer et al., 2025; Xia et al., 2025), though one study dosed up to 460 mg/kg/day (Tallon and Child, 2023), all using high purity (>99%) CBD isolates. Despite being independent tests, the results are remarkably similar. Most endpoints were unaffected by the CBD treatment up to the high dose. In-life findings (body weight, food consumption, overall health status of the test system) were similar and demonstrated overall acceptable tolerance to the treatments. Organ weight changes were observed and were mainly stress-dependent effects such as higher weight of the adrenals and thyroid, with reduced weight of the spleen as compared to the control groups. There was consistently higher liver weight among the studies, also associated hepatocyte hypertrophy. These findings recovered following the post-exposure recovery period in all cases and were consistent with xenobiotic-induced microsome enzyme induction (Hall et al., 2012). Hence, these macro- and microscopic findings were considered as adaptive changes of no toxicological relevance by the pathologist. As comparison, when less purified hemp extracts were evaluated, slight differences in the overall no-observed-adverse-effect-levels (NOAELs) were reported (Marx et al., 2018; Dziwenka et al., 2020).

Importantly, the study also revealed that sub-lethal concentrations of CBD in vitro—again, independent of source or purity—impaired key hepatic functions. Albumin synthesis and secretion were significantly reduced at concentrations well below the cytotoxic threshold. At 15–20 μM, where ATP levels remained near baseline, albumin secretion dropped by 20%–60% compared to solvent controls. This functional impairment, in the absence of overt cytotoxicity, suggests that CBD may interfere with protein synthesis or secretion pathways. Supporting this hypothesis, Kosgodage et al. (Kosgodage et al., 2018) reported that CBD inhibits exosome and microvesicle release in cancer cells, a mechanism that could similarly affect albumin export in hepatocytes.

In parallel, CBD exposure led to a marked, concentration-dependent suppression of cytochrome P450 (CYP) enzyme activities. All six major CYP isoforms tested (CYP1A2, 2A6, 2C9, 2C19, 2D6, and 3A4) exhibited dose-dependent inhibition, with no observable differences between the four CBD APIs. Even at 5 µM—well below cytotoxic levels—enzyme activities were reduced by 5%–30%, and at 25 μM, inhibition reached 60%–95%. These findings are consistent with prior reports of CBD as a potent, reversible inhibitor of multiple CYP isoforms (Stout and Cimino, 2014; Nasrin et al., 2021). The reversibility of this inhibition, as demonstrated by the recovery of CYP activities upon CBD removal, suggests a non-permanent, likely competitive mechanism. While our study observed dose-dependent inhibition of major CYP isoforms at micromolar concentrations, other reports suggest that lower, nanomolar concentrations or repeated CBD dosing in humans can lead to enzyme induction, at least for specific CYPs such as CYP3A4 (Morrison et al., 2018; Balhara et al., 2025). These findings could imply that CBD’s modulation of hepatic metabolism may follow a biphasic pattern, with inhibitory effects predominating at acute high concentrations and possible induction at lower, and repeated dosing, but for substantiation, additional translatable data will be needed.

Mechanistically, the data indicates that the CBD APIs, regardless of their manufacturing process or botanical origin, possess a comparable cytotoxic potency in human liver cells in vitro as well as the potential to decrease albumin secretion and inhibit the activity of major CYP enzymes. The consistency of these effects across all four APIs underscores that the effects observed are a fundamental property of CBD itself, rather than a consequence of impurities or origin. Although the in vitro hepatotoxicity assessments demonstrated potential effects of CBD, consistent with findings from other studies (Li et al., 2023; Chen et al., 2024), such effects were only observed at very high concentrations within a narrow dose-response window, and their toxicological significance under physiological conditions remains to be fully elucidated. In human subjects repeatedly administered exceptionally high doses of CBD (1,500 mg/day), the plasma concentrations achieved under both fasted and fed conditions were 1.07 µM and 5.18 µM, respectively (Taylor et al., 2018). The reported plasma concentration of 5.18 µM in humans following high-dose oral CBD administration is substantially lower than those required in our study to induce hepatotoxic effects in cultured liver cells: the in vitro EC₅₀ values (∼27–31 µM) are equivalent to 7,819–8,977 mg/day CBD in human, the in vitro sub-cytotoxic concentration (e.g., 15 μM) with about 50% decrease of albumin is equivalent to 4,344 mg/day CBD in human. Possible explanations for the observed in vitro “toxicity” include: (i) accumulation of metabolites such as 7-COOH-CBD in the static assay system, (ii) the absence of organotypic architecture and lack of metabolic support from communicating cell types present in vivo, and/or (iii) the use of exaggerated concentrations of CBD and its metabolites which is inherent to the typical design of in vitro assays, i.e., the need to increase the dosing until a substantial effect is achieved. In this context, it is also crucial to consider the potential for CBD accumulation during repeated administration, which could result in elevated plasma concentration over time. Additional risk factors include high CBD usage (≥1,000 mg/day or ≥20 mg/kg/day) and concomitant antiepileptic drug use (Lo et al., 2023), pediatric population, individuals with pre-existing liver impairment, elderly patients and polypharmacy as well as other factors that could impact the pharmacokinetics of CBD.

In this study, we used the same CBD source and bioanalytical method as the reported intravenous data (Sandmeier et al., 2025). These methodological approaches minimize variability due to CBD products or assay differences, thereby strengthening the validity and reliability of the bioavailability calculation. Under fed state and current study set-up, the CBD bioavailability is 25.6%. FDA/EMA-approved CBD formulation (100 mg/mL oral solution in a sesame-oil-based vehicle) had an estimated bioavailability of 6% under fasting state in humans (Millar et al., 2018; Perucca and Bialer, 2020). Co-administration with high-fat meals resulted in improved bioavailability compared with fasting state. This is demonstrated by the higher peak plasma concentration (C_max) and the overall exposure (AUC) when CBD was taken with food (Taylor et al., 2018; Bergeria et al., 2022; Jang et al., 2024; Saals et al., 2025). This effect is likely due to lipid digestion, action of bile and enzymes for micelle formation which can improve dissolution of CBD and lymphatic transport (Charman et al., 1997; Birnbaum et al., 2019). Furthermore, experimental PK study in rats demonstrated higher CBD systemic bioavailability in lipid-based excipients compared to formulations without lipids with increased absorption of CBD in the presence of lipids by intestinal lymphatic transport (Zgair et al., 2016). In male rats under fed conditions (Supplementary Figure 1), the dose-normalized C_max and AUC_last values observed after oral administration of synthetized CBD in sesame oil were comparable to those obtained with hemp-derived CBD isolate formulated in olive oil (Xia et al., 2025). These results demonstrate that highly purified CBDs exhibited similar in vivo pharmacokinetic profiles following oral administration, regardless of whether it is synthetized or derived from hemp. The potential influence of other cannabinoids, e.g., in distillates, on PK of CBD needs to be further explored.

Consistent with the impact of food and lipid-based formulations to improve bioavailability of CBD, there was approximately 2-fold higher AUC_inf value in our study conducted under fed state compared to that of Feng et al. (Feng et al., 2021a; Feng et al., 2021b) where CBD in sesame oil formulation was administered to fasted rats. Our results are also consistent with higher bioavailability of sesame oil-based formulations compared to those containing medium-chain fatty acid (capric acid, C10:0) (Kok et al., 2022), potentially due to more efficient incorporation into chylomicrons for lymphatic uptake (Feng et al., 2022).

Considerable inter-subject variability in the plasma CBD concentrations and hence the PK parameters are noted following single oral administration of sesame oil-based CBD formulations (Zgair et al., 2016; Zgair et al., 2017; Feng et al., 2021a; Feng et al., 2021b). Co-administration with high-fat meals also resulted in reduced inter-subject variability compared with fasting state (Perucca and Bialer, 2020). Consistent with the literature, we observed lower inter-subject variability of key PK parameters including the AUC_last, C_max and T_max following repeated dosing compared to single dosing. Repeated oral dosing also resulted in a shorter T_max especially at higher CBD dose (Taylor et al., 2018; Child and Tallon, 2022; Xia et al., 2025). Based on the understanding of the impact of feeding and lipid composition of the formulations on the oral bioavailability, the repeated oral dosing with lipid rich formulation potentially impacted the physiological state of the animal such as its gastric and intestinal luminal contents, lipid transporter activity and/or gut blood flow, which overall improved the emulsification and absorption of CBD (Wall et al., 1976; Fabritius et al., 2012; Daublain et al., 2017; Feng et al., 2022).

A limitation of the present study is that the in vivo PK evaluation was conducted using only one representative synthesized CBD API and was therefore not intended to establish comparative PK equivalence across all four CBD sources. The head-to-head comparison was restricted to the in vitro toxicology endpoints. Selection of API2 for PK was based on its GMP-grade status, high purity, and the absence of meaningful API-dependent differences across the in vitro assays. Consequently, the PK data should be interpreted as providing exposure and accumulation context for repeated oral administration of a representative purified CBD API, rather than as evidence that all tested CBD sources would necessarily exhibit identical PK behavior in vivo.

Systemic accumulation of CBD and the main metabolites upon repeated dosing were observed in the in vivo part of this study. The tested CBD dose of 26.5 mg/kg is equivalent to 4.27 mg/kg human dose (extrapolating based on body surface area with a factor of 6.2) or 214 mg/day for a 50 kg subject. In the in vivo PK study, repeated dosing resulted in the accumulation of CBD and its primary metabolites. The calculated fold increase in C_max_D was 2.5 (Supplementary Table 6). Although the percentage extrapolation for AUC_INF exceeds 15% (at 20.8% in Table 3) relative to AUC_last on Day 21, which implies AUC_last values being slightly underestimated, this has minimal impact on the calculated fold accumulation in AUC as reflected by the closely similar fold increase of AUC_last_D (i.e. 2.9) and AUC_inf_D (i.e. 3.1) (Supplementary Table 6) upon repeated dosing. The literature has reported mixed findings, likely due to variations in group sizes and dosing regimens. In a reported 90-day oral toxicology study, both C_max and AUC_last increased following repeated administration of CBD at doses of 5, 15, and 150 mg/kg in male and female rats (Xia et al., 2025). In a separate study with rats receiving 115 mg/kg of CBD for 28 days, an increase in AUC_last was observed only in females (Child and Tallon, 2022). In contrast, no significant increases in CBD C_max or AUC_last were observed after 14 days of repeated inhalation exposure (Schwotzer et al., 2023). The 2.9 to 3.1-fold accumulation of CBD (Supplementary Table 6) observed in this study in male rats was closely similar to that in humans (1.8- and 2.6-fold after 750 and 1,500 mg/day) following 7-day repeated twice per day dosing (Taylor et al., 2018) for CBD. However, the accumulation of 7-COOH-CBD was more prominent (4.5 to 9.6-fold) in humans compared to only about 2-fold accumulation in the rats, potentially linked to the higher 7-COOH-CBD to CBD ratios of the C_max and AUC_last (30 and 47, respectively at day 7) in humans compared to 2.5 and 2.3, respectively at Day 21 in the rats. Trough CBD plasma levels reached a maximum concentration within about 6 days in this study upon once daily dosing. This was longer as compared to 2 days upon twice-daily dosing in humans (Taylor et al., 2018), potentially due to shorter dosing interval (12 h vs. 24 h) in the 7-day human PK study as well as interspecies differences. CBD exhibits multi-phasic elimination phase and is characterized by long terminal elimination half-life in humans (Taylor et al., 2018). We observed a long terminal elimination phase of 41.9 h and lower K_e (0.017 h-1) upon repeated dosing, which contrasted with the unchanged K_e values reported in an oral repeated dosing study in rats (Child and Tallon, 2022). The differences between the 2 rat studies could possibly be due to short (24-h) post-exposure phase in Child and Tallon’s study which may miss the later slow clearance phase of CBD when CBD was slowly released from storage sites.

CBD accumulates to different extents in various tissues. Sesame oil promoted intestinal lymphatic transport (Zgair et al., 2016; Feng et al., 2021a; Feng et al., 2021b) and distribution at high levels to the mesenteric lymph nodes at 1.5–2.5-h following oral dosing (Feng et al., 2021a; Feng et al., 2021b). Adipose tissue tends to have the highest CBD levels, followed by liver and muscle (Child and Tallon, 2022; Dehner et al., 2025). Our study has also consistently demonstrated highest CBD accumulation in adipose tissue followed by the MLN and the liver but no accumulation in the brain. While the exact tissue concentrations and fold-accumulation of CBD in various tissues could differ between studies, potentially due to differences in fasting states, older rats and larger fat depot mass in heavier rats, slower release of CBD from adipose tissue compared to other tissues, and different time points when tissues were harvested following dosing. Brain distribution studies indicated that CBD reaches peak concentrations in the brain within 1–2 h post-oral/IP administration, with specific formulations potentially enhancing CBD’s brain bioavailability (Brookes et al., 2023; Binova et al., 2025; Feng et al., 2025). The reported brain-to-plasma ratios differed and ranged either <1 (Deiana et al., 2012) or >1 (Brookes et al., 2023), suggesting the influence of formulations in permeability and accumulation in the brain. The brain-to-plasma ratio in our study, obtained from a single time point (72-h), suggested no accumulation of CBD in the brain during the clearance phase of the repeated dosing study. Further work will need to be performed to assess the tissue PK and tissue-to-plasma ratios at steady state condition. Due to their more polar nature, both 7-COOH-CBD and 7-OH-CBD were much less abundant in the adipose tissue and MLN compared to CBD and were not detected in the brain, liver and lung. In addition to the above-described factors, understanding how feeding status and repeated dosing affect CBD pharmacokinetics is essential for improving the effectiveness of CBD-based treatments.