Investigation into the oral toxicity profile of TruCBN™

Abstract

Floraworks TruCBN™ is a proprietary form of cannabinol (CBN), a high-purity CBN isolate processed to be substantially free of THC. CBN is derived from the Cannabis sativa plant, and is unique among cannabinoids, as it is not produced directly through the cannabis plant’s metabolic processes; instead, it forms as an oxidative decomposition product of cannabidiol (CBD) and tetrahydrocannabinol (THC). CBN acts as a low affinity partial agonist at both CB₁ and CB₂ receptors, with a greater affinity for CB₂. Therapeutic properties of CBN include potential efficacy in the treatment of ocular disease, neuroprotection, antimicrobial, anti-inflammatory, and immunosuppressive effects, as well as ongoing investigations for relevance to sleep quality as an alternative to traditional sleep aids, such as melatonin. The safety of TruCBN™ was examined comprehensively through GLP studies conducted to assess the safety profile, including its toxicologic potential. In 14-day and 90-day repeat-dose oral toxicity studies, at dose levels of 0 (vehicle control), 100, 200, and 400 mg/kg/day in male and female Sprague-Dawley rats, no adverse effects were observed. Specifically, there were no clinical signs, body weight, food consumption, functional observations, or motor activity effects, and no clinical pathology, macroscopic, or microscopic changes attributable to the administration of the test substance. The 90-day toxicity study supports a NOAEL for TruCBN™ of 400 mg/kg/day.

1. Introduction

Cannabinol (CBN) is derived from the Cannabis sativa plant, and is unique among cannabinoids, as it is not produced directly through the cannabis plant’s metabolic processes; instead, it forms as an oxidative decomposition product of cannabidiol (CBD) and tetrahydrocannabinol (THC) [1]. CBN acts as a low affinity partial agonist at both CB₁ and CB₂ cannabinoid receptors, with a greater affinity for CB₂ [2], [3]. TruCBN™ is a proprietary form of CBN, a high-purity CBN isolate processed to be substantially free of THC [4]. Therapeutic properties of CBN include potential efficacy in treatment of ocular disease [5], neuroprotection [6], [7], [8], [9], antimicrobial, anti-inflammatory, and immunosuppressive effects [10], as well as sleep induction, as an alternative to traditional sleep aids, such as melatonin [11], [12], [13], [14]. The primary commercial use of cannabinol (CBN) is promoted as a natural sleep aid in the health and wellness industry. These beneficial qualities make CBN ideal for inclusion both alone and in conjunction with other cannabinoids as supplements for natural pain relief, facilitation for relaxation and sleep, as well as nascent neuroprotective products [5], [15].

Cannabinoid receptors, primarily CB₁, which are found in high concentrations in the brain and spinal cord, are where most neurological effects of cannabinoids transpire [16]. CBN has been suggested to regulate cell cycle-associated genes in motor neurons, which may contribute to its neuroprotective capabilities, by influencing stress response and neuroplasticity pathways. More specifically, NSC−34 motor neuron cells demonstrate the lack of cytotoxicity while influencing the downregulation of specific Cdkn genes, known to suppress the cell cycle, as well as the upregulation of cell cycle progression activators, with no effects on the expression of genes involving cell death or tumorigenesis [7]. However, CBN activity independent of cannabinoid receptor pathways associated in the process of cell death can be considered for its potential neuroprotective properties. CBN has been shown to inhibit the non-apoptotic, iron-dependent, oxytosis/ferroptosis cell death pathway in a rodent hippocampal cell line, HT22, which do not possess CB₁ and CB₂ receptors. Evidence suggests that in vitro CBN pretreatment can prevent cellular morphological changes associated with cell death from treatment with glutamate or RSL3 [8]. Moreover, CBN has been investigated for its potential inhibition of neuroblastoma tumorigenesis to further support a neuroprotective role [6]. Evidence suggests that in vitro CBN pretreatment can prevent the morphological and biochemical hallmarks of cell death induced by glutamate or the ferroptosis inducer RSL3 in mouse hippocampal HT22 neurons, inhibiting the non-apoptotic, iron-dependent oxytosis/ferroptosis pathway by directly targeting mitochondria and preserving redox balance, calcium handling, membrane potential and mitochondrial dynamics independently of CB₁/CB₂ receptors [8]. Consistent with a broader cytoprotective profile, fragment-based SAR studies have validated CBN as a unique mitochondrial-targeted inhibitor of oxytosis/ferroptosis in neuronal cells. In parallel, CBN has been shown to preserve mitochondrial and synaptic function under conditions of oxidative stress, supporting a role in stress-adaptive and survival signaling. Moreover, CBN suppresses neuroblastoma tumorigenesis by attenuating proliferation, invasion, and angiogenesis through miR−34α-associated regulatory pathways, underscoring its capacity to modulate survival and stress-related signaling in neuronal-derived cells (6, 8, and 9). Complementary in vitro and in vivo data in glaucoma models demonstrate that CBN protects retinal ganglion-like cells from pressure-induced toxicity and improves pattern electroretinogram responses while lowering intraocular pressure, further supporting a neuroprotective role in CNS-derived tissues [5].

In vivo studies were conducted with TruCBN™ with the aim to evaluate general toxicological safety and further characterize its suitability as a human oral supplement, as well as for other oral applications. Subacute and subchronic studies were conducted, including preliminary tolerability and range-finding studies followed by a 90-day oral gavage toxicity study in rats.

2. Materials and methods

All in vivo procedures were conducted in accordance with applicable Animal Welfare Act Regulations, the Guide for the Care and Use of Laboratory Animals, and AVMA Guidelines on Euthanasia. The test facility was AAALAC-accredited, and all procedures were designed to minimize animal discomfort, distress, and pain. Animal procedures were approved by the Institutional Animal Care and Use Committee under protocol identification P710N.01 FLO and were conducted in accordance with established animal-care standards [17].

2.1. Test substance/TruCBN™ and preparation

TruCBN™ was supplied as an off-white powder consisting of 99.7% cannabinol (CAS No. 521–35–7) and was stored under ambient conditions protected from light. TruCBN™ was formulated as weight/volume mixtures in medium-chain triglyceride (MCT) oil. Control groups received MCT oil alone. All reported concentrations and dose levels reflect the amount of TruCBN™ administered.

2.2. Analytical support and method validation

An HPLC method was developed and validated under GLP conditions for analysis of TruCBN™ in MCT oil. The method demonstrated acceptable linearity, precision, accuracy, specificity, system suitability, homogeneity, and freezer stability. Dose formulations were prepared in MCT oil and analyzed to confirm concentration, homogeneity, and stability.

2.3. Production and synthesis

TruCBN™ (cannabinol isolate; FloraWorks, Milwaukie, OR, USA; FWCBN077–20230127; ≥99.7% by GC-FID) was produced by the manufacturer using a patented one-pot, halogen-catalyzed conversion of cannabidiol to tetrahydrocannabinol isomers, followed by in situ oxidative aromatization to cannabinol (CBN; Fig. 1). The material was purified by distillation and crystallization and determined to be substantially free of THC isomers by analytical detection limits. Full reaction conditions are disclosed in Jensen and Hartung, “Methods of Preparing Cannabinoids or Derivatives Thereof” (U.S. Patent No. 12,371,412 B2).

Fig. 1.

Partial synthesis of cannabinol CBN [1] by conversion of hemp-derived cannabidiol (CBD) [2] via cyclized intermediates including tetrahydrocannabinol (D9-THC & D8-THC) (3&4), along with their isomers. TruCBN™ is a high-purity CBN isolate processed to be substantially free of THC.

2.4. Maximum tolerated dose study

A maximum tolerated dose study was conducted in Sprague Dawley CD IGS rats, approximately eight weeks of age, using a single oral gavage dose of 3500 mg/kg TruCBN™ in 2 animals/sex/group [18]. Animals were observed for viability, clinical signs, gross toxicity, and behavioral changes. Body weights were recorded prior to dosing and prior to sacrifice, and gross necropsies were performed.

A subsequent seven-day repeat-dose tolerability phase was conducted to support dose selection for longer-duration studies. Rats were assigned to 0, 850, 1750, or 3500 mg/kg/day TruCBN™ at 5 animals/sex/group and dosed once daily by oral gavage for seven consecutive days. Animals were monitored daily for health status, clinical signs, and tolerability.

2.5. 14-Day dose range-finding study

A 14-day oral gavage dose range-finding study was conducted in Sprague Dawley CD® IGS rats, approximately eight weeks of age, at dose levels of 0, 100, 200, and 400 mg/kg/day, with 5 animals/sex/group [19]. Animals were evaluated for viability, clinical observations, body weight, body weight gain, food consumption, food efficiency, gross necropsy findings, and selected organ and tissue weights. At terminal sacrifice, selected organs and tissues were collected, weighed, and preserved.

2.6. 90-Day oral gavage toxicity study

A 90-day repeat-dose oral gavage toxicity study with a 28-day recovery period was conducted in Sprague-Dawley CD® IGS rats [20].

2.6.1. Animal care and acclimation

Animals were group housed in cages meeting applicable size recommendations [17], maintained under controlled environmental conditions with a 12-hour light/dark cycle, and acclimated for seven days prior to dosing. Certified rodent diet and filtered tap water were provided ad libitum. Feed and water were periodically analyzed, and no contaminants were identified that were expected to interfere with study results.

2.6.2. Dose formulations and test administration

TruCBN™ was formulated daily in MCT oil at concentrations of 20, 40, or 80 mg/mL to achieve dose levels of 100, 200, or 400 mg/kg/day, respectively. Control animals received MCT oil alone. Individual doses were calculated weekly based on the most recent body weight and administered by oral gavage at 5 mL/kg.

A total of 100 rats were assigned to four treatment groups, with 10 animals/sex/group assigned to the main study and 5 animals/sex assigned to control and high-dose recovery groups. Animals were dosed once daily for at least 90 days, with recovery animals maintained for a non-dosing period of an additional 28 days.

2.6.3. Observations and in-life endpoints

Animals were observed at least twice daily for viability and once daily for cage-side clinical observations. Detailed clinical observations were performed prior to dosing and weekly thereafter. Body weights and food consumption were recorded during acclimation, prior to dosing, and at approximately weekly intervals. Ophthalmic examinations were conducted during acclimation and near the end of the dosing phase.

2.6.4. Functional observational battery and motor activity

Functional observational battery assessments were performed prior to dosing, during Week 1, monthly thereafter, and at the end of the recovery period for recovery animals. Evaluations included handling observations, open-field observations, sensorimotor responses, grip strength, foot splay, and rotarod performance using established methods [21], [22]. Motor activity was assessed using an automated photobeam activity system over a one-hour evaluation period divided into six 10-minute intervals.

2.6.5. Clinical pathology assessments

Clinical pathology evaluations were performed on surviving main study and recovery animals at scheduled termination. Assessments included hematology, coagulation, clinical chemistry, urinalysis, and thyroid hormone analysis. Blood and urine samples were collected and analyzed using validated laboratory methods and standard instrumentation. Thyroid hormone analysis included triiodothyronine, thyroxine, and thyroid-stimulating hormone.

2.6.6. Necropsy, organ weights, and histopathology assessments

At scheduled termination, surviving main study and recovery animals were euthanized by exsanguination under isoflurane anesthesia and subjected to complete gross necropsy. Selected organs were weighed, and tissues were preserved in appropriate fixatives for histopathological evaluation. Animals found dead or sacrificed moribund were necropsied promptly, and tissues were collected and preserved when possible.

2.6.7. Statistical methods

Quantitative data were summarized using means and standard deviations, with males and females evaluated separately. Statistical analyses were conducted using Provantis™, INSTAT, and GraphPad Prism. Repeated-measure endpoints, including body weight, food consumption, motor activity, and selected FOB parameters, were analyzed by two-way ANOVA with repeated measures over time [23], followed by post hoc comparisons to control when appropriate [24], [25].

Single-measure continuous endpoints, including organ weights and clinical pathology parameters, were assessed for variance homogeneity and normality when group size allowed [26], [27]. Parametric data were analyzed by one-way ANOVA followed by Dunnett’s test where appropriate [24], [25]. Nonparametric data were analyzed using Kruskal-Wallis testing followed by appropriate multiple comparisons, including Dunn’s test [28], [29]. For recovery comparisons involving only control and high-dose groups, t-tests were used.

3. Results

3.1. MTD and range-finding studies

A single oral administration of TruCBN™ at 3500 mg/kg resulted in adverse clinical signs and mortality or humane sacrifice in male and female rats. Clinical signs attributed to TruCBN™ included hypoactivity at varying levels, ano-genital staining, hunched posture, piloerection, coldness to touch, and irregular respiration.

In the seven-day repeat-dose component, adverse effects were observed at 1750 and 3500 mg/kg/day. Several males and females at these dose levels were either found dead or sacrificed for humane reasons by Days 4 or 5. Clinical signs preceding death or sacrifice included slight to moderate hypoactivity, ano-genital staining, soft feces, irregular respiration, prone or hunched posture, coldness to touch, and piloerection. These findings indicated that dose levels of 1750 and 3500 mg/kg/day exceeded the tolerable range for repeated administration.

In the 14-day dose range-finding study, no mortality occurred at dose levels of 100, 200, or 400 mg/kg/day. Clinical observations attributed to TruCBN™ included hunched posture, hypoactivity, ano-genital staining, and irregular respiration, primarily at 200 and 400 mg/kg/day. These findings were not associated with macroscopic abnormalities at necropsy.

Male rats showed statistically significant decreases in mean absolute body weight and daily body weight gain compared with controls. These changes were associated with decreased food consumption and demonstrated a dose-dependent trend, with the greatest effect at 400 mg/kg/day. Female rats did not show statistically significant body weight changes, although slight dose-dependent decreases in food consumption were observed. No macroscopic findings were noted at necropsy. In males, decreased absolute organ weights were generally considered secondary to reduced body weight, while no significant absolute or relative organ weight changes were observed in females.

3.2. 90-Day oral gavage toxicity study

3.2.1. Analytical support and method validation

The analytical method for TruCBN™ in MCT oil was demonstrated to be acceptable for use in the study. Dose formulations were homogeneous, and the test substance was stable under the conditions of storage. Homogeneity results showed relative standard deviations of 1.1% and 1.5% at bracketed concentrations of 5 and 100 mg/mL, respectively (Fig. 2).

Fig. 2.

Representative HPLC chromatogram demonstrating the marker peak used for identification/quantitation of TruCBN™ in MCT oil, for analytical support during the 90-day oral toxicity study. Neat test substance stability was assessed by comparing samples collected at study initiation and again at the end of the study.

Neat test substance stability was confirmed over the course of the study, with measured values of 100.09% of expected concentration on Day 1 and 99.68% on Day 96. The overall change in active concentration was −0.4%, corresponding to 99.6% stability. Concentration verification results for the 20, 40, and 80 mg/mL formulations ranged from 103.5% to 112.0% of target across Weeks 1, 6, and 13. These results confirmed that animals received the intended TruCBN™ dose levels.

3.2.2. Mortality, cage-side observations, and detailed clinical examination

Two animals died during the 90-day study: one control recovery male on Study Day 81 and one high-dose male on Study Day 83. Both deaths were attributed to gavage-related complications rather than physiological effects of TruCBN™. The control male had findings consistent with esophageal damage and subsequent sepsis, while the high-dose male had findings consistent with esophageal perforation and secondary tissue effects.

Clinical signs attributed to TruCBN™ included hypersalivation and slight to moderate hypoactivity. Hypersalivation was considered consistent with dose administration or a local response. Hypoactivity was observed primarily as a post-dose cage-side finding and was not associated with corresponding detailed clinical observations or histopathological correlates. No other clinical observations were attributed to TruCBN™ administration.

3.2.3. Ophthalmology

Both eyes of all animals on test were examined by focal illumination and indirect ophthalmoscopy prior to study initiation and near completion of the dosing phase of the study for all surviving Main Study animals. On Day 93, one mid dose (200 mg/kg/day) male was observed with an incipient cataract in the right eye. These findings were not attributed to exposure to the test substance; thus, the test substance was not considered an ocular toxicant. All other surviving Main Study animals were normal upon ophthalmic exam.

3.2.4. Functional observational battery and motor activity

There were no adverse test substance-related changes in any of the parameters evaluated in the functional observational battery over the course of the study. Any decreases in quantitative measurements or increases in incidence of open field measurements were minimal and not associated with a constellation of findings that would support a toxicologically significant behavioral change.

There were no adverse, significant changes in motor activity observed over the course of this study. Mean total movements (Fig. 3) observed over the course of the study were considered comparable to that of the control levels. Any highly variable changes or trends of increased activity in treatment groups from control over the testing time points without statistical significance were interpreted to be of no toxicological significance due to the lack of any correlating toxicologically significant findings.

Fig. 3.

Summary of motor activity (total movements) collected during the Functional Observational Battery (FOB) evaluations across the study timepoints for TruCBN™ treated and vehicle control rats. Motor activity was considered comparable to control levels across dose groups, with no toxicologically meaningful trends (Supporting timepoint/sex-specific diagrams are provided, as Supplementary Fig. S1-S4).

3.2.5. Body weight and food consumption

Decreases in mean weekly body weights were observed at dose levels of 200 and 400 mg/kg/day in male rats, reaching statistical significance at Week 3, and continuing over the course of the study (Fig. 4). Daily body weight gain for the same male dose groups were significantly lower than control Group 1 in the first two weekly intervals and recovered to control levels for the remainder of the study. Decreases in body weight parameters were consistent with lower food consumption observed in male rats at 200 and 400 mg/kg/day treatment groups. Absolute mean body weight in high dose males demonstrated a recovery trend to control levels with discontinued treatment over the 28-day recovery period.

Fig. 4.

Mean weekly body weights over the dosing and recovery phases (where applicable) for male and female rat administered TruCBN™ (0, 100, 200, or 400 mg/kg/day) Statistically significant decreases were observed primarily in males at 200 and 400 mg/kg/day during early study intervals, with recovery to control values throughout the remainder of the study, including the recovery period.

Decreases in mean daily food consumption were noted for Groups 3 and 4 male rats, as compared to control Group 1, over the course of the study. Significant changes in mean daily food consumption returned to control levels prior to and throughout the 28-day recovery period. Decreases in food consumption consequently resulted in corresponding decreases in body weight and is considered attributable to the animal variation noted in organ weight findings.

3.2.6. Clinical pathology

Administration of TruCBN™ to male and female rats did not induce any adverse test substance-related changes in hematology, coagulation, clinical chemistry, urinalysis, and thyroid hormone parameters. Statistically significant changes if noted in hematology, coagulation, clinical chemistry, or urinalysis were present without any histopathological correlate, and were within historical control ranges, therefore not considered to be adverse findings. Thyroid stimulating hormone (TSH) changes in male and female rats at the high dose level (400 mg/kg/day) were within historical control values (Fig. 5). Furthermore, there was no histopathological finding in the thyroid gland or other tissues involved in thyroid function, nor were changes in T₃ or T₄ hormones indicative of TSH dysregulation in the thyroid gland.

Fig. 5.

Serum thyroid hormone results (TSH, T₃ and T₄) for male and female rats following repeat-oral administration of TruCBN™ (up to 400 mg/kg/day). Observed values were within historical control ranges and were not associated with histopathologic correlates in thyroid or related tissues (individual analyte tables are provided as Supplementary Tables S8-S12).

3.2.7. Macroscopic observations, organ weights, and histopathology

Compared with the vehicle control, TruCBN™ produced no adverse effects on survival, absolute or relative organ weights, or macroscopic and microscopic findings. Microscopic observations were considered spontaneous or incidental because they occurred at low incidence, were distributed randomly across groups, including concurrent controls, and/or were typical for rats of this strain and age. Therefore, these findings were considered unrelated to TruCBN™ administration [30], [31], [32]. The lung hemorrhage and the esophageal fibrosis and perforation were considered procedure-related and associated with euthanasia or oral gavage.

Statistically significant (**p < 0.01, *p < 0.05) effects detected on mean absolute (liver, male rats; spleen, male rats; prostate, seminal vesicles, and coagulating glands) and mean relative organ to body weight (adrenals and brain, male and female rats; testes) and mean relative organ to brain ratio (kidneys, male rats; liver, male rats; prostate, seminal vesicles, and coagulating glands) in rats from Groups 2, 3, and/or Group 4 appeared to likely be due to animal variation and unrelated to TruCBN™ administration, as they were non-dose dependent, appeared in one sex (kidney, spleen), and/or lacked correlate when evaluated microscopically.

Statistically significant (*p < 0.05) effects were detected on the mean relative adrenal to body weight ratio (38% increased change) in male rats from Group 3 (at 200 mg/kg/day) and in female rats in Groups 3 and 4 (at 200 mg/kg/day and 400 mg/kg/day, respectively). Rats from Group 3 were not evaluated in histopathology. Female rats in Group 4 had no correlation in histopathology.

Highly statistically significant (**p < 0.01) effects were detected on the mean relative brain to body weight ratio in male rats from Group 3 (16% increased change at 200 mg/kg/day) and Group 4 (15% increased change at 400 mg/kg/day). Statistically significant (*p < 0.05) related effects were detected on the mean relative brain to body weight ratio in female rats from Group 3 (15% increased change at 200 mg/kg/day). Rats from Group 3 were not evaluated in histopathology. Male rats in Group 4 had no correlation in histopathology.

Statistically significant (**p < 0.01) effects were detected on the mean absolute and mean relative liver to brain weight ratio in male rats from Group 3 (19% and 17% decrease change, respectively, at 200 mg/kg/day). Rats from Group 3 were not evaluated in histopathology.

Statistically significant (*p < 0.05) effects were detected on the mean relative kidney to brain weight ratio in male rats in Groups 3 (11% decrease at 200 mg/kg/day). Rats from Group 3 were not evaluated in histopathology.

While statistically significant (**p < 0.01) effects were detected on the mean absolute and mean relative spleen to brain weight ratio in male rats treated at 400 mg/kg/day (20% and 19% decrease change, respectively), there were no correlations in histopathology.

Statistically significant (*p < 0.05) effects were detected on the mean relative testes to body weight ratio (16% increased change) in male rats at 200 mg/kg/day.

Statistically significant (*p < 0.05) effects were detected on the mean absolute and relative brain to prostate, seminal vesicles, and coagulating glands (combined) weight from Group 3 (16% and 15% decrease, respectively, at 200 mg/kg/day). Highly statistically significant (**p < 0.01) effects were detected on the mean absolute and mean relative brain to prostate, seminal vesicles, and coagulating glands (combined) weight from Groups 2 (18% decrease for both mean absolute and mean relative weights at 100 mg/kg/day) and 4 (19% decrease for both mean absolute and relative weights at 400 mg/kg/day). Rats from Group 2 and 3 were not evaluated in histopathology. Rats in the high dose group had no correlations in histopathology.

The non-statistically significant variations in the mean group organ weights, absolute and relative to brain and body weight were considered spontaneous and/or incidental because there were no dose-dependent trends, no similar trends between sexes, and/or were related to sexual maturation. Therefore, they were considered unrelated to test substance administration (Table 1).

Table 1.

90-Day absolute and relative organ weights.

Test Substance Dose Levels (mg/kg/day)
	0				400
Parameter	Males	N	Females	N	Males	N	Females	N
Terminal BW	584.0 ± 69.2	10	304.4 ± 33.6	10	502.9 ± 67.8	9	299.0 ± 29.4	9
Adrenals (g)	0.0628 ± 0.0126	9	0.0714 ± 0.0138	10	0.0648 ± 0.0101	9	0.0786 ± 0.0092	9
Adrenals-BW Ratio	0.1112 ± 0.0301	9	0.2096 ± 0.0346	10	0.1298 ± 0.0218	9	0.2666 ± 0.0478**	9
Adrenals-Br Ratio	0.0279 ± 0.0057	9	0.0349 ± 0.0066	10	0.0291 ± 0.0047	9	0.0392 ± 0.0056	10
Brain (g)	2.242 ± 0.096	10	2.046 ± 0.050	10	2.228 ± 0.135	9	2.013 ± 0.146	9
Brain-BW Ratio	3.881 ± 0.423	10	6.059 ± 0.554	10	4.474 ± 0.411**	9	6.755 ± 0.774	10
Heart (g)	1.646 ± 0.250	10	1.119 ± 0.100	10	1.451 ± 0.235	9	1.053 ± 0.110	9
Heart-BW Ratio	2.823 ± 0.295	10	3.302 ± 0.308	10	2.898 ± 0.382	9	3.522 ± 0.341	9
Heart-Br Ratio	0.733 ± 0.099	10	0.547 ± 0.043	10	0.650 ± 0.086	9	0.526 ± 0.075	10
Kidneys (g)	3.511 ± 0.404	10	2.127 ± 0.258	10	3.354 ± 0.509	9	2.078 ± 0.176	9
Kidneys-BW Ratio	6.060 ± 0.806	10	6.253 ± 0.535	10	6.677 ± 0.485	9	6.905 ± 0.636	9
Kidneys-Br Ratio	1.562 ± 0.128	10	1.039 ± 0.119	10	1.500 ± 0.141	9	1.035 ± 0.096	10
Liver (g)	15.498 ± 2.321	10	9.397 ± 0.716	10	13.823 ± 1.801	9	8.616 ± 1.005	9
Liver-BW Ratio	26.520 ± 2.442	10	27.754 ± 2.530	10	27.537 ± 1.441	9	28.669 ± 1.532	9
Liver-Br Ratio	6.901 ± 0.914	10	4.595 ± 0.365	10	6.192 ± 0.556	9	4.294 ± 0.530	10
Pituitary Gland (g)	0.0158 ± 0.0018	10	0.0211 ± 0.0020	10	0.0143 ± 0.0027	9	0.0196 ± 0.0013	9
Pituitary Gland-BW Ratio	0.0027 ± 0.0004	10	0.0062 ± 0.0007	10	0.0029 ± 0.0005	9	0.0066 ± 0.0009	9
Pituitary Gland-Br Ratio	7.0353 ± 0.5827	10	10.3119 ± 0.9142	10	6.4413 ± 1.2476	9	9.7609 ± 0.6369	10
Spleen (g)	0.905 ± 0.143	10	0.633 ± 0.083	10	0.728 ± 0.118**	9	0.587 ± 0.095	9
Spleen-BW Ratio	1.547 ± 0.157	10	1.857 ± 0.124	10	1.452 ± 0.183	9	1.959 ± 0.214	9
Spleen-Br Ratio	0.403 ± 0.061	10	0.309 ± 0.039	10	0.325 ± 0.037**	9	0.293 ± 0.049	10
Thymus (g)	0.3046 ± 0.1104	10	0.2999 ± 0.0703	10	0.2911 ± 0.1060	9	0.2617 ± 0.0592	9
Thymus-BW Ratio	0.5146 ± 0.1387	10	0.8792 ± 0.1831	10	0.5783 ± 0.1950	9	0.8842 ± 0.2005	9
Thymus-Br Ratio	0.1350 ± 0.0458	10	0.1464 ± 0.0330	10	0.1295 ± 0.0429	9	0.1309 ± 0.0332	10
Thyroid-Parathyroid (g)	0.0267 ± 0.0073	10	0.0245 ± 0.0047	10	0.0286 ± 0.0074	9	0.0213 ± 0.047	9
Thyroid-Parathyroid-BW Ratio	0.46268 ± 0.13211	10	0.72482 ± 0.15074	10	0.57596 ± 0.16908	9	0.74826 ± 0.21731	9
Thyroid-Parathyroid-Br Ratio	0.01192 ± 0.00323	10	0.01196 ± 0.00220	10	0.01289 ± 0.00364	9	0.01064 ± 0.00253	10
Epididymides (g)	1.6004 ± 0.2297	10	-		1.5098 ± 0.1321	9	-
Epididymides-BW Ratio	2.7691 ± 0.4653	10	-		3.0437 ± 0.4349	9	-
Epididymides-Br Ratio	0.7151 ± 0.1054	10	-		0.6789 ± 0.0617	9	-	-
Pro, SV, & CG (g)	4.100 ± 0.600	10	-		3.302 ± 0.436**	9	-
Pro, SV, & CG-BW Ratio	0.007 ± 0.001	10	-		0.007 ± 0.001	9	-
Pro, SV, & CG-Br Ratio	1.834 ± 0.300	10	-		1.483 ± 0.193**	9	-
Testes (g)	3.861 ± 0.476	10	-		3.868 ± 0.353	9	-
Testes-BW Ratio	6.700 ± 1.158	10	-		7.798 ± 1.123	9	-
Testes-Br Ratio	1.727 ± 0.234	10	-		1.740 ± 0.174	9	-
Ovaries-Oviduct (g)	-		0.1379 ± 0.0286	10	-		0.1279 ± 0.0152	9
Ovaries-Oviduct-BW Ratio	-		0.4075 ± 0.0893	10	-		0.4294 ± 0.0351	9
Ovaries-Oviduct-Br Ratio	-		0.0674 ± 0.0138	10	-		0.0638 ± 0.0089	10
Uterus (g)	-		1.004 ± 0.360	10	-		0.780 ± 0.392	9
Uterus-BW Ratio	-		2.958 ± 1.081	10	-		2.221 ± 0.405	9
Uterus-Br Ratio	-		0.490 ± 0.172	10	-		0.387 ± 0.186	10

BW – Body Weight.

Br – Brain.

N = Number of Animals.

** = p < 0.01.

Pro, SV, & CG - Prostate Gland, Seminal Vesicle and Coagulating Gland.

4. Discussion

Cannabis sativa, the botanical source of cannabinol (CBN), has a long history of human use for both medicinal and recreational purposes [33]. Hemp, a variety of Cannabis sativa, is the predominant source of commercial cannabidiol (CBD) and may be used as feedstock for the manufacture of commercial CBN. TruCBN™ is a purified cannabinol isolate manufactured and supplied by FloraWorks. The increasing commercial use of CBN-containing products, particularly for sleep-related applications, supports the need for a well-characterized toxicological profile for high-purity CBN ingredients intended for oral consumption.

The available clinical and preclinical literature supports continued interest in CBN as a bioactive cannabinoid with potential relevance to sleep modulation, neuroprotection, and immunomodulatory activity. TruCBN™ has been evaluated clinically and was reported to improve measures of sleep quality without major side effects [11]. In addition, CBN and other phytocannabinoids have been reported to influence melatonin exposure through inhibition of CYP1A2-mediated metabolism, supporting potential interaction with sleep-related pathways when used alone or in combination with melatonin-containing products [34], [35]. Preclinical studies have also described neuroprotective and anti-inflammatory properties of CBN, including suppression of oxidative stress, modulation of microglial inflammatory responses, preservation of mitochondrial function, and potential activity in models relevant to neurodegeneration and glaucoma [5], [8], [9], [10], [36], [37]. While these findings support the broader biological interest in CBN, they also emphasize the importance of defining an appropriate safety margin for repeated oral exposure.

The present toxicology program was designed to evaluate the oral tolerability and subchronic safety of TruCBN™ in rats. Initial tolerability and dose range-finding studies were used to identify dose levels appropriate for the definitive 90-day oral gavage study. The maximum tolerable dose assessment demonstrated that very high acute and short-term repeat-dose levels were not tolerated, as evidenced by mortality, humane sacrifice, and adverse clinical signs at dose levels substantially above those selected for the definitive study. These findings were important for dose selection, as they established an upper bound of tolerability and supported the need for adjustment to a lower dose range of 100, 200, and 400 mg/kg/day for the subsequent 14-day and 90-day studies. The absence of mortality at these lower dose levels in the 14-day range-finding study supported their use in the definitive subchronic study.

In the 14-day dose range-finding study, clinical observations such as hunched posture, hypoactivity, ano-genital staining, and irregular respiration were noted primarily at 200 and 400 mg/kg/day. These observations were considered to be not adverse without the presence of corresponding gross pathological findings or other evidence of systemic toxicity. Male body weight and food consumption decreases were observed, particularly at the higher dose levels, and were considered relevant for dose selection. However, these findings did not progress to overt toxicity and were not associated with macroscopic abnormalities at necropsy. Organ weight changes in males were generally interpreted as secondary to lower body weight rather than direct target-organ toxicity. Collectively, the 14-day findings supported 400 mg/kg/day as an appropriate high dose for the definitive 90-day study because it produced some evidence of physiological response without toxicity.

In the 90-day oral gavage toxicity study, TruCBN™ was administered daily to male and female Sprague-Dawley rats at dose levels of 100, 200, and 400 mg/kg/day. The study included a recovery period, allowing assessment of persistence or reversibility of any findings. Overall, repeated oral administration of TruCBN™ did not result in adverse toxicological effects at any dose level tested. There were no treatment-related mortalities. The deaths that occurred during the study were attributed to gavage-related technical complications rather than systemic toxicity of the test substance. This distinction is important because gavage-associated esophageal injury and secondary inflammatory changes can occur independently of test substance toxicity and should not be interpreted as evidence of intrinsic toxicity when supported by necropsy and histopathological findings.

Clinical observations in the 90-day study were limited primarily to hypersalivation and occasional hypoactivity. Hypersalivation is commonly observed in oral gavage studies and may reflect palatability, local irritation, viscosity, taste, or anticipatory response to dosing rather than systemic toxicity. In this study, hypersalivation was not associated with adverse clinical pathology, gross pathology, or microscopic findings. Hypoactivity was observed as a post-dose clinical sign but was not accompanied by functional observational battery abnormalities, motor activity deficits, or neuropathological correlates. Given the known pharmacological activity of cannabinoids and the absence of persistent or functionally significant findings, the observed hypoactivity was interpreted as a transient pharmacological or procedure-associated response rather than an adverse toxicological effect.

Body weight and food consumption changes were among the principal findings observed in the 90-day study. Decreases in mean body weight were most evident in males at 200 and 400 mg/kg/day and were associated with reduced food consumption during portions of the dosing phase. However, body weight gain generally recovered toward control levels during the study, and high-dose males demonstrated recovery after cessation of treatment. These findings suggest an adaptive or reversible response rather than progressive systemic toxicity. Importantly, the body weight and food consumption findings were not associated with adverse clinical signs, clinical pathology changes, histopathological lesions, or persistent impairment during the recovery period. Although previous data suggest that CBN may increase food consumption in short-term rodent models [38], the present study did not show increased food intake. This observation may reflect differences in study design, dose level, duration of exposure, formulation, animal model, or the physiological context of repeated gavage administration.

The functional observational battery and motor activity assessments provided additional support for the absence of adverse neurobehavioral toxicity. Despite occasional clinical observations of hypoactivity, there were no adverse test substance-related effects on sensorimotor responses, grip strength, foot splay, rotarod performance, or automated motor activity. This is an important interpretive point because isolated cage-side observations can be difficult to distinguish from transient pharmacological effects or procedural responses. The lack of corresponding changes in structured neurobehavioral endpoints indicates that TruCBN™ did not produce persistent or functionally meaningful neurotoxicity under the conditions of this study.

Clinical pathology findings also did not indicate adverse systemic toxicity. Hematology, coagulation, clinical chemistry, urinalysis, and thyroid hormone evaluations did not reveal treatment-related adverse changes. Any statistically significant differences were considered non-adverse because they lacked dose-response consistency, were within historical control expectations, were not associated with microscopic correlates, or resolved during the recovery period. Thyroid-stimulating hormone changes were specifically interpreted as non-adverse because they were within historical control ranges and were not accompanied by corresponding changes in T₃ or T₄ or microscopic findings in the thyroid gland or related tissues. The absence of concordant endocrine, clinical chemistry, and histopathological changes supports the conclusion that the thyroid axis was not adversely affected by TruCBN™ administration.

Gross pathology, organ weight, and histopathology findings further supported the lack of target-organ toxicity. No adverse macroscopic or microscopic findings were attributed to TruCBN™. Organ weight differences were observed in selected organs, including adrenal glands, brain-to-body weight ratios, liver, spleen, kidney, testes, and male accessory sex organs. However, these changes were generally not considered adverse because they were not consistently dose-dependent, were often limited to one sex, lacked corresponding histopathological correlates, or were plausibly secondary to differences in terminal body weight. From a toxicological perspective, organ weight changes are most meaningful when supported by dose-response patterns, biological plausibility, clinical pathology changes, or microscopic lesions. In the present study, these supporting indicators were absent. Therefore, the organ weight findings were interpreted as incidental, secondary to body weight differences, or within expected biological variability rather than direct evidence of organ toxicity.

The recovery data further strengthened the interpretation of the findings. Changes in body weight and food consumption showed evidence of reversibility following cessation of dosing, and no delayed toxicity emerged during the recovery period. The absence of persistent clinical signs, functional deficits, clinical pathology changes, or histopathological findings after recovery supports the conclusion that the effects observed during dosing were not adverse or progressive. This is particularly relevant for the high-dose group, as recovery evaluation provides additional support that 400 mg/kg/day did not produce lasting toxicological effects.

Based on the results of the definitive 90-day oral gavage toxicity study, the no-observed-adverse-effect level (NOAEL) for TruCBN™ was established at 400 mg/kg/day, the highest dose tested. This NOAEL is supported by the absence of adverse effects on survival, clinical signs, ophthalmology, neurobehavioral function, motor activity, body weight recovery, clinical pathology, organ weights, gross pathology, and histopathology. While some treatment-related or treatment-associated observations were noted, they were not considered adverse because they were transient, reversible, lacked pathological correlates, or did not impair overall animal health.

The NOAEL was also used to estimate the margin of safety for expected human exposure. Comparison of the 400 mg/kg/day rat NOAEL with an initial human clinical dose of 0.833 mg/kg/day yields a margin of safety of approximately 480-fold. This exceeds the generally accepted 200-fold margin of safety commonly applied in European regulatory food safety evaluations when appropriate compound-specific data are available [39]. Therefore, the calculated margin of safety provides additional support for the conclusion that TruCBN™ does not present a toxicological safety concern at the evaluated human intake level.

5. Conclusion

The results of this toxicology program support the oral safety of TruCBN™ under the conditions tested. The high-dose level of 400 mg/kg/day was tolerated in the 90-day study without adverse effects, and the observed findings were either non-adverse, reversible, procedure-related, or not supported by corroborating clinical pathology or histopathology. Taken together, the absence of target-organ toxicity, the recovery of body weight-related changes, the lack of neurobehavioral deficits, and the favorable margin of safety support the conclusion that TruCBN™ is safe for its intended use at the tested intake levels. With expected human doses at 50 mg per day (assuming a 60 kg human), the highest dose administered targeted approximately 482 times the expected human oral intake. This margin of safety supports the conclusion that TruCBN™ is safe for its intended use as a sleep aid [11].

CRediT authorship contribution statement

Matthew Roberts: Writing – review & editing, Supervision, Investigation. Michael McCormick: Writing – review & editing, Supervision, Investigation. Robert Paul Jensen: Writing – review & editing, Supervision, Investigation. Mark R. Bauter: Writing – review & editing, Writing – original draft, Methodology, Investigation, Formal analysis, Data curation, Conceptualization. Celeste Dunn: Writing – review & editing, Writing – original draft. Vaughn Hartung: Writing – review & editing, Writing – original draft, Supervision, Investigation, Conceptualization. Denise DiCarlo-Emery: Writing – review & editing, Methodology, Formal analysis, Data curation.

Author’s Contributions

Mark Bauter contributed to in vivo study conception and design, acquisition, analysis, and interpretation; drafted the manuscript; and critically revised the manuscript.

Vaughn Hartung, Robert Paul Jensen, Michael McCormick contributed to drafted manuscript, and critically revised manuscript.

Celeste Dunn contributed to the drafted manuscript and critically revised manuscript.

Denise DiCarlo-Emery contributed to study conception and design, acquisition, analysis, and interpretation.

Funding

The work reported in this paper was conducted during the normal course of employment. This work was funded by Floraworks. The authors have the sole responsibility for the writing, content, and conclusions of this article.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Handling Editor: Dr. L.H. Lash

Appendix A. Supplementary material

Supplementary material

Data Availability

Data will be made available on request.