Toxicological properties of Δ9-tetrahydrocannabinol and cannabidiol

Abstract

Cannabis sativa L. contains more than 100 phytocannabinoids that can interact with cannabinoid receptors CB1 and CB2. None of the cannabinoid receptor ligands is entirely CB1- or CB2-specific. The effects of cannabinoids therefore differ not just because of different potency at cannabinoid receptors but also because they can interact with other non-CB1 and non-CB2 targets, such as TRPV1, GPR55, and GPR119. The most studied phytocannabinoid is Δ9-tetrahydrocannabinol (THC). THC is a partial agonist at both cannabinoid receptors, but its psychotomimetic effect is produced primarily via activation of the CB1 receptor, which is strongly expressed in the central nervous system, with the noteworthy exception of the brain stem. Although acute cognitive and other effects of THC are well known, the risk of irreversible neuropsychological effects of THC needs further research to elucidate the association. Unlike THC, phytocannabinoid cannabidiol (CBD) does not appear to have psychotomimetic effects but may interact with some of the effects of THC if taken concomitantly. CBD administered orally has recently undergone well-controlled clinical trials to assess its safety in the treatment of paediatric epilepsy syndromes. Their findings point to increased transaminase levels as a safety issue that calls for postmarketing surveillance for liver toxicity. The aim of this review is to summarise what is known about acute and chronic toxicological effects of both compounds and address the gaps in knowledge about the safety of exogenous cannabinoids that are still open.

Full text

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Review DOI: 10.2478/aiht-2020-71-3301
Toxicological properties of Δ9-tetrahydrocannabinol and
cannabidiol
Katarina Černe
Department of Pharmacology and Experimental Toxicology, Faculty of Medicine, University of Ljubljana, Ljubljana,
Slovenia
[Received in June 2019; Similarity Check in June 2019; Accepted in March 2020]
Cannabis sativa L. contains more than 100 phytocannabinoids that can interact with cannabinoid receptors CB1 and CB2.
None of the cannabinoid receptor ligands is entirely CB1- or CB2-specic. The effects of cannabinoids therefore differ
not just because of different potency at cannabinoid receptors but also because they can interact with other non-CB1 and
non-CB2 targets, such as TRPV1, GPR55, and GPR119. The most studied phytocannabinoid is Δ9-tetrahydrocannabinol
(THC). THC is a partial agonist at both cannabinoid receptors, but its psychotomimetic effect is produced primarily via
activation of the CB1 receptor, which is strongly expressed in the central nervous system, with the noteworthy exception
of the brain stem. Although acute cognitive and other effects of THC are well known, the risk of irreversible
neuropsychological effects of THC needs further research to elucidate the association. Unlike THC, phytocannabinoid
cannabidiol (CBD) does not appear to have psychotomimetic effects but may interact with some of the effects of THC if
taken concomitantly. CBD administered orally has recently undergone well-controlled clinical trials to assess its safety
in the treatment of paediatric epilepsy syndromes. Their ndings point to increased transaminase levels as a safety issue
that calls for postmarketing surveillance for liver toxicity. The aim of this review is to summarise what is known about
acute and chronic toxicological effects of both compounds and address the gaps in knowledge about the safety of exogenous
cannabinoids that are still open.
KEY WORDS: acute toxicity; animal studies; cannabidiol; CB1; CB2; CBD; chronic toxicity; clinical trials; Δ9-
tetrahydrocannabinol; phytocannabinoids; THC
Černe K. Toxicological properties of Δ9-tetrahydrocannabinol and cannabidiol
Arh Hig Rada Toksikol 2020;71:1-11
With the growing interest in the use of cannabinoids for
medicinal purposes grows a need for a systematic review
of their toxicological properties. There are still many
uncertainties and contradictions remaining from the
increasing number of published cannabinoid safety studies.
This is because these studies vary to extremes in their
methodology and quality, rendering results difcult to
compare. Moreover, toxicity is not systematically covered,
and there are no chronic toxicity data from well-dened
exposure settings. Higher quality toxicological data are
available for cannabinoid-based medicines that are
manufactured today as approved drugs. However, the main
indications for their use are serious and/or rare diseases,
mostly after all other treatment has failed, so their
toxicological prole is less detailed than that of the drugs
of rst choice (1).
Cannabinoid receptor ligands are a varied group of over
100 chemical compounds isolated from Cannabis sativa L.
(2). The best-characterised cannabinoids found in the
cannabis plant are Δ9-tetrahydrocannabinol (THC) and
cannabidiol (CBD). They can interact with two types of
Corresponding author: author: Katarina Černe, Department of
Pharmacology and Experimental Toxicology, Faculty of Medicine, University
of Ljubljana, Korytkova 2, Ljubljana, Slovenia
E-mail: katarina.cerne@mf.uni-lj.si
cannabinoid receptors – cannabinoid type 1 (CB1) and
cannabinoid type 2 (CB2) – that both belonging to the
superfamily of G protein-coupled, seven-transmembrane
(7TM) domain receptors (3). None of the cannabinoid
receptor ligands, however, are entirely CB1- or CB2-specic.
Each of these ligands therefore differs in effect, not only
because they have different potency at cannabinoid
receptors but also because they can interact with other non-
CB1/non-CB2 targets, such as transient receptor potential
channel, vanilloid subfamily member 1 (TRPV1, aka
capsaicin or vanilloid receptor), G protein-coupled receptors
(GPR55 and GPR119), voltage-gated ion channels, and
neuronal transporters of catecholamines (4–6). Despite such
diversity, there are only four cannabinoid-based medicines
currently on the market: nabiximols (Sativex®), nabilone
(Cesamet® or Canemes®), dronabinol (Marinol® or
Syndros®), and cannabidiol (Epidiolex®) (7). Still being
developed are selective synthetic cannabinoid receptor
agonists, antagonists, and modulators, metabolism
inhibitors [such as fatty acid amide hydrolase (FAAH)
inhibitors] or inhibitors of endocannabinoid reuptake (8).
The aim of this review is to summarise what is known
about acute and chronic cannabinoid toxicity, primarily
based on animal and clinical studies of medicinal product
safety (9). Particular attention will be paid to identifying
future studies that could ll in current gaps in knowledge

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Černe K. Toxicological properties of Δ9-tetrahydrocannabinol and cannabidiol
Arh Hig Rada Toksikol 2020;71:1-11
and uncertainties surrounding the safety of exogenous
cannabinoids. This review will discuss the toxicology of
chemically defined, single compounds that are either
synthetic, semisynthetic, or plant-derived. We will also
discuss why the combination of THC with CBD has fewer
adverse effects than THC alone.
What this review will not discuss is the toxicology of
medicinal or recreational cannabis use or the health issues
associated with contaminants in plant extracts obtained
from uncontrolled sources.
CANNABINOID RECEPTORS
THC shares the ability of endocannabinoid ligands
anandamide (AEA) and 2-arachidonoylglycerol to activate
both the CB1 and CB2 receptor. It is their partial agonist, as
it binds to them with Ki values in the low nanomolar range.
Both receptors are coupled through Gi/o proteins, negatively
to adenylate cyclase and positively to mitogen-activated
protein kinase (3). CB1 receptors are mainly located at the
terminals of central and peripheral neurons, where they
usually mediate inhibition of neurotransmitter release. CB1
is one of the G protein-coupled receptors expressed at the
highest level in the central nervous system, with the notable
exception of the brain stem (4, 10). This may be why THC
is not associated with sudden death due to respiratory
depression, which indicates its low acute toxicity. In the
brain, CB1 receptors are particularly concentrated in the
hippocampus and cerebral cortex (areas involved in memory
and cognition), olfactory areas, basal ganglia and cerebellum
(areas involved in motor activity and posture control),
hypothalamus (area involved in appetite regulation and
energy homeostasis), limbic cortex (area involved in
sedation), and neocortex (area involved in the executive
function). CB1 is also found in peripheral nervous organs
(lungs, liver, bowel, thyroid, uterus, placenta, and testicles).
Therefore, these sites can also be the targets of cannabinoid
effects. CB2 receptors are primarily associated with cells
governing the immune function, such as splenocytes,
macrophages, monocytes, microglia, and B- and T-cells.
Recently, CB2 receptors have also been reported in other
cells, often up-regulated under pathological conditions (5).
The functions of these receptors include modulation of
cytokine release and immune cell migration. CB2 receptors
are expressed in the brain by microglia, blood vessels, and
by some neurons (4, 10). However, their action has not been
elucidated.
In contrast to THC, CBD does not seem to be
psychoactive and has low afnity for CB1 and CB2 receptors
(4). This is why its research has focused on non-CB1/non-
CB2 targets (see THC/CBD interactions below). When
interpreting the effects of cannabinoids, we should bear in
mind that cannabinoid receptors are members of the
rhodopsin-like family of 7TM receptors, at which,
according to Kenakin (11), the efcacy of agonist depends
on cell type and its condition. Therefore, it is difcult to
predict the therapeutic behaviour of cannabinoid receptor
agonists. This is probably why higher release of
endocannabinoids can be protective in one and damaging
in another case.
TOXICOLOGICAL PROPERTIES OF THC
Apart from natural THC, the most reliable toxicological
data available to date are for synthetic THC dronabinol and
synthetic THC analogue nabilone. Nabilone has a similar
chemical structure and is twice as potent as THC at the CB1
and CB2 receptors (12). The main indication for dronabinol
and nabilone is nausea and vomiting in adult patients
receiving chemotherapy when conventional antiemetics fail
to do the job. Dronabinol is also indicated for anorexia in
adults with AIDS. There are no safety proles for dronabinol
and nabilone in paediatric (<18 years) and elderly (>65
years) populations. The starting dose of dronabinol is
2.5 mg, administered twice daily as capsules for oral use.
The maximum recommended dosage is 20 mg/day (4–6
doses a day). Dronabinol is also administered as a 5 mg/
mL oral solution. The usual nabilone dose is 1 or 2 mg twice
a day, and the maximum recommended dosage is 6 mg/day,
administered as capsules for oral use (13, 14). Since both
are used short-term, data on chronic effects in humans are
not available.
Pharmacokinetics/toxicokinetics of THC
The bioavailability of dronabinol is low (4–20 %)
because of its high lipid solubility and extensive rst-pass
hepatic metabolism (15, 16). Its effects do not show clear
dose dependence (17). Due to lipid solubility, the apparent
volume of distribution is high (10 L/kg). Dronabinol is
extensively metabolised in the liver, primarily by
cytochrome P450 enzymes CYP2C9 and CYP3A4.
CYP2C9 is probably responsible for the formation of the
primary active metabolite hydroxy-Δ9-THC.
Pharmacogenomics studies indicate two to three times
higher plasma THC in individuals with a less active form
of CYP2C9, so adverse drug reaction in these individuals
may be more frequent and/or severe. The major route of
excretion is faeces (65 %), and the minor is urine (20 %)
(16). Urinary metabolites of dronabinol are identical to
those of marijuana and may be excreted over long time (18).
Nabilone has better bioavailability (at least 60 %) than
dronabinol and demonstrates dose linearity (15, 19).
Multiple cytochrome P450 enzymes extensively metabolise
nabilone to various metabolites, which have not been fully
characterised yet. Two major metabolic pathways are
probably involved in the biotransformation of nabilone: 1)
enzymatic reduction of the 9-keto group to form carbinol
metabolites; and 2) direct enzymatic oxidation of the
aliphatic side-chain to produce carboxylic and hydroxylic
analogues. The formation of carbinol metabolites is a major

3
nabilone metabolic pathway in dog. Hydroxylic analogues
appear to be more important in rhesus monkey and man.
Carbinols are long-lived metabolites that accumulate in the
plasma and concentrate in the brains of treated dogs over
time (see chronic toxicity) (20). Nabilone and its metabolites
are primarily eliminated in faeces (~65 %) and to a lesser
extent in urine (~20 %) (14, 17). Although no accumulation
of nabilone was observed after repeated doses, some
accumulation was observed for its metabolites (21).
Non-clinical toxicity of THC
Acute oral toxicity of THC in rats is lower in males
(LD50=1910 mg/kg) than in females (LD50=1040 mg/kg)
(22). The LD50 of oral nabilone is >1000 mg/kg in rats of
both sexes (21). The signs of acute toxicity of THC and
nabilone are similar and include lower respiratory rate,
ataxia, decreased activity, catatonia, hypothermia,
hypersensitivity to touch, and generalised body twitching.
Death was reported to be due to respiratory arrest (21, 22).
Sub-chronic and chronic effects of THC (5, 15, 50, 150,
and 500 mg/kg/day) administered by gavage were assessed
in rats in a 13-week study followed by a 9-week recovery
period and in a 2-year study (12.5, 25, and 50 mg/kg/day)
(23). Briey, THC-treated rats had lower body weight than
controls and exhibited convulsions, hyperactivity, and
changes in the reproductive organs of both male and female
rats. Reduced body weight was notable even at low dose
exposure and was attributed to metabolic changes caused
by THC. Weight loss was not associated with lower feed
consumption but with increased energy consumption
(evidenced by higher plasma corticosterone levels) needed
for hyperactivity, adaptation, and detoxication from THC.
Convulsions and hyperactivity were observed at all doses.
The onset and frequency of convulsions were also dose-
related. However, Chan et al. (23) observed no histological
changes in brain tissue of rats with a history of THC-related
convulsion or seizures. Luthra et al. (24) reported
generalised depression, followed by hyperactivity,
irritability, aggressiveness, and convulsion in rats treated
with THC for 119 days. The highest dose of THC in a sub-
chronic study in rats induced testicular atrophy and uterine
and ovarian hypoplasia (23). This study also found higher
serum FSH and LH at all doses.
Nabilone was assessed in two chronic toxicity studies
(21). The one in beagle dogs (0.5, 1.0, 2.0 mg/kg/day) was
planned to last one year but was terminated after seven
months due to high mortality. Most deaths were preceded
by convulsions, and toxicity was attributed to accumulation
of carbinol metabolites in the brain over time. In contrast
to dogs, nabilone chronic toxicity was minimal in rhesus
monkeys receiving doses of up to 2.0 mg/kg/day for one
year. Transient periods of anorexia, emesis, and ataxia were
observed only at the highest dose.
Chan et al. (23) also evaluated THC carcinogenicity in
rats and mice and found no evidence in rats at doses of up
Černe K. Toxicological properties of Δ9-tetrahydrocannabinol and cannabidiol
Arh Hig Rada Toksikol 2020;71:1-11
to 50 mg/kg/day [~20 times the maximal human
recommended dose (MHRD)]. In mice, THC produced
thyroid follicular cell adenoma (a common benign neoplasm
of the thyroid) in both sexes, but the effect was not dose-
dependent, as the hyperplasia was increased compared to
control at all doses and in both sexes. It is unclear what
these ndings mean. Carcinogenicity studies have not been
performed with nabilone.
Genotoxicity
THC and nabilone have no mutagenic potential (11–13,
23). Positive Ames and skin test results in mice for THC in
some in vitro tests are attributed to cytotoxic rather than
mutagenic action (25).
Reproductive toxicity
THC was evaluated in an oral embryo-foetal
developmental study in rats (at doses ranging from 12.5 to
50 mg/kg/day) (26) and in rabbits (0.5, 1.5, 5 and 15 mg/
kg/day) (27). No teratogenic effects were observed in rats.
Increased foetal mortality and early resorption were
associated with maternal toxicity, which manifested itself
as lower weight gain. In rabbits, one third of the foetuses
in the high-dose group had multiple anomalies (such as
acrania and spina bida). In a single-generation reproductive
study (28), male and female rats received 0.5, 1.5, and 5 mg/
kg/day of THC by gavage. Offspring to mothers receiving
1.5 and 5 mg/kg/day showed a dose-related drop in survival
at day 12 of lactation and at weaning.
A reproduction study of nabilone in rats (1.4, and 12 mg/
kg/day) and rabbits (0.7, 1.6, and 3.3 mg/kg/day) (29)
showed no teratogenic effects. However, it did nd dose-
related developmental toxicity, such as embryo death, foetal
resorption, decreased foetal weight, and disrupted
pregnancy. Another study in rats (24) revealed postnatal
developmental toxicity of nabilone at 1.4 and 12 mg/kg/
day), manifested by smaller litter size and lower survival
as well as lower initial body weight and hypothermia in
pups from the high-dose group.
There are no sufcient data on pregnancy outcomes in
women exposed to dronabinol (THC) or nabilone.
THC toxicity in clinical trials
Safety data on dronabinol come from 10 randomised,
double-blind, placebo-controlled clinical trials. In one trial
(30) patients with AIDS-related anorexia (N=139) were
receiving dronabinol as appetite stimulant (5 mg/day), and
in nine trials patients with cancer (N=454) were receiving
dronabinol as antiemetic in the dose range of 2.5–40 mg/
day (31–39) for no longer than six weeks. The most
frequently reported adverse events (33 %) in patients with
AIDS were euphoria, dizziness, somnolence, and thinking
abnormalities. The most common adverse events in patients
receiving the antiemetic dronabinol were drowsiness,
dizziness and transient impairment of sensory and

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Černe K. Toxicological properties of Δ9-tetrahydrocannabinol and cannabidiol
Arh Hig Rada Toksikol 2020;71:1-11
perceptual functions. Patients from both studies (24% in
antiemetic and 8% in appetite stimulant) reported dose-
related “highs” (elation, laughter, and heightened
awareness). The frequency of adverse effects on the central
nervous system (CNS) increased with doses, and their
severity greatly varied between patients. After oral
administration, dronabinol had an action onset of
approximately 30 min to one hour and a peak effect at two
to four hours (40). Psychoactive effects lasted four to six
hours. Other than those affecting the nervous system, the
most frequent adverse effects were gastrointestinal
(abdominal pain, nausea, and vomiting) and cardiovascular
(palpitation, tachycardia, vasodilatation/facial ush) (30–
39). The following were the most serious adverse effects
of dronabinol: neuropsychiatric, haemodynamic instability,
seizure, paradoxical nausea, vomiting, and abdominal pain.
Dronabinol should be discontinued in patients experiencing
a psychotic reaction or showing cardiovascular effects
(tachycardia, transient changes in blood pressure) and used
with caution in patients with a history of epilepsy or
recurrent seizures (13).
Nabilone has systematically been evaluated in
controlled clinical trials that lasted up to nine weeks (41–
43). The lowest nabilone dose (2 mg) had a few adverse
effects, whereas a 3–5 mg dose closely mirrored dronabinol’s
(25 mg) effects (18).
THC addiction and dependence
High levels of CB1 receptors are found in the brain areas
that are part of the mesocorticolimbic dopaminergic
pathway and are implicated in motivational and reward
processes (44). Being partial CB1 receptor agonists, THC
and its analogues should be tested for their addictive
potential (45). Many abused drugs that can lead to addiction
increase synaptic dopamine levels in the human limbic
striatum. The same was reported for THC in human studies
in healthy participants (46–48). Dopamine release was small
compared to amphetamine, cocaine, alcohol (10–15 %),
and nicotine (~10 %).
First studies in monkeys (49, 50) failed to show the
rewarding effects of THC, but newer studies with
intravenous dronabinol injection (1–6 µg/kg) conrmed it
in squirrel monkeys (51, 52). Another widely used predictor
of a reinforcing (and therefore addictive) effect is the
conditional place preference (CCP) test, in which a
compartment in a cage is associated (paired) with a tested
substance. Lepore et. al. (53) reported that CCP depended
on the dose and intervals between administration and that
dronabinol doses of 2 or 4 mg/kg every 24 h produced a
reliable shift in favour of the dronabinol-paired compartment.
Reinforcing effects have also been observed in humans
(12). Nabilone (4–8 mg/day) and dronabinol (10–20 mg/
day) produced stronger marijuana-like subjective effects,
such as feeling good, feeling “high”, and feeling “stoned”
than placebo. Nabilone had a slower onset of the peak
subjective effects.
Chronic therapy with dronabinol can lead to physical
dependence. One human study (17) showed that dronabinol
doses of 210 mg/day (~10 times higher than MHRD)
administered for 12 to 16 consecutive days produced
withdrawal syndrome within 12 h after discontinuation.
Initial symptoms were irritability, insomnia, and restlessness.
By hour 24 of discontinuation, withdrawal symptoms
intensied to include “hot ashes”, sweating, rhinorrhoea,
loose stool, hiccoughs, and anorexia. We still do not know
whether nabilone can also lead to physical dependence.
Patients that participated in clinical trials for up to ve days
showed no withdrawal symptoms after discontinuation of
dosing (54).
TOXICOLOGICAL PROPERTIES OF CBD
As a 99 % pure extract from C. sativa, active substance
cannabidiol was first approved in June 2018 under
proprietary name Epidiolex® (55). The United States Food
and Drug Administration (US FDA) and European
Medicines Agency (EMA) approved it for the treatment of
seizures associated with Lennox-Gestaut (LGS) and Dravet
syndrome (DS) in patients two years of age or older.
Epidiolex® is administered as a 100 mg/mL oral solution.
The starting dose is 2.5 mg/kg twice a day and the maximum
recommended dose is 10 mg/kg twice a day (20 mg/kg/day)
(55, 56). Considering that Epidiolex® has been approved
for treatment in children, CBD has become the most
extensively toxicologically tested cannabinoid, and thus the
most reliable source of toxicological data. However,
because of the seriousness of the indications and failure of
patients to respond to existing medication, Epidiolex® was
approved in spite of certain deficiencies in the safety
assessment (e.g., inadequate safety assessment of major
human metabolite 7-COOH-CBD). Additional studies listed
in Table 1 should therefore be carried out as part of post-
marketing surveillance to obtain a complete safety prole
of CBD. Furthermore, no clinical trial with Epidiolex® has
been conducted in patients older than 55 years, so its safety
prole does not cover the elderly population. General
recommendation is to start with the lowest dose (56).
Since CBD is derived from C. sativa, Table 2 presents
a thorough assessment of the abuse and dependence
potential of Epidiolex® (4, 57–59). A human study (58)
found marginal abuse potential at a higher therapeutic dose
(1500 mg/day) and supratherapeutic dose (4500 mg/day),
but there is little other evidence that CBD could cause
addiction. The results of a human dependence study of CBD
were negative (59).
Pharmacokinetics/toxicokinetics of CBD
Plasma CBD concentrations show a nonlinear increase
with dose and 6.5 % bioavailability at a 3000-mg dose (60).

5
Černe K. Toxicological properties of Δ9-tetrahydrocannabinol and cannabidiol
Arh Hig Rada Toksikol 2020;71:1-11
CBD absorption increases three times with a high-fat meal
and six times with new oral delivery system for lipophilic
active compounds (61, 62). Its high estimated volume of
distribution (18,800—30,959 L) indicates accumulation of
CBD in body fat (63). CBD is extensively metabolised in
the liver and gut, mainly by the CYP2C19, CYP3A4,
UGT1A7, UGT1A9, and UGT2B7 enzymes (64). Drug
interaction trials to assess the effect of CBD on these
enzymes in healthy volunteers will be conducted during the
post-marketing period (Table 1) (55, 56). The metabolism
of CBD is very complex, especially in hepatocytes. The
main human metabolite is 7-carboxy-cannabidiol (7-COOH-
CBD; ~90 % of all drug-related substances measured in the
plasma) (64). Its toxicological profile has not been
investigated because experimental animals for toxicological
studies (mice, rats, and dogs) do not metabolise CBD to a
comparable extent as humans (65). The major concern with
7-COOH-CBD could be its reactive acyl-glucuronide (66)
The primary excretion route of CBD is through faeces
(84 %), followed by urine (8 %) (63).
Non-clinical toxicology of CBD
In a study of acute effects in rhesus monkeys (67),
intravenous CBD caused death by respiratory arrest and
cardiac failure at doses above 200 mg/kg (LD50=212 mg/
kg). At the lower dose of 150 mg/kg, survivors recovered
in one to three days, and liver weights increased from 19
to 142 %. In the part of the study investigating subchronic
effects (after 90 days of oral administration), the authors
reported inhibition of spermatogenesis at the highest oral
dose of 300 mg/kg (67).
Animal studies of CBD alone described below make
part of the Epidiolex® European Public Assessment Report
(EPAR, EMA’s scientic monography) (56). To the best of
my knowledge, they have not been published and therefore
no further detail or original references are currently
available. All these studies were conducted in accordance
with medicinal product safety standards and protocols and
reviewed by the EMA committee (9).
Two oral chronic toxicity studies (referred to in 56) have
assessed CBD in Wistar rats (receiving 15, 50, or 150 mg/
kg/day for 6 months) and Beagle dogs (receiving 10, 50,
100 mg/kg/day for 9 months). In both species the primary
target organ was the liver. Hepatocellular hypertrophy was
detected at all doses, accompanied by an increase in alanine
transferase (ALT) and alkaline phosphatase (ALP).
A 104-week oral carcinogenicity study in Wistar rats
(referred to in 56) revealed no drug-related neoplastic
findings. However, the study had several drawbacks,
including impure active substance, excessive effect of body
weight, and unknown exposure to the two major human
metabolites.
Table 1 Recommended post-marketing studies to obtain a complete safety prole of cannabidiol (CBD)
Non-clinical toxicity studies
Toxicity studies with CBD metabolite 7-COOH-cannabidiol in rat:
- embryo-foetal developmental study
- pre- and postnatal developmental study
- juvenile animal toxicity study
- 2-year carcinogenicity study with gavage
Toxicity studies with CBD
- 2-year carcinogenicity study in mouse
- 2-year carcinogenicity study in rat with gavage
Clinical studies
- Potential for chronic liver injury
- Effect on glomerular ltration rate
- Pregnancy outcome study
- QT interval prolongation trial at the maximum tolerable dose
Drug-drug interaction trials in healthy volunteers
CBD effect on the pharmacokinetics of:
- caffeine
- sensitive CYP2B6* and CYP2C9 substrate
- sensitive UGP1A9** and UGTB7 substrate
Strong CYP3A inhibitor effects on pharmacokinetics of CBD
Strong 2C9 inhibitor effects on pharmacokinetics of CBD
Rifampin effects on pharmacokinetics of CBD
* cytochrome P450; ** UDP-glucuronosyltransferase

6
The genotoxic potential of CBD was also investigated
in a standard battery of tests, but their results were negative
for mutagenicity and clastogenicity (referred to in 56).
A full battery of oral reproductive and developmental
studies has been conducted with purified CBD. In an
embryo-foetal development study in Wistar rats, litter loss
was noted at the highest applied dose of 250 mg/kg. In a
prenatal and postnatal development study (referred to in
56) rat exposure to the highest doses (150 and 200 mg/kg/
day) affected reproductive organs (smaller testes in males,
reduced fertility index in females). A high dose of 125 mg/
kg also reduced foetal body weight in New Zealand white
rabbit, which was related to maternal toxicity. The
developmental toxicity in rabbits occurred at maternal
plasma concentration similar to human at therapeutic doses
(referred to in 56). In rats these concentrations were much
higher. No adequate data are available on pregnancy
outcome in women exposed to CBD.
A juvenile toxicity study in Wistar rats (referred to in
56) showed neurobehavioral decits and delayed sexual
maturation in males. A no observed effect level (NOAEL)
was 150 mg/kg/day.
Clinical toxicology of CBD
Safety data on Epidiolex® were obtained from four
randomised, double-blind, placebo-controlled multicentre
trials with exposure to CBD doses of 5, 10, and 20 mg/kg/
day (68–70). These phase II studies were conducted in 2 to
55 year-old patients with LGS (N=235) and DS (N=88) for
up to 14 weeks.
Additional non-controlled safety data have been
obtained from an ongoing open-label Phase III study (Study
1415) in LGS and DS patients (N=644), which is being
conducted at 38 sites in the USA and Australia. Since this
trial is not nished, an interim analysis of long-term safety
was conducted (71, 72).
The most common adverse events in CBD-treated
patients affected the following systems: CNS (somnolence,
sedation), gastrointestinal tract (lower appetite, diarrhoea),
liver (higher transaminase), and the lungs (pneumonia). The
severity of these events was generally mild to moderate.
Diarrhoea, weight loss, higher ALT, and somnolence/
sedation/lethargy were all dose-related. There were two
serious cases of transaminase elevation, two severe events
with rash (one consistent with a hypersensitivity reaction)
and three severe cases of appetite loss. The CBD-treated
and the placebo group did not differ in the rate of respiratory
failure. Children had lower weight, which was associated
to a certain extent with appetite loss (68–71).
Treatment with CBD is clearly associated with an
increased risk of hepatotoxicity (68–71). Higher doses of
CBD and concomitant use of valproate increase the risk of
transaminase elevation in patients. Two patients
concomitantly treated with valproate experienced toxic
Černe K. Toxicological properties of Δ9-tetrahydrocannabinol and cannabidiol
Arh Hig Rada Toksikol 2020;71:1-11
Table 2 Cannabidiol (CBD) abuse potential
TYPE OF STUDY RESULTS
Receptor binding studies
- cannabinoid receptors no signicant afnity
- opioid receptors no signicant afnity
Non-clinical studies evaluating general behaviour (similarity to THC)
- tetrad test no meaningful abuse related signal
- drug discrimination study no meaningful abuse related signal
- self-administration study no meaningful abuse related signal
Clinical studies evaluating efcacy and safety in patients with LGS* or DS*
- Phase I clinical study no euphoria or other abuse-related signals
- Phase II/III studies could not be evaluated***
Phase I human abuse potential (HAP) study (N=40, with 35 completers)
randomized, double blind, placebo-controlled trial
subjects: healthy recreational poly-drug users
positive control: THC (10, 30 mg), alprazolam (2 mg)
negative control: placebo
mean DRUG LIKING SCORE
lower therapeutic dose: 750 mg/day not signicantly different
higher therapeutic dose: 1500 mg/day signicantly different (very small increase)
supra-therapeutic dose: 4500 mg/day signicantly different (very small increase)
Human physical dependence study following chronic administration
3 days after discontinuation no withdrawal signs and symptoms
*Lennox-Gastaut syndrome; **Dravet syndrome; ***concomitant use of other seizure drugs and limited capacity of patients

7
hepatocellular injury, metabolic acidosis, and
encephalopathy. There appears to be no pharmacokinetic
interaction between CBD and valproate, although a
pharmacodynamical interaction is currently being
investigated. The potential of CBD to cause chronic liver
injury should be evaluated in the post-marketing period (55,
56) (Table 1).
MECHANISMS OF THC/CBD
INTERACTIONS
In spite of its low afnity for the CB1 and CB2 receptors,
CBD can interfere with some THC adverse effects,
particularly in the brain, without interfering with the
intended THC effects, such as muscle relaxation (73).
Understanding pharmacodynamic interactions between
THC and CBD can be quite a challenge. CBD is a ligand
with very low afnity for the CB1 receptor but can still
increase CB1 constitutional or endocannabinoid activity (5),
which has been conrmed by. thermodynamic ndings that
CBD increases membrane uidity and thereby the activity
of the CB1 receptor (74). Another mechanism of action is
that CBD increases the levels of primary endocannabinoids
AEA and 2-arachidonyl-glycerol (2-AG) (5). CBD may
also interfere with THC through interaction with other non-
CB1 receptors and enzymes in the ‘expanded
endocannabinoid system’ (5). In their systematic review
McPartland et al. (5) propose several non-CB1 receptor
mechanisms of CBD antagonising or potentiating THC
effects. For example, CBD may attenuate the anxiogenic
effect of THC by acting as a direct or indirect agonist on
serotonin 1A receptors (5-HT1A). In contrast, it can
potentiate THC action on CB1 receptors by reducing
peripheral hyperalgesia via TRPV1 channels (75). Sativex®,
as a mixture of THC and CBD, consequently provided better
antinociception than THC given on its own 76).
In terms of pharmacokinetic CBD/THC interaction,
CBD may impair THC hydrolysis by CYP450 enzymes
(77). The inhibition of THC metabolism may vary with
species, timing of administration (CBD pre-administration
vs co-administration), and CYP isoenzymes. In rats or mice
THC effects are potentiated when CBD is administered
30 min to 24 h before THC but mitigated if co-administered
(78). In humans, no pharmacokinetic interactions between
THC and CBD at clinically relevant doses have been
reported (79). Co-administration of CBD with THC in one
study (80) yielded similar maximum plasma levels of THC
as when it was administered alone. Whether CBD will
antagonise or potentiate THC effects also seems to depend
on their administration ratio, and this ratio varies with
species (5).
TOXICOLOGY OF CBD+THC
COMBINATIONS
The combination of THC and CBD in a 1:1 ratio makes
the active substance nabiximols of the cannabinoid-based
medicine Sativex® (81). It is an oromucosal spray approved
for the treatment of multiple sclerosis-associated spasticity
in adult patients when all other treatment has failed. There
is no safety prole of nabiximols in children (>18 years)
and the elderly, even though clinical trials included patients
up to 90 years of age. Elderly patients may be more
susceptible to some adverse effects in the CNS. The
oromucosal (e.g. sub-lingual) route resolves the problem
of variable bioavailability (typically 6 to 20 %) of orally
administered cannabinoids due to rst-pass metabolism.
Each 100 µL spray contains 2.7 mg THC and 2.5 mg CBD.
The starting dose is two sprays per day and the maximum
dose is 10–12 sprays per day (corresponding to 32.4 mg
THC and 30 mg CBD) (81).
A study using a rat model of Huntington’s disease
showed that nabiximols can up-regulate CB1 gene
expression (82). CBD increases the levels of the primary
endocannabinoids AEA and 2-arachidonyl-glycerol (2-AG)
(6).
The most common adverse effects of nabiximols in
clinical trials conducted in patients with multiple sclerosis
were dizziness, fatigue and gastrointestinal disorders (e.g.
nausea, vomiting, diarrhoea) (82–92). These adverse effects
and poor efcacy were the main reasons for some patients
to discontinue therapy (88, 90). In patients with multiple
sclerosis the risk of accidental injury may be increased (83,
87, 92–94). There is little evidence of abuse (addiction) or
dependence, and the risk of either to develop is small.
However, trials to date have mainly used therapeutic doses,
and it is possible that supratherapeutic doses could cause
addiction and/or dependence (85, 87, 92–94).
CONCLUSION
In spite of uncertainties about the safety of cannabinoids,
there are no doubts about the acute neurological and
cardiovascular effects of THC. However, THC is not
associated with sudden death due to respiratory depression
as is the case with opioid analgesics. Long-term cognitive,
psychological, and endocrine effects of THC are still being
investigated.
As for CBD, it can be toxic to the liver and increases
the risk of somnolence and sedation, but the most commonly
observed adverse events in controlled clinical trials were
mild to moderate. However, these clinical trials included a
small number of subjects and some aspects require
continued pharmacovigilance. Regardless of different views
on the subject, cannabinoid-based medicines need to be
assessed just as any other substance in terms of quality,
efcacy, and safety.
Černe K. Toxicological properties of Δ9-tetrahydrocannabinol and cannabidiol
Arh Hig Rada Toksikol 2020;71:1-11

8
Conicts of interest
None to declare.
Acknowledgements
This work was supported by research grants from the
Slovenian Research Agency (P3-067). I wish to thank
Nevenka Dolžan for technical assistance and Martin
Cregeen for translating sections of the text into English.
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Toksikološke lastnosti kanabinoidov
Iz rastline Cannabis sativa L. so do sedaj izolirali že več kot 100 tokanabinoidov, poleg njih pa obstaja več kot 550
sintetičnih spojin, ki delujejo na kanabinoidne receptorje CB1 in CB2. Prav tako je treba omeniti, da nobeden od ligandov
kanabinoidnih receptorjev ni popolnoma CB1- ali CB2-specičen. Zato se učinki vsakega od njih razlikujejo ne le zaradi
različne moči na kanabinoidnih receptorjih, ampak tudi zato, ker lahko delujejo na druga ne-CB1 in ne-CB2 prijemališča.
Najpogosteje proučevani kanabinoid je Δ9-tetrahidrokanabinol (THC). THC je delni agonist na obeh kanabinoidnih
receptorjih, vendar je njegov psihoaktivni učinek povezan predvsem z aktivacijo receptorjev CB1. Receptor CB1 je eden
izmed metabotropnih receptorjev z največjo ekspresijo v osrednjem živčevju, z izjemo možganskega debla. Čeprav so
akutni učinki na osrednji živčni sistem THC jasno opredeljeni, je tveganje za ireverzibilne nevropsihološke učinke THC
kot neodvisnega dejavnika potrebno nadalje raziskati za pojasnitev povezave. Za razliko od THC, tokanabinoid kanabidiol
(CBD) nima psihoaktivnih učinkov, vendar lahko pri sočasni uporabi vpliva na nekatere učinke THC. CBD, ki nima
pomembne anitete za CB1 in CB2, aktivira ali zavira številne uveljavljene in domnevne farmakološke tarče. CBD je kot
aktivna snov v zdravilu Epidiolex® pred kratkim opravil nadzorovana klinična preskušanja, da so ocenili njegovo varnost
pri zdravljenju redkih epileptičnih sindromov pri otrocih. Največjo zaskrbljenost glede varnosti so predstavljale povišane
vrednosti transaminaz. Zato je treba izvesti postmarketinški nadzor toksičnosti za jetra. Članek bo povzel kar je znano o
akutnih in kroničnih toksikoloških učinkih, katere študije še manjkajo in kaj so negotovosti v zvezi z varnostjo eksogenih
kanabinoidov.
KLJUČNE BESEDE: Δ9-tetrahidrokanabinol; akutna in kronična toksičnost; kanabidiol; podatki od ljudi; študije na živalih