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Safety Assessment of a Hemp Extract using Genotoxicity and Oral Repeat-Dose Toxicity Studies in Sprague-Dawley Rats

Authors:
  • RW Coppock, DVM, Toxicologist and Assoc
  • Charlotte's Web Inc.

Abstract and Figures

Cannabinoids are extracted from Cannabis sativa L. and are used for a variety of medicinal purposes. Recently, there has been a focus on the cannabinoid Cannabidiol (CBD) and its potential benefits. This study investigated the safety of a proprietary extract of C. sativa, consisting of 9% hemp extract (of which 6.27% is CBD) and 91% olive oil. The mutagenic potential of the hemp extract was evaluated with the AMES assay inclusive of a hepatic drug metabolizing mix (S9) rich in CYP enzymes. The test article did not elicit evidence of bacterial mutagenicity. GLP compliant 14-day and a 90-day toxicity study were conducted. Olive oil was used as a control. The 90-day study had a 28-day recovery period. Treatments for the 14-day non-recovery range-finding study were 0, 1000, 2000 and 4000 mg test article/kg body weight (bw)/day for 14 days. There was a non-statistically significant (p > 0.05) decrease in body weights for the male and female rats receiving the test article. Hypoactivity, hyperactivity, reduced food consumption and piloerection were observed in the rats receiving 4000 mg test article/kg bw. Histopathology showed an increase in the size of liver cells (hypertrophy) around the central vein (centrilobular) in Groups 3 (3/10) and 4 (5/10) that correlated with increased liver weights. In the 90-day study, 8 groups of rats were dosed with 0, 200, 400 and 800 mg test article/kg bw/day. Groups 5 to 8 had a 28-day recovery. There were no test article-linked changes in clinical observations, physical examinations, Functional Observation Battery, ophthalmology, Motor Activity Assessment, hematology, clinical chemistries and macropathology (all groups). With the exception of the liver and adrenal gland, no test article-linked pathology was observed. For all rats receiving the test article, histopathology showed hypertrophy of liver cells around the central vein. The increase of liver weight is most likely caused by hypertrophy due to up-regulation of the hepatic drug metabolizing enzymes. The hepatocellular hypertrophy was completely reversed in 28 days and was not considered to be an adverse effect. Vacuolization of the adrenal zona fasciculata was observed in the control and 800 mg test article/kg bw groups. The vacuolization of the zona fasciculata was of the same incidence and severity in treatment and control male rats and correlated with an increased in the weights of the adrenal glands. In addition, a statistically significant increase (p
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Toxicology Reports
journal homepage: www.elsevier.com/locate/toxrep
Safety Assessment of a Hemp Extract using Genotoxicity and Oral Repeat-
Dose Toxicity Studies in Sprague-Dawley Rats
Margitta Dziwenka
a,
*, Robert Coppock
b
, Alexander McCorkle
c
, Eddie Palumbo
c
, Carlos Ramirez
c
,
Stephen Lermer
c
a
Toxalta Consulting Ltd, Box 8, Vegreville, AB, T9C 1R1 Canada
b
RW Coppock, DVM, Toxicologist and Associates Ltd, PO Box 2031, Vegreville, AB T9C 1T2 Canada
c
Charlottes Web Inc., 2425 55
th
Street, Suite 100, Boulder, CO 80301 United States
ARTICLE INFO
Keywords:
Cannabis sativa
cannabinoids
cannabidiol
CBD
toxicology
olive oil
hemp extract
CYP
liver
mutagenicity
CW hemp
Charlottes Web, Inc.
ABSTRACT
Cannabinoids are extracted from Cannabis sativa L. and are used for a variety of medicinal purposes. Recently,
there has been a focus on the cannabinoid Cannabidiol (CBD) and its potential benets. This study investigated
the safety of a proprietary extract of C. sativa, consisting of 9% hemp extract (of which 6.27% is CBD) and 91%
olive oil. The mutagenic potential of the hemp extract was evaluated with the AMES assay inclusive of a hepatic
drug metabolizing mix (S9) rich in CYP enzymes. The test article did not elicit evidence of bacterial muta-
genicity. GLP compliant 14-day and a 90-day toxicity study were conducted. Olive oil was used as a control. The
90-day study had a 28-day recovery period. Treatments for the 14-day non-recovery range-nding study were 0,
1000, 2000 and 4000 mg test article/kg body weight (bw)/day for 14 days. There was a non-statistically sig-
nicant (p> 0.05) decrease in body weights for the male and female rats receiving the test article.
Hypoactivity, hyperactivity, reduced food consumption and piloerection were observed in the rats receiving
4000 mg test article/kg bw. Histopathology showed an increase in the size of liver cells (hypertrophy) around
the central vein (centrilobular) in Groups 3 (3/10) and 4 (5/10) that correlated with increased liver weights. In
the 90-day study, 8 groups of rats were dosed with 0, 200, 400 and 800 mg test article/kg bw/day. Groups 5 to 8
had a 28-day recovery. There were no test article-linked changes in clinical observations, physical examinations,
Functional Observation Battery, ophthalmology, Motor Activity Assessment, hematology, clinical chemistries
and macropathology (all groups). With the exception of the liver and adrenal gland, no test article-linked pa-
thology was observed. For all rats receiving the test article, histopathology showed hypertrophy of liver cells
around the central vein. The increase of liver weight is most likely caused by hypertrophy due to up-regulation of
the hepatic drug metabolizing enzymes. The hepatocellular hypertrophy was completely reversed in 28 days and
was not considered to be an adverse eect. Vacuolization of the adrenal zona fasciculata was observed in the
control and 800 mg test article/kg bw groups. The vacuolization of the zona fasciculata was of the same in-
cidence and severity in treatment and control male rats and correlated with an increased in the weights of the
adrenal glands. In addition, a statistically signicant increase (p < 0.05) in adrenal-to-body weight ratios was
observed for females receiving 800 mg test article/kg bw. This increase in adrenal-to-body weight ratio did not
correlate with any of the pathology ndings. The NOAEL for the test article is 800 mg/kg bw/day for female and
400 mg/kg bw/day for male Sprague Dawley rats.
1. Introduction
Humans have been utilizing the Cannabis sativa L. plant for mil-
lennia for both medicinal and recreational purposes. The C. sativa L.
plant originates from Central Asia and has recently seen an increase in
interest likely because of its many applications due to the large phy-
tochemical content as well as being a rich source of both cellulosic and
woody bers [1]. Two preparations of marijuana for recreational use
are hashish (resinous) and marijuana (leaves and owers) [2]. Syn-
thetic cannabinoids are emerging as psychoactive substances and have
recreational use [3]. Recreational use of marijuana, hashish synthetic
cannabinoids are associated with ischemic and other types of strokes
[2]. The cannabinoids, which are oxygen containing aromatic hydro-
carbon compounds, are one of the most researched groups of all the
https://doi.org/10.1016/j.toxrep.2020.02.014
Received 21 September 2019; Received in revised form 18 February 2020; Accepted 18 February 2020
Corresponding author.
E-mail addresses: dziwenka.toxalta@gmail.com (M. Dziwenka), r.coppock@toxicologist.ca (R. Coppock).
Toxicology Reports 7 (2020) 376–385
Available online 20 February 2020
2214-7500/ © 2020 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/BY-NC-ND/4.0/).
T
phytochemicals in C. sativa L. and include at least 70 compounds, of
which delta-9-tetrahydrocannabinol (THC) and cannabidiol (CBD) are
some of the most well-known [4]. THC and synthetic cannabinoids have
anity for the cannabinoid receptors. CBD does not have anity for
the cannabinoid 1 receptor (CB1R) and the cannabinoid 2 receptor
(CB2R) and there is animal model evidence to show it modulates the
adverse eects of ischemic stroke and likely acts on the sigma1 re-
ceptor [57]. Additionally, CBD, in laboratory animal models, has been
shown to be a benecial treatment in substance use disorder including
protection of the liver from alcohol damage [8,9]. The US government
recently passed the Agriculture Improvement Act which included
changes to the production and marketing of hemp and derivatives of
cannabis with extremely low concentrations of delta-9-tetra-
hydrocannabinol (THC). These changes removed hemp from the Con-
trolled Substances Act, but preserved the US Food and Drug Adminis-
trations(FDA) authority to regulate cannabis and cannabis-derived
compounds. This study is investigating the toxicology of a proprietary
CBD rich hemp extract.
With the increasing interest in using products containing CBD in
humans, it is essential to fully evaluate the safety of CBD consumption.
While the published oral toxicological studies on CBD and hemp ex-
tracts are limited, the current available data suggests CBD is safe for
human consumption, though additional studies need to be conducted. A
review by Bergamaschi et al. [10] described in vivo and in vitro reports
of CBD administrations at a variety of dose levels. The authors con-
cluded that several studies support the conclusion that CBD is well
tolerated and safe for humans at high doses and with chronic use, but
there is evidence of potential drug metabolism interactions (pharma-
cokinetics), cytotoxicity, and decreased receptor activity (pharmaco-
dynamics). Therefore, the authors also stated additional studies are
needed to further evaluate the safety of CBD. A more recent review was
conducted by Iand and Grotenhermen [11] to build on the Berga-
maschi et al. [10] review regarding CBD safety and any potential side
eects. This review also concluded that numerous studies show that
CBD is well tolerated and safe in humans at high doses and with chronic
use. However, in order to further understand CBD and validate these
ndings, additional studies evaluating the safety of CBD are needed.
The objective of the current studies was to assess the genotoxicity and
preclinical safety of a proprietary hemp extract and to contribute sig-
nicant safety data on CBD to the currently limited available data.
2. Material and Methods
2.1. GLP, OECD, and National Research Council compliances
Three Ames tests, one on the extract diluted in olive oil and two on
undiluted extracts, and two oral (gavage) dosing studies in rats were
completed. The preclinical studies included a 14-day range nding
study (14-day study) and a 90-day study with a 28-day recovery period
(90-day study). All studies were compliant with the US FDA Good
Laboratory Practices, and the preclinical studies were also compliant
with the OECD Principles of Good Laboratory Practices, the US FDA
Toxicological Principles for the Safety Assessment of Food Ingredients
[Redbook 2000, Revised 2007 IV.C. 4. a. Subchronic Toxicity Studies
with Rodents (2003)] and the OECD Guidelines for Testing of
Chemicals [Section 4 (Test No. 408): Health Eects, Repeated Dose 90-
Day Oral Toxicity Study in Rodents (1998)] [1215]. Animal housing
and care was in compliance with the Guide for the Care and Use of La-
boratory Animals [16]. The current state of scientic knowledge does
not provide acceptable alternatives to the use of live animals to ac-
complish the objective of this study.
Table 1
Specications for the Test Article.
Parameter Specication Testing Method
Identication
Visual and Aroma Olive oil aroma
Dark brown color
Free of foreign material
No visual inconsistencies
No haze to slightly hazy appearance
Organoleptic
Density As reported NIST Handbook 133
Potency
Hemp Extract Concentration NLT 43 mg/serving (0.5 ml) Calculated
THC As reported HPLC
THC-A As reported HPLC
Total THC + THC-A NMT 3 mg/ml HPLC
CBD 50-65 mg/mL HPLC
CBD-A NMT 3 mg/mL HPLC
Microbiology
Salmonella spp. Absent AOAC 2016.01/USPS2022
Escherichia Coli < 10 CFU/mL CMMEF 8.933/AOAC 991.14
Total aerobic plate count < 10
4
CFU/mL BAM Ch. 8/USPC2021
Yeast As reported CMMEF 5
th
21.51/USPM2021
Molds As reported CMMEF 5
th
21.51/USPM2021
Total yeast and molds < 10
3
CFU/mL CMMEF 5
th
21.51/USPM2021
Total coliforms < 10
2
CFU/mL CMMEF 8.933/AOAC 991.14
Heavy Metals
Inorganic arsenic NMT 6.67 ppm 2011.19 and 993.14 AOAC International
Cadmium NMT 2.73 ppm 2011.19 and 993.14 AOAC International
Lead NMT 333 ppb 2011.19 and 993.14 AOAC International
Mercury NMT 200 ppb 2011.19 and 993.14 AOAC International
Residual Solvents
Class 3 NMT 5000 ppm USP chapter 467
Pesticides
Bifenthrin NMT 50 ppb AOAC Ocial method 2007.01
Bifenazate NMT 300,000 ppb AOAC Ocial method 2007.01
Pyrethrin NMT 1000 ppb AOAC Ocial method 2007.01
CBD cannabidiol; CBD-A = cannabidiolic acid; THC delta-9-tetrahydrocannabinol; THC-A = tetrahydrocannabinolic acid; HPLC = high pressure
liquid chromatography; NLT = not less than; NMT = not more than; USP United States Pharmacopeia.
M. Dziwenka, et al. Toxicology Reports 7 (2020) 376–385
377
2.2. Test material
The test article was supplied by Charlottes Web, Inc. (2425 55
th
Street, Suite 100, Boulder, CO 80301) and is a proprietary blend of 9%
hemp extract and 91% organic extra virgin olive oil, which is produced
by an isopropanol extraction method under current Good
Manufacturing Practices (CGMP). Fatty acids comprise approximately
88.70% of this extract, while the phytocannabinoid content is 6.96% (of
this, 6.27% is CBD); the remaining 4.34% consists of fatty alkanes,
sterols, terpenes and tocopherols. Therefore, approximately 100% of
the constituents of this proprietary hemp extract are accounted for. An
Ames test was conducted on this test article and two additional Ames
tests were conducted on undiluted extract, one an isopropanol extract
and the other a supercritical CO
2
extract. This was done to determine
the impact, if any, of the olive oil on the results. Additionally, this
product meets the Federal requirements for hemp products under the
Agriculture Improvement Act in regard to THC. The test article used in
these studies met the specications outlined in Table 1 and the can-
nabinoid content is listed in Table 2. For the 14-day study, concentra-
tion verications were conducted on study day 1. For the 90-day study,
concentration verication analysis samples were collected from the
preparations on day 1, day 46 and Day 94, and assayed for the hemp
extract.
2.2.1. Test material preparation
The test article, for both the 14-day study and the 90-day study was
mixed, weight to volume (w/v), in olive oil (Sigma-Aldrich, St. Louis,
MO and O-Live & Co, Norwalk, CT) to obtain the desired concentra-
tions. Fresh formulations containing 200, 400, and 800 mg/mL of the
test article in olive oil were prepared daily. The formulations were
stirred at ambient temperature to achieve a homogenous mixture. For
the 90-day study, there were no analytical dierences between the neat
test article collected at the beginning of dosing regimens and the test
article collected at the end of the dosing regimens. For the Ames tests,
the same test article which was used for the animal studies was tested as
well as undiluted extract produced using two dierent manufacturing
methods; isopropanol extraction and supercritical CO
2
extraction.
2.3. Animals
Sprague-Dawley male and female rats (Charles River CD®
1
IGS,
Stone Ridge, NY and Raleigh, NC) were used in the 14-day and the 90-
day studies. For both studies, the rats were 6 weeks of age at the start of
the conditioning interval. The acclimation period was 6 days for the 14-
day study and 12 days for the 90-day study. Criteria used for selecting
animals for both studies were adequate body weight gain, absence of
clinical signs of disease or injury, and a body weight within ± 20% of
the mean within a sex. For the 14-day study, 40 rats were distributed to
treatment groups according to stratication by body weight so that
there was no statistically signicant dierence among group body
weight means within a sex (Table 3). Sixty male rats weighing 224-
286 g and 60 female rats weighing 170-218 g were distributed to
treatment groups stratied by body weight among the dose and control
groups (Table 4). For both the 14-day and the 90-day studies, body
weights were recorded twice during the acclimation period and weekly
for the duration of the study. Feed intake was determined at the same
day body weights were determined. Filtered potable water and feed
(2016CM Certied Envigo Teklad Global Rodent Diet
2
) were provided
ad libitum. Feed and water were assayed for detrimental substances and
none were found at levels that would alter study results. In the 90-day
study, sentinel rats were kept in the animal rooms. Serology done on
samples collected at the end of the study from the sentinel rats were
negative for Rat Parvovirus, Toolans Virus (H-1), Kilham Rat Virus, Rat
Minute Virus, Parvovirus NS-1, Rat Coronavirus, Rat Theilovirus, and
Pneumocystis carinii.
2.4. Clinical exams
The animals in the 14-day and the 90-day study were observed daily
for clinical evidence of ill health and given physical exams weekly
corresponding to body weight determinations. The physical exam in-
cluded observing for changes in skin, fur, eyes, and mucous membranes,
occurrence of secretions and excretions and autonomic activity (e.g.,
lacrimation, piloerection, pupil size and unusual respiratory pattern).
The exam also included changes in gait, posture, and response to
handling, as well as the presence of clonic or tonic movements, ste-
reotypies (e.g., excessive grooming, repetitive circling), or bizarre be-
havior (e.g., self-mutilation, walking backwards). All abnormal ob-
servations were recorded. Rats in the 90-day study (during week 12)
received a Functional Observation Battery in an open eld for excit-
ability, autonomic function, gait and sensorimotor coordination (open
eld and manipulative evaluations), reactivity and sensitivity (elicited
behavior) and other abnormal clinical signs including, but not limited
to convulsions, tremors, unusual or bizarre behavior, emaciation, de-
hydration and general appearance. Additionally, during week 12 rats in
the 90-day study underwent a Motor Activity Assessment using a
Photobeam Activity System [San Diego Instruments, Inc (San Diego,
CA)] following recommended procedures. Investigators doing the
physical examinations, Functional Observation Battery, and Motor
Activity Assessment were blind to the treatments the rats were re-
ceiving.
2.5. Ophthalmologic exam
Ophthalmic examinations were done on all rats in Groups 1-4 in the
90-day study by a veterinary ophthalmologist
3
. The evaluations were
done once in the pretrial period and on study day 88. The examinations
were done using focal illumination, slit lamp biomicroscopy, and in-
direct ophthalmoscopy.
2.6. Treatment
For both studies, individual doses were calculated using the most
recent weekly body weights. All doses were adjusted with the olive oil
vehicle and all rats received a volume of 5 mL/kg. The formulated test
substances were administered orally at approximately the same time
( ± 2 hours) each day by gavage using an accepted procedure.
Treatments for the 14-day non-recovery range-nding study were 0,
1000, 2000 and 4000 mg test article/kg body weight (bw)/day for 14
days (Table 3). The control groups received 5 ml/kg bw of the olive oil
vehicle. For the 90-day study, the rats were dosed with 0, 200, 400 and
800 mg test article/kg bw/day (Table 4). In the 90-day study, rats in
Table 2
Cannabinoid content of the Test Article.
Cannabinoid 14- Day Study
Result (mg/mL)
90- Day Study
Result (mg/mL)
THC 2.1 2
THC-A 0 0
CBD 55.0 60
CBD-A 0 0
CBD cannabidiol; CBD-A = cannabidiolic acid; THC delta-9-tetra-
hydrocannabinol.
THC-A = tetrahydrocannabinolic acid.
1
®Charles River.
2
®Envigo Teklad, Inc.
3
Diplomat, American College of Veterinary Ophthalmologists (DACVO).
M. Dziwenka, et al. Toxicology Reports 7 (2020) 376–385
378
Groups 5 to 8 had a 28-day recovery period before being sacriced. In
the 90-day study, male rats in Groups 1-8 were administered the test
article daily for 93 days and female rats in Groups 1-8 were adminis-
tered test article daily for 94 days. The recovery period was 30 and 31
days for the female and male rats, respectively.
2.7. Pathologic methods
2.7.1. Hematology and clinical chemistry
The clinical chemistry parameters for the 14-day and 90-studies are
given in Table 5. For the 14-day study, blood, after overnight fasting,
was collected before necropsy (study day 15) from the inferior vena
cava while the rats were anesthetized with isourane. For the 90-day
study, blood was collected from all groups for hematology and clinical
chemistry on study day 94 for males and study day 95 for females in
Groups 1 to 4 (90-day sacrice) and on study day 124 for Groups 5 to 8
(recovery sacrice). Blood samples for hematology (except coagulation
samples) and clinical chemistry were collected by sublingual bleeding
after the rats were anesthetized with isourane. Approximately 500 μL
of blood was collected for hematologic parameters in a pre-calibrated
tube containing Potassium EDTA
4
anticoagulant and 1000 μL of whole
blood was collected in tubes (no anticoagulant) for clinical chemistry
parameters (Table 5). Whole blood samples were kept cold until ex-
amined in the laboratory using standard hematology methods. For
clinical chemistry, blood was allowed to coagulate, and the samples
were centrifuged in a refrigerated centrifuge. The serum supernatant
was harvested and placed in cryotubes, and frozen and stored at -80 °C
until thawed and assayed. Hematology parameters were determined on
an ADVIA 120 Hematology System (Siemens, Erlangen, Germany) and
clinical chemistry parameters were determined on a COBAS C311 au-
toanalyzer (Roche, Rotkreuz, Switzerland). Blood samples used to de-
termine the prothrombin time and activated partial thromboplastin
time were collected immediately before terminal sacrice by veni-
puncture of the inferior vena cava during anesthesia with isourane.
Approximately 1.8 mL of blood was collected in a pre-calibrated tube
containing anticoagulant (3.2% sodium citrate). These samples were
centrifuged in a refrigerated centrifuge and the plasma was transferred
to labeled tubes. Plasma samples were frozen and stored in a -80 °C
freezer until thawed and analyzed on a Sysmex CA620 (Siemens, Er-
langen, Germany). The day before collection of samples for the clinical
chemistry evaluations, the animals were placed in metabolism cages.
Food was withheld for at least 15 hours prior to blood collection, and
voided urine was collected from each animal. Urine samples were re-
frigerated until analyzed (Table 5). Urine volume was measured, the
appearance was recorded, chemical parameters were measured by
Multistix®10 SG Reagent Strips (Siemens, Erlangen, Germany) and
urine sediment was evaluated by light microscopy.
Table 3
Treatment groups for the 14-day study.
Group No. Males/
Females
Dose of Test Article (mg/kg bw/
day)
Sacrice Day
Male/Female
1 5/5 0 15/15
2 5/5 1000 15/15
3 5/5 2000 15/15
4 5/5 4000 15/15
Dose is mg test article/kg body weight/day.
Table 4
Treatment groups for the 90-day study with recovery.
Group No. Males/
Females
Dose of Test Article (mg/kg bw/day Sacrice Day
Male/Female
1 10/10 0 93/94
2 10/10 200 93/94
3 10/10 400 93/94
4 10/10 800 93/94
5 5/5 0 124/124
6 5/5 200 124/124
7 5/5 400 124/124
8 5/5 800 124/124
Dose is mg test article/kg body weight/day.
Table 5
Clinicopathology parameters and tissues collected for histopathology.
Parameter Test Tissues Collected
For Histopathology
Hematology
(90-Day study
only)
Red blood cell count Adrenals
1,2,3,4
Red blood cell indices Brain
1
(medulla/pons,
cerebellum, cerebral cortex)
4
Hematocrit Spinal cord (cervical, mid-
thoracic, lumbar)
4
, sciatic nerve
4
Hemoglobin Epididymies
1,2,4
Platelet count Testes
1,2,4
White blood cell count Prostate
4
White blood cell
dierential count
Seminal vesicles
4
Abnormal morphology Ovary and oviducts
1,2,4
Clinical chemistry Prothrombin time Vagina
4
, uterus
1,4
, cervix
4
Activated partial
thromboplastin time
Mammary gland
4
Aspartate
aminotransferase
Heart
1,4
Alanine
aminotransferase
Aorta
4
Sorbitol dehydrogenase Kidneys
1,2,3,4
Alkaline phosphatase Urinary bladder
4
Urea nitrogen Pancreases
4
Creatinine (blood) Liver
1,3,5
Glucose (after 15 hours
of fasting)
Esophagus
4
, stomach
4
,
duodenum,
4
ileum with GULT
4,6
,
jejunum
4
, colon
4
, cecum
4
,
rectum
4
Triglycerides Salivary glands (sublingual,
submandibular, parotid)
4
Total protein Spleen
1,4
Albumin Thymus
1,4
Globulin Lymph nodes (mandibular,
mesenteric)
4
Phosphorous
(inorganic)
Sternum
4
Calcium Femur
4
(bone)
Sodium Bone marrow (femur and
sternum)
4
Potassium Pituitary gland
4
Chloride Thyroid
4
Urinalysis Quality Parathyroid gland
4
Volume Nose
4
Clarity Nasal turbinates
4
color Pharynx
4
pH Larynx
4
Specic gravity Trachea
4
Blood Lungs
4
Glucose Eyes
4
Protein Skeletal muscle
4
Ketones Skin
4
Bilirubin Harderian gland
4
Urobilinogen Eye ball
4
and optic nerve
4
Microscopic exam
(sediment)
Necropsy lesions
7
1
Relative organ weight determined on 90-day study.
2
Combined weight.
3
Histopathology - 14-day study.
4
Histopathology - 90-day study, Groups 1 and
4.
5
All animals in 90-day study.
6
Gut associated lymphoid tissue.
7
All lesions
observed during necropsy.
4
Potassium ethylenediaminetetraacetic acid.
M. Dziwenka, et al. Toxicology Reports 7 (2020) 376–385
379
2.7.2. Macroscopic and Histopathology (14-day and 90-day studies)
A full necropsy was done on each study animal including animals
removed from the studies. Included in the necropsy were examination
of the external body surface, body orices, and the thoracic, abdominal
and cranial cavities inclusive of contents. All surviving animals were
weighed, anesthetized with isourane and exsanguinated from the ab-
dominal aorta. All gross lesions were recorded. Absolute and normal-
ized organ weights (organ weight/body weight) were determined on
selected tissues (Table 5). The eyes, epididymides, optic nerve and
testes were xed in modied Davidsonsxative and then stored in
ethanol. All other tissues were xed in 10% neutral buered formalin.
Specied tissues were embedded in wax, thin sections cut and stained
with hematoxylin and eosin, and examined by light microscopy for
histopathology (Table 5). For the 14-day study, liver and adrenal glands
from all treatment and control animals, and the kidneys from Groups 1
and 4 were examined by histopathology. For the 90-day study, tissues
from all animals removed from the study, tissues from Groups 1 and 4
and the livers from Groups 2 and 3 and groups 5 to 8 were examined for
histopathologic changes by light microscopy (Table 5). All gross lesions
observed were described, the tissues taken and examined by histo-
pathology. All pathology procedures were under the supervision of a
veterinary pathologist
5
.
2.8. Statistical analyses (14-day and 90-day studies)
Mean and standard deviations were calculated for all quantitative
data. For all in-life endpoints that were identied as multiple mea-
surements of continuous data over time (e.g. body weight, body weight
gain, food consumption, and food eciency), treatment and control
groups were compared using a two-way analysis of variance (ANOVA),
testing the eects of both time and treatment, with methods accounting
for repeated measures in one independent variable [17]. Signicant
interactions observed between treatment and time, as well as main ef-
fects, were further analyzed by a post hoc multiple comparisons test; e.g.
Dunnetts test [18,19] of the individual treated groups to control. When
warranted by sucient group sizes, all endpoints with single mea-
surements of continuous data within groups (e.g., organ weight and
relative organ weight) were evaluated for homogeneity of variances
[20] and normality [21]. Where homogeneous variances and normal
distribution was observed, treated and control groups were compared
using a one-way ANOVA. When one-way ANOVA was signicant, a
comparison of the treated groups to control was performed with a
multiple comparisons test, e.g., Dunnetts test [18,19]. Where variance
was considered signicantly dierent, groups were compared using a
nonparametric method, e.g., Kruskal-Wallis non-parametric analysis of
variance [22]. When non-parametric analysis of variance was sig-
nicant, a comparison of treated groups to control was performed, e.g.,
Dunns test [23]. Signicance was a probability value of p< 0.05.
For hematology and clinical chemistry, the data from male and fe-
male rats were analyzed separately. Means and standard deviations
were calculated for all quantitative clinical pathology parameters using
Pristima®version 7 (Statistical Analysis, Xybion Corporation,
Lawrenceville, NJ). These data were analyzed in a sequential manner.
First, Bartletts test for homogeneity and Shapiro-Wilk test for normality
was done. If the Bartletts test for homogeneity and Shapiro-Wilk test
for normality were not signicant, a one-way analysis of variance fol-
lowed with Dunnett's test was performed. If the Bartletts test for
homogeneity and Shapiro-Wilk test for normality were signicant then
data transformations to achieve normality and variance homogeneity
were done. The order of transformations attempted was log, square
root, and rank-order. If the log and square root transformations fail, the
rank-order was used. When an individual observation was recorded as
being less than a certain value, e.g., below the lower limit of
quantitation, calculations were performed on one-half of the recorded
value. For example, if bilirubin was reported as < 0.1 or 0.1, then
0.05 was used for all calculations performed with that bilirubin data.
When an individual observation was recorded as being greater than a
certain value, e.g., above the upper limit of quantitation, then a greater
value was used in place of the recorded value. For example, if specic
gravity was reported as > 1.100 or 1.100, then 1.100 was used for all
calculation performed using that specic gravity value. For all statis-
tical testing, signicance was a probability value of p< 0.05.
2.9. Bacterial reverse mutation assay (Ames assay)
The mutagenicity potential of the test article as well as undiluted
extracts were evaluated in the Bacterial Reverse Mutation Assay in
accordance with FDA GLP (21 CFR Part 58, 1987) and US FDA Redbook
2000 (IV.C.1.a, 2007) and ICH guidelines [14,24,25]. Four strains of
Salmonella typhimurium (TA98, TA100, TA1535 and TA1537) and one
strain of Escherichia coli (WP2 uvrA) were used. The studies were con-
ducted in the presence and absence of a metabolic activation system
from male Sprague-Dawley rats which had been induced with pheno-
barbital and benzoavone (Moltox Inc, USA). The overlay agar and
minimal glucose agar plates were purchased (Moltox Inc, USA). The
fresh bacterial suspension cultures in the nutrient broth were prepared
so that they were in the late exponential phase of growth when used.
The test article in olive oil was formulated as a solution in dimethyl
sulfoxide (DMSO) to provide the required dose levels of up to
76,335 μg/plate to account for the 6.55% of active ingredient (6.27%
CBD). For the undiluted extract prepared by isopropanol or super-
critical CO
2
extraction, the extract was formulated as a solution in
DMSO to provide the required dose levels up to 5000 μg/plate. Positive
controls were used, both in the presence and absence of a metabolic
activation system. The positive control substances included were so-
dium azide, ICR 191, daunomycin and methyl methanesulfonate for S.
typhimurium strains TA100 and TA1535, TA1537, TA98 and E. coli WP2
uvrA, respectively in the absence of metabolic activation and 2-ami-
noanthracene for all strains in the presence of metabolic activation. The
initial test for all test articles utilized the plate incorporation method in
which the following materials were mixed and poured onto the minimal
agar plate; 100 μL of the prepared test substance solutions/negative
control/positive control substance, 500 μL of S9 mix or substation
buer, 100 μL bacterial suspension or 2000 μL overlay agar (at 45 °C).
The plates were then incubated at 37 °C until the growth was adequate
for enumeration. A conrmatory test for all test articles was conducted
utilizing the pre-incubation method. The test or control substances,
bacterial suspensions and the S9 mix or substitution buer were in-
cubated under agitation for approximately 30 minutes at 37 °C prior to
mixing with the overlay agar and pouring onto the minimal agar plates
and proceeding as for the initial test. The strains used and dose levels
were the same as that in the initial test for all test articles. The plates for
both tests were prepared in triplicate for each experimental point. The
nal doses utilized for the extract diluted in olive oil were 0.24, 0.76,
2.41, 7.633, 24.12, 76.33, 241.22, 763.33, 2,412.2, 7,633.5, 24,122
and 76,355 μg/plate. For the undiluted isopropanol extract, the nal
doses utilized for both the initial and conrmatory tests were 1.58, 5.0,
15.8, 50, 158, 500, 1580 and 5000 μg/plate. For the undiluted super-
critical CO
2
extract, the nal doses utilized were 1.58, 5.0, 15.8, 50,
158, 500, 1580 and 5000 μg/plate for the initial test and 0.5, 2.5 and
25 μg/plate for the conrmatory test. Due to toxicity noted for strains
TA100 and TA1537 with the supercritical CO
2
extract, a supplemental
test was conducted to ensure ve concentrations could be assessed
without toxicity. Both the plate incorporation and pre-incubation
methods were used as previously described at nal doses of 0.5, 2.5 and
25 μg/plate. Following incubation, the number of colonies per plate was
counted manually and/or with the aid of a plate counter. The mean and
standard deviation were calculated for each set of triplicate plates. The
test was considered valid if the control plates had normal background
5
Diplomate, American college of Veterinary Pathologist (DACVP).
M. Dziwenka, et al. Toxicology Reports 7 (2020) 376–385
380
Table 6
Eect of 90-Day oral administration of test article on hematological parameters in male and female rats (n = 60/sex).
Parameter Units Group and Dose (mg/kg bw/day)
G1 (0)
n=10
G2 (200)
n=10
G3 (400)
n=10
G4 (800)
n=10
G5 (0)
n=5
G6 (200)
n=5
a
G7 (400)
n=5
G8 (800)
n=5
Males
WBC x10^3/μL 12.003 ± 2.5464 12.181 ± 2.6785 12.824 ± 2.6230 12.613 ± 2.4870 10.212 ± 1.3324 10.746 ± 2.3541 10.602 ± 2.1115 10.173 ± 2.5306
RBC x10^6/μL 9.145 ± 0.2419 8.975 ± 0.4785 9.063 ± 0.2436 9.064 ± 0.3084 9.050 ± 0.1869 8.966 ± 0.2845 8.988 ± 0.4482 8.683 ± 0.3308
HGB g/dL 15.71 ± 0.415 15.48 ± 0.614 15.48 ± 0.380 15.41 ± 0.567 15.06 ± 0.627 15.28 ± 0.838 15.20 ± 0.515 14.85 ± 0.500
HCT % 50.76 ± 1.356 49.97 ± 2.048 49.83 ± 1.443 50.28 ± 1.915 48.86 ± 2.503 50.58 ± 2.359 48.64 ± 1.581 47.83 ± 0.971
MCV fL 55.52 ± 1.316 55.96 ± 1.407 54.98 ± 0.997 55.48 ± 1.576 54.36 ± 1.733 56.44 ± 1.582 54.12 ± 0.983 55.13 ± 1.024
MCH pg 17.20 ± 0.481 17.25 ± 0.635 17.10 ± 0.610 16.99 ± 0.524 16.74 ± 0.586 17.06 ± 0.684 16.88 ± 0.383 17.13 ± 0.287
RDW % 13.35 ± 0.698 12.65 ± 0.519 12.39 ± 0.703 12.31 ± 0.576 14.22 ± 1.055 13.58 ± 1.270 13.78 ± 0.841 14.40 ± 1.080
PLT x10³/μL 1102.2 ± 94.23 1086.8 ± 116.78 997.0 ± 120.12 1115.8 ± 130.17 1063.8 ± 64.93 1107.4 ± 75.33 1090.2 ± 63.05 1220.5 ± 90.89
ANEU x10³/μL 1.344 ± 0.3022 1.670 ± 0.4869 1.678 ± 1.0030 1.274 ± 0.5797 1.612 ± 0.4010 1.536 ± 0.7282 1.860 ± 0.7529 1.795 ± 0.8626
ALYM x10³/μL 9.924 ± 2.2637 9.812 ± 2.2488 10.487 ± 2.6969 10.738 ± 2.0499 8.074 ± 1.1539 8.610 ± 2.0187 7.988 ± 1.3494 7.975 ± 1.6805
AMON x10³/μL 0.236 ± 0.0682 0.245 ± 0.0636 0.247 ± 0.0638 0.222 ± 0.0675 0.290 ± 0.0700 0.364 ± 0.0934 0.290 ± 0.1032 0.210 ± 0.0796
AEOS x10³/μL 0.135 ± 0.0538 0.145 ± 0.0599 0.127 ± 0.0403 0.113 ± 0.0271 0.124 ± 0.0251 0.140 ± 0.0255 0.142 ± 0.0630 0.118 ± 0.0206
ABAS x10³/μL 0.157 ± 0.0634 0.123 ± 0.0702 0.151 ± 0.0692 0.115 ± 0.1300 0.032 ± 0.0110 0.034 ± 0.0195 0.072 ± 0.0268 0.080 ± 0.0432
ALUC x10³/μL 0.210 ± 0.1175 0.143 ± 0.0538 0.138 ± 0.0563 0.150 ± 0.0650 0.084 ± 0.0297 0.062 ± 0.0249 0.060 ± 0.0200 0.053 ± 0.0189
ARET ^10³/μL 174.64 ± 30.227 162.53 ± 21.656 154.13 ± 25.008 153.24 ± 38.475 226.70 ± 49.313 208.22 ± 45.992 202.16 ± 37.096 282.15 ± 78.425
%RET % 1.911 ± 0.3349 1.815 ± 0.2654 1.698 ± 0.2491 1.693 ± 0.4258 2.506 ± 0.5445 2.334 ± 0.5865 2.266 ± 0.5042 3.278 ± 1.0448
MCHC g/dL 30.98 ± 0.469 30.84 ± 0.615 31.09 ± 0.775 30.78 ± 0.294 30.84 ± 0.643 30.62 ± 0.432 31.20 ± 0.442 31.05 ± 0.387
Females
WBC x10^3/μL 7.393 ± 3.0714 7.684 ± 1.8437 9.415 ± 3.2499 10.953 ± 4.5153 5.458 ± 0.6154 8.803+D ±2.1248 5.794 ± 1.3307 7.054 ± 1.5227
RBC x10^6/μL 8.494 ± 0.1585 8.476 ± 0.4019 8.969 ± 0.3346 8.564 ± 0.3825 8.480 ± 0.3836 8.203 ± 0.5986 8.426 ± 0.7084 8.504 ± 0.3164
HGB g/dL 14.93 ± 0.422 14.85 ± 0.728 15.36 ± 0.497 15.01 ± 0.649 14.74 ± 0.673 14.78 ± 0.695 14.60 ± 0.837 15.28 ± 0.396
HCT % 47.16 ± 1.243 47.00 ± 2.552 48.82 ± 1.755 47.84 ± 2.097 47.50 ± 2.437 45.73 ± 1.898 45.60 ± 3.415 47.24 ± 1.135
MCV fL 55.52 ± 1.229 55.34 ± 1.585 54.45 ± 1.252 55.87 ± 0.943 55.98 ± 0.881 55.90 ± 2.578 54.18 ± 1.316 55.58 ± 1.628
MCH pg 17.56 ± 0.395 17.48 ± 0.374 17.14 ± 0.433 17.50 ± 0.269 17.42 ± 0.342 18.03 ± 0.714 17.34 ± 0.627 17.96 ± 0.559
RDW % 11.44 ± 0.422 11.15 ± 0.438 11.31 ± 0.574 11.13 ± 0.377 11.88 ± 0.277 11.90 ± 0.891 11.96 ± 0.207 11.50 ± 0.339
PLT x10³/μL 1063.0 ± 115.60 1104.7 ± 148.69 1136.5 ± 92.72 1086.6 ± 140.98 1130.2 ± 124.43 1132.8 ± 53.38 1196.2 ± 184.23 1079.0 ± 90.42
ANEU x10³/μL 0.777 ± 0.2868 0.948 ± 0.4079 1.098 ± 0.6354 1.041 ± 0.4468 1.074 ± 0.1604 1.310 ± 0.6002 0.808 ± 0.4494 1.022 ± 0.3292
ALYM x10³/μL 6.117 ± 2.6226 6.359 ± 1.4719 7.762 ± 2.8518 9.198 ± 3.8399 4.074 ± 0.5327 5.648 ± 2.1077 4.526 ± 0.8009 5.660 ± 1.5465
AMON x10³/μL 0.146 ± 0.0941 0.131 ± 0.0453 0.187 ± 0.1058 0.211 ± 0.1042 0.154 ± 0.0503 0.248 ± 0.1056 0.148 ± 0.0342 0.152 ± 0.0476
AEOS x10³/μL 0.111 ± 0.0331 0.095 ± 0.0443 0.145 ± 0.0510 0.141 ± 0.0569 0.116 ± 0.0451 1.045 ± 1.8237 0.144 ± 0.0673 0.106 ± 0.0313
ABAS x10³/μL 0.089 ± 0.0666 0.055 ± 0.0276 0.111 ± 0.0479 0.088 ± 0.0909 0.012 ± 0.0084 0.068 ± 0.0350 0.052 ± 0.0409 0.066 ± 0.0182
ALUC x10³/μL 0.094 ± 0.0608 0.097 ± 0.0365 0.119 ± 0.0443 0.158 ± 0.1033 0.024 ± 0.0114 0.508 ± 0.08954 0.030 ± 0.0071 0.032 ± 0.0130
ARET ^10³/μL 144.89 ± 27.439 134.71 ± 28.594 128.37 ± 35.153 117.06 ± 22.355 165.06 ± 21.450 159.40 ± 18.481 156.48 ± 25.886 156.76 ± 47.490
7.%RET % 1.706 ± 0.3266 1.584 ± 0.3151 1.429 ± 0.3741 1.372 ± 0.2820 1.948 ± 0.2406 1.948 ± 0.2105 1.848 ± 0.1571 1.850 ± 0.5735
MCHC g/dL 31.64 ± 0.414 31.56 ± 0.392 31.49 ± 0.398 31.34 ± 0.336 31.14 ± 0.508 32.30*D ±0.497 32.02 ± 0.829 32.34*D ±0.559
Values are mean ± standard deviation. ABAS =absolute basophil; AEOS= absolute eosinophil; ALUC =absolute large unstained cell; ALYM=absolute lymphocyte; AMON = absolute monocyte; ANEU = absolute
neutrophil (all forms); ARET = absolute reticulocyte; HCT = hematocrit; HGB = hemoglobin; MCH = mean corpuscular hemoglobin; MCHC = mean corpuscular hemoglobin concentration; MCV = mean corpuscular
volume; PLT = platelet count; RBC = red blood cell count; RDW = red cell distribution width; WBC = white blood cell count.
a
N=4 for group 6 females.
M. Dziwenka, et al. Toxicology Reports 7 (2020) 376–385
381
lawn; the mean revertant colony counts for each strain treated with
vehicle was close to or within the expected laboratory historical control
range or published values; and the positive controls should produce
substantial increases in revertant colony numbers with the appropriate
bacterial strain. The plates were also evaluated for cytotoxicity which is
indicated by the partial or complete absence of a background lawn on
non-revertant bacteria or a substantial dose-related reduction in re-
vertant bacteria.
3. Results
3.1. Concentration verication
For the 14-day study, the concentration verication analysis for Day
1 averaged 249.2 and 1003.4 mg/mL, which were 124.6 and 125.4% of
the target concentrations of 200 and 800 mg/mL for Groups 2 and 4,
respectively. For the 90-day study, the concentration compliance
ranged from 103.3% to 125.4%. The Day 1 samples averaged 117.4,
125.4, and 110.5%, the Day 46 samples averaged 103.3, 104.7 and
106.7%, and the Day 94 samples averaged 109.6, 108.2 and 106.8% of
the target concentrations of 40, 80 and 160 mg/mL, respectively
3.2. Mortalities
There were no mortalities in the 14-day and 90-day studies that
were linked to administration of the test article or olive oil vehicle.
3.3. Body weights
In the 14-day study there was a non-statistically signicant
(p> 0.05) decrease in body weights for the male and female rats in
Groups 2-4. For the female rats in the 90-day study, there were no test
article-related changes (p> 0.05) in mean weekly body weights, daily
body weight gain, food consumption, or food eciency. For the male
rats in Groups 3 and 4, a statistically signicant (p< 0.05) dose-de-
pendent decrease in mean weekly body weights was observed that
correlated with signicant decreases (p< 0.05) in mean daily body
weight gain and food eciency for Groups 3 and 4 as well as food
consumption for Groups 2-4. At the end of the recovery period, dose-
dependent decrease in mean weekly body weights was still observed for
Table 7
Eect of 90-Day oral administration of test article on clinical chemistry parameters in male and female rats (n = 60/sex).
Parameter Units Group and Dose (mg/kg bw/day)
G1 (0)
n=10
G2 (200)
n=10
G3 (400)
n=10
G4 (800)
n=10
G5 (0)
n=5
G6 (200)
n=5
G7 (400)
n=5
G8 (800)
n=5
Males
Na mmol/L 140.2 ± 2.66 139.8 ± 2.15 140.7 ± 2.00 140.9 ± 2.60 142.8 ± 0.84 142.2 ± 0.84 142.6 ± 1.52 140.3 ± 3.59
K mmol/L 5.563 ± 0.4536 5.097 ± 0.4437 5.093 ± 0.3864 5.030 ± 0.7194 5.324 ± 0.4663 5.236 ± 0.2078 5.322 ± 0.3023 5.353 ± 0.2716
6Cl mmol/L 99.32 ± 2.433 98.88 ± 1.075 98.44 ± 1.661 99.30 ± 1.928 101.44 ± 0.688 101.28 ± 0.653 101.70 ± 1.693 99.98 ± 3.470
ALB g/dL 3.86 ± 0.272 3.86 ± 0.217 4.06 ± 0.300 4.10 ± 0.236 3.62 ± 0.164 3.64 ± 0.152 3.52 ± 0.239 3.43 ± 0.126
AST U/L 80.2 ± 9.94 76.1 ± 14.99 76.8 ± 5.24 83.4 ± 25.36 76.2 ± 16.15 71.2 ± 10.47 77.2 ± 25.05 63.3 ± 11.44
ALT U/L 31.8 ± 6.09 33.3 ± 5.10 35.0 ± 4.90 34.3 ± 3.20 34.4 ± 1.52 36.2 ± 10.62 38.6 ± 6.88 32.8 ± 6.70
ALKP U/L 90.8 ± 18.68 108.3 ± 17.99 96.0 ± 22.15 99.3 ± 6.33 63.6 ± 15.82 71.8 ± 12.81 79.2 ± 9.86 70.0 ± 12.08
BUN mg/dL 11.5 ± 1.58 11.1 ± 1.20 12.4 ± 1.01 13.3 ± 2.58 11.6 ± 1.52 12.2 ± 1.92 12.6 ± 1.82 12.3 ± 0.96
CA mg/dL 9.84 ± 0.320 9.84 ± 0.435 10.28 ± 0.563 9.98 ± 0.529 10.38 ± 0.277 10.34 ± 0.627 10.42 ± 0.259 10.40 ± 0.469
CHOL mg/dL 72.2 ± 15.45 70.9 ± 10.90 72.9 ± 15.66 66.7 ± 13.30 99.6 ± 16.77 100.6 ± 24.82 104.4 ± 31.01 86.3 ± 28.69
CREAT mg/dL 0.185 ± 0.0398 0.196 ± 0.0237 0.209 ± 0.0289 0.205 ± 0.0372 0.148 ± 0.0335 1.90 ± 0.0520 0.162 ± 0.0286 0.183 ± 0.0403
GLU mg/dL 111.0 ± 24.07 103.6 ± 16.47 96.1 ± 12.14 102.1 ± 15.87 119.8 ± 9.86 120.8 ± 15.40 133.8 ± 17.25 120.0 ± 19.92
PHOS mg/dL 6.67 ± 0.397 6.56 ± 0.479 6.72 ± 0.370 6.41 ± 0.384 5.94 ± 0.261 6.12 ± 0.487 6.02 ± 0.559 6.30 ± 0.245
TP g/dL 6.21 ± 0.260 6.35 ± 0.398 6.68 ± 0.578 6.55 ± 0.360 6.70 ± 0.224 6.74 ± 0.313 6.68 ± 0.110 6.63 ± 0.443
TBIL mg/dL 0.074 ± 0.0313 0.073 ± 0.0291 0.071 ± 0.0183 0.067 ± 0.0134 0.108 ± 0.0239 0.100 ± 0.0245 0.112 ± 0.0383 0.088 ± 0.0222
TRIG mg/dL 132.1 ± 57.66 95.1 ± 32.10 83.2 ± 23.24 61.2 ± 18.84 116.2 ± 28.62 95.2 ± 28.37 129.4 ± 53.87 92.5 ± 35.72
SDH U/L 4.18 ± 2.046
a
4.07 ± 1.599 4.12 ± 1.987 3.37 ± 1.830
b
6.62 ± 1.867 11.00 ± 6.605 9.14 ± 3.634 6.60 ± 3.017
TBA μmol/L 26.0 ± 14.92 32.3 ± 19.73 52.7 ± 44.71 22.7 ± 10.29 16.7 ± 15.00 25.3 ± 12.11 73.7 ± 48.98 34.7 ± 23.66
GLOB g/dL 2.35 ± 0.118 2.49 ± 0.321 2.62 ± 0.390 2.45 ± 0.207 3.08 ± 0.249 3.10 ± 0.187 3.16 ± 0.251 3.20 ± 0.346
Females
Na mmol/L 139.6 ± 1.43 139.7 ± 1.83 140.1 ± 2.08 140.4 ± 2.51 140.2 ± 1.64 140.4 ± 1.82 141.6 ± 1.14 141.6 ± 1.52
K mmol/L 4.589 ± 0.5311 4.669 ± 0.2851 4.593 ± 0.3708 4.488 ± 0.4549 4.702 ± 0.1758 5.024 ± 0.9254 4.658 ± 0.3737 4.992 ± 0.2335
Cl mmol/L 99.10 ± 1.778 99.96 ± 1.773 99.08 ± 1.346 99.63 ± 2.680 100.54 ± 1.274 100.38 ± 2.780 101.04 ± 0.902 101.00 ± 1.739
ALB g/dL 5.03 ± 0.359 4.71 ± 0.378 4.93 ± 0.359 4.99 ± 0.276 4.74 ± 0.207 4.04 ± 1.547 4.80 ± 0.292 4.74 ± 0.472
AST U/L 63.9 ± 10.52 61.1 ± 6.85 70.6 ± 7.01 65.3 ± 7.23 86.4 ± 28.25 127.0 ± 84.03 128.0 ± 142.62 68.4 ± 9.76
ALT U/L 30.4 ± 5.68 28.9 ± 3.07 32.8 ± 5.37 31.8 ± 4.49 47.2 ± 16.27 48.2 ± 35.29 40.6 ± 27.30 37.4 ± 5.59
ALKP U/L 56.8 ± 21.13 60.4 ± 10.88 69.3 ± 19.52 67.6 ± 24.59 35.6 ± 11.10 102.6 ± 145.11 46.8 ± 19.70 40.4 ± 15.90
BUN mg/dL 12.6 ± 1.90 13.6 ± 2.12 17.0 ± 2.45 15.1 ± 2.98 14.6 ± 4.72 15.0 ± 3.74 13.2 ± 2.39 16.2 ± 1.64
CA mg/dL 10.63 ± 0.890 10.47 ± 0.460 10.63 ± 0.797 10.47 ± 0.510 10.50 ± 0.255 9.96 ± 1.122 10.48 ± 0.676 10.52 ± 0.164
CHOL mg/dL 76.3 ± 21.45 84.8 ± 13.89 82.9 ± 17.31 90.9 ± 19.77 117.8 ± 22.44 92.4 ± 39.30 106.8 ± 32.07 109.0 ± 20.04
CREAT mg/dL 0.259 ± 0.0357 0.264 ± 0.0241 0.270 ± 0.0383 0.276 ± 0.0555 0.254 ± 0.0607 0.230 ± 0.0543 0.232 ± 0.0383 0.274 ± 0.0385
GLU mg/dL 108.8 ± 12.28 120.3 ± 14.50 127.8 ± 16.19 118.2 ± 19.43 130.4 ± 12.16 113.4 ± 36.29 123.0 ± 10.37 109.6 ± 7.99
PHOS mg/dL 4.94 ± 0.773 4.90 ± 0.527 5.69 ± 0.479 5.87 ± 0.875 4.44 ± 0.577 5.00 ± 0.775 5.00 ± 0.529 5.62 ± 0.804
TP g/dL 7.12 ± 0.349 6.88 ± 0.402 7.15 ± 0.528 7.37 ± 0.436 5.78 ± 0.455 6.98 ± 1.361 7.44 ± 0.662 7.62 ± 0.502
TBIL mg/dL 0.082 ± 0.0239 0.083 ± 0.0226 0.114 ± 0.0406 0.094 ± 0.0375 0.118 ± 0.0370 0.142 ± 0.0687 0.108 ± 0.0179 0.114 ± 0.0279
TRIG mg/dL 73.1 ± 23.96 77.1 ± 20.56 78.3 ± 20.91 71.9 ± 20.76 76.0 ± 20.87 82.2 ± 32.16 65.0 ± 6.44 79.2 ± 15.94
SDH U/L 4.22 ± 1.624 4.04 ± 1.329 4.38 ± 1.873 4.24 ± 1.071 8.74 ± 3.314 15.60 ± 24.280 13.72 ± 18.836 5.50 ± 1.609
TBA μmol/L 34.6 ± 20.95 32.9 ± 42.40 52.6 ± 52.00 79.7 ± 62.13 26.6 ± 13.24 40.3 ± 48.94 55.6 ± 70.69 56.4 ± 72.21
GLOB g/dL 2.09 ± 0.338 2.17 ± 0.125 2.22 ± 0.274 2.38 ± 0.377 2.84 ± 0.321 2.94 ± 0.336 2.64 ± 0.541 2.88 ± 0.130
Values are mean ± standard deviation. ALB = albumin; ALKP = alkaline phosphatase; ALT = alanine aminotransferase; AST = aspartate aminotransferase;
BUN = urea nitrogen; CA = calcium; CHOL = cholesterol; Cl = chloride; CREAT = creatinine; GLOB = globulin; GLU = glucose; K = potassium; NA = sodium;
PHOS = inorganic phosphorous; SDH = sorbitol dehydrogenase; TBIL = total bilirubin; TP = total protein; TRIG = triglycerides.
a
N=8
b
N=9
M. Dziwenka, et al. Toxicology Reports 7 (2020) 376–385
382
the male rats in Groups 6-8 with correlating signicant decrease
(p< 0.05) in mean daily body weight gain and food consumption for
Groups 6-8 as well as food eciency for the male rats in Group 8.
3.4. Clinical observations
Group 4 animals in the 14-day study had clinical signs consisting of
hypoactivity, hyperactivity, reduced food consumption and piloerec-
tion that are directly attributable to test article administration. For the
90-day study, there were no adverse clinical observations that were
consistent across treatment groups and these observations were not
linked with pathological observations.
3.5. Ophthalmology, Functional Observation Battery and Motor Activity
Assessment using a Photobeam Activity System
For all treatment groups in the 90-day study, there were no con-
sistent abnormal ndings in the ophthalmological, Functional
Observation Battery and Motor Activity Assessment examinations.
3.6. Pathology
3.6.1. Hematology and clinical chemistry
Treatment-linked changes in hematology and clinical chemistry
values, for Group 4 male and female rats in the 14-day study were in-
creased blood urea nitrogen and serum creatinine. For the 90-day study,
there were no signicant changes (p< 0.05) between groups in the
hematology, prothrombin and activated partial thromboplastin times,
urinalysis, and the clinical chemistries (Tables 6 and 7).
3.6.2. Necropsy observations
There were no macroscopic lesions observed in the 14-day and 90-
day studies that were linked to the administration of the test article.
3.6.3. Organ weights and histopathology
A board-certied veterinary pathologist (DACVP) evaluated the
tissues for histopathology. In the 14-day study, centrilobular hepato-
cellular hypertrophy (increased cell size) in Groups 3 (3/10) and 4 (5/
10) was seen that correlated with an increase in liver weights. Adrenal
cortical vacuolation was observed and was mild in all Group 4 animals,
minimal to mild in 3/5 of the Group 3 males and 4/5 of the Group 3
females and minimal in 1/5 for the Group 2 males. For the 90-day
study, test article related histopathology changes were limited to he-
patocellular hypertrophy of centrilobular hepatocytes. This lesion was
seen in the male and female animals in Groups 2 to 4. The hepatocel-
lular hypertrophy was associated with dose-dependent increases in
absolute liver weight for Group 2 to 4 females, liver-to-body weight
ratios for Group 3 females, and liver-to-body/brain weight ratios for
Group 4 females. Signicant (p< 0.05) increase in liver-to-body
weight ratios for Group 3 females and liver-to-body/brain weight ratios
for Group 4 females were seen. The increases in liver weight and ratios
correlated with the microscopic nding of hepatocellular hypertrophy
at all dose levels. Non-signicant (p> 0.05) dose-dependent increase
in absolute liver weight was observed for Group 2-4 females. The
centrilobular hepatocellular hypertrophy and increased liver weights
were not seen in recovery groups at the end of the 28-day recovery
period indicating the hepatocellular hypertrophy was reversible.
Vacuolization of the zona fasciculata at the same incidence and
severity was observed in the adrenal glands of treatment and control
(Groups 1 and 4) male rats and correlated with an increase in the
weights of the adrenal glands. In addition, a statistically signicant
increase (p < 0.05) in adrenal-to-body weight ratios was observed for
Group 4 females that did not correlate with any adrenal histopathology.
3.7. Bacterial Reverse Mutation Assay
There was no concentration related or substantial test article related
increases in the number of revertant colonies for each of the strains
tested in the presence or absence of metabolic activation (S9 mix), in
either the plate incorporation or the pre-incubation methods (data not
shown). Precipitation which interfered with lawn evaluation was noted
for all strains at doses 7,633.5 μg/plate but did not obscure counts in
the test with the diluted test article. Precipitation which obscured lawn
evaluation was seen in all strains with the supercritical CO
2
extract at
doses 1580 μg/plate with and without S9 in both the plate in-
corporation and pre-incubation methods. Toxicity was evident for
strains TA 98, TA 1535, TA 1537 and E. coli WP2 uvrA at 50 μg/plate,
with and without S9, in the plate incorporation and/or pre-incubation
tests. Precipitation which obscured lawn evaluation was seen in all
strains with the isopropanol extract at doses 1580 μg/plate with and
without S9 in both the plate incorporation and pre-incubation methods.
Toxicity was noted for strains TA 1537 and TA 100 at 500 and/or
1580 μg/plate without S9 in the pre-incubation method. The studies
were considered valid as the mean revertant colony counts for vehicle
controls were close to or within the expected range based on the la-
boratory historical controls and/or published values and the positive
control substances resulted in the expected substantial increases in re-
vertant colony counts.
The mutagenicity testing showed that the extract diluted with olive
oil as well as the extracts produced with an isopropanol and super-
critical CO
2
extraction method were not mutagenic to bacteria in the
Ames assay.
4. Discussion
Recently, there has been an increasing interest regarding the health
benets of CBD and other phytocannabinoids and with this increased
interest, more research is also being conducted to assess the safety of
these compounds for human consumption. The current studies were
performed to better understand the toxicological prole of a CBD rich
proprietary hemp extract and to assess the results in tandem with in-
formation currently available regarding the toxicity and safety of CBD.
Marx et al. [4] reports on a battery of GLP compliant toxicological
studies which were conducted on a supercritical CO
2
extract of the
aerial parts of the C. sativa plant. Assay of the extract was 61% edible
fatty acids, 26% phytocannabinoids (approximately 96% is CBD, < 1%
THC) and 13% other plant chemicals including fatty alkanes, plant
sterols, triterpenes, and tocopherols. In the 14-day repeated oral dose-
range nding study reported by Marx et al. [4], a No Observed Adverse
Eect Level (NOAEL) could not be determined, however, the results of a
90-day repeated dose study with a 28-day recovery period in Wistar rats
was also reported. In this study, doses of 0 (sunower oil vehicle), 100,
360 and 720 mg extract/kg bw per day were used. Signicant decreases
in body weight, body weight gain, and dierences in various organ
weights, compared to controls, were reported at the mid and high dose
levels, but the authors concluded that many of the ndings were re-
versible as they were trending towards normal at the end of the re-
covery period. A NOAEL for the hemp extract in Wistar rats in the 90-
day study was determined to be 100 mg/ kg bw per day and 360 mg/kg
bw per day for males and females, respectively.
In the 90-day study being reported here, test article related sig-
nicant changes in body weights, daily body weight gain and feed ef-
ciency were seen in the males in all treatment groups which was still
noted at the end of the recovery period. The magnitude of the sig-
nicant change in body weights, daily body weight gain and feed ef-
ciency in the low and mid dose groups was less than 10% and showed
signs of obvious recovery and were therefore considered to be not
toxicologically relevant. The eect in the males receiving 800 mg/kg/
day was > 10% and was still evident at the end of the recovery period
and was considered toxicologically relevant.
M. Dziwenka, et al. Toxicology Reports 7 (2020) 376–385
383
Reported rodent studies have diering ndings on hepatotoxicity
when CBD is orally administered in high doses [4,26]. Hepatocellular
hypertrophy with a centrilobular pattern was observed in rat livers in
the study being reported. This pattern of hepatocellular hyperplasia is
frequently observed in rats and other animals exposed to agents that
induce the CYP family of enzymes and can be associated with activation
of peroxisome proliferator-activated receptors (PPAR) [27]. THC has
anity for PPARα, and CBD has very low to no anity for PPARαand
high anity for PPARγ[28]. Interaction with the PPARγis one of the
mechanisms of action for CBD. In our study, we did not show the me-
chanism of action for the hepatocellular hypertrophy. We did show that
the activities of liver enzymes in serum were not signicantly changed
by treatment with the test article and the hepatocellular hypertrophy
was reversed during the 28-day recovery period. In the study reported
by Marx et al. [4], no histopathological changes were observed in the
livers from the treated and control rats and the liver weights in the male
and female rats in the 360 and 720 mg/kg body weight/day were sig-
nicantly increased (p< 0.05) at 90 days. The 28-day recovery males
and females receiving 720 mg/kg/day retained the signicantly in-
creased in hepatic weights. The induction of hepatic drug metabolizing
enzymes (HDMEs) can be associated with increased liver weights, and
hepatocellular hypertrophy and hyperplasia (increased number of cells)
and elevation of hepatic-source enzymes in serum. The evidence in the
scientic literature supports a conclusion that the centrilobular pattern
of hepatocellular hypertrophy and increased liver weights observed in
our study was due to induction of HDMEs and/or peroxisomes. No
hepatocellular necrosis and changes in the clinical chemistries occurred
which is evidence that liver damage did not occur. This conclusion is
further supported by not observing hepatocellular hypertrophy and
increased liver weights in the 28-day recovery groups that received the
test article. Studies in laboratory animals have shown CBD to protect
the liver from toxic insults [8,29,30].
In the study being reported both the treated and control male rats
had the same incidence and severity of vacuolization of the adrenal
zona fasciculata and the adrenal weights were signicantly increased in
the Group 4 females. The vacuolization of the adrenal zona fasciculata
and increased adrenal weights were not observed in Groups 5 to 8. The
histopathological lesions noted in the adrenal glands in the current
study was seen in both control and high dose males and is not con-
sidered to be due to treatment with test article and not toxicologically
relevant.
The hemp extract in these studies was shown to be non-mutagenic
in a bacterial test system used to evaluate mutagenicity. Marx et al. [4]
reported on a GLP-compliant study that concentrations of 5,000 μg/
plate of a CO
2
supercritical extract of C. sativa were not mutagenic in a
bacterial test system. Our GLP-compliant mutagenicity testing on the
diluted extract showed that concentrations of 76,355 μg/plate were not
mutagenic with and without the S9 metabolic activation. The extracts
produced by isopropanol extraction and supercritical CO
2
extraction
were not mutagenic with and without S9 metabolic activation at con-
centrations up to 5000 μg/plate. The bacterial test system with the S9
mix did cause mutagenicity providing evidence that mutagenic meta-
bolites were not produced with any of the extracts. The two additional
Ames tests conducted on the undiluted extracts produced by two dif-
ferent extraction methods, were conducted to determine if the method
of production or the olive oil diluent impacted the results of the Ames
assay. No mutagenicity was noted in any of the tests conducted. Other
botanical extracts have been evaluated for mutagenicity. Mutagenic
studies on extracts from the plant Euphorbia triaculeata showed that it is
not mutagenic and provides protection from the mutagenic eects of
cyclophosphamide [31]. A study on a novel taste modulating powder
derived from Cordyceps sinensis showed this product was not mutagenic
in the Ames test and these results were supported in the micronucleus
assay [32]. In a study on the genotoxicity of CBD in Caco-2 cells, 10 μM
of CBD did not signicantly cause DNA damage after 24 hours of in-
cubation, and CBD was also shown in the comet assay to protect Caco-2
cells from hydrogen peroxide-induced DNA damage [33]. CBD at an
oral dose of 1 mg/kg was shown to signicantly (P < 0.05) reduce
azoxymethane-induced colonic aberrant crypt foci, colonic polyps and
tumors [33].
In summary, the test article, both undiluted and diluted in olive oil,
was not mutagenic in a bacterial reverse mutation assay and the NOAEL
in the 90-day study was concluded to be 800 mg/kg bw/day and
400 mg/kg bw/day for female and male Sprague Dawley rats, respec-
tively. This assessment adds signicant data to the currently available
literature as to the safety and toxicology of CBD rich hemp extracts.
Given the potential of CBD for a variety of human uses and the limited
data currently available, these results support that hemp extracts are
likely safe human consumption and additional studies should be con-
ducted to validate this conclusion.
Declaration of Competing Interest
The authors declare that they have no conicts of interest with the
exception.
CRediT authorship contribution statement
Margitta Dziwenka: Conceptualization, Writing - original draft,
Writing - review & editing. Robert Coppock: Writing - original draft,
Writing - review & editing. Alexander McCorkle: .Eddie Palumbo: .
Carlos Ramirez: .Stephen Lermer: .
Appendix A. Supplementary data
Supplementary material related to this article can be found, in the
online version, at doi:https://doi.org/10.1016/j.toxrep.2020.02.014.
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... With the increase in consumer consumption of hemp extracts there has been an increased interest in and demand for determining the safety of these extracts. There are recently published studies in both humans and laboratory animals evaluating the safety of orally consumed hemp extracts, however with the variation in the composition of these extracts, comparison of the information must be conducted in tandem with a detailed evaluation of the extract composition [5][6][7]. The bioactivity or potential for hemp extracts to cause toxicity may be influenced by method of manufacture or slight differences in the chemical profile. ...
... Similar to what is observed with other hemp extracts [5,6], increases in liver weight and fatty changes were observed in high dose animals; however, these changes were not accompanied by increases in ALT, AST, ALP or BIL and did not occur in the recovery group, indicating reversibility. These changes were therefore not considered to be adverse. ...
... Changes in adrenal glands such as increased weight, pale appearance and diffuse cytoplasmic vacuolation of the cortical cells of the adrenal glands have been observed in rats given hemp extracts [5,6]; therefore, the changes in the adrenals observed in the current study are likely related to administration of the test substance. High dose main study males exhibited increased absolute adrenal weight, adrenal to body weight and adrenal to brain weight ratios, and mid dose males exhibited increased adrenal to body weight. ...
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VOHO Hemp Oil (Verdant Nature LLC (in collaboration with HempFusion)) is an extract of the aerial parts of hemp ( Cannabis sativa L) manufactured using a supercritical CO 2 extraction process. The results of four safety studies are reported here including a bacterial reverse mutation assay, an in vivo mammalian micronucleus study, a maximum tolerated dose study in rats and a 90-day repeat dose subchronic toxicity study in rats. VOHO Hemp oil can contain up to 30% phytocannabinoids and less than 0.2% is tetrahydrocannabinol (THC). VOHO Hemp Oil was found to be non-mutagenic in the bacterial reverse mutation assay and was negative for inducing micronuclei in the rat bone marrow micronucleus assay. The maximum tolerated dose in male and female Wistar rats was 2250 mg/kg bw/day. A 90-day repeat dose study was conducted in male and female Wistar rats according to OECD Guideline 408 and included a 21-day recovery period. The doses used in the study were 0, 25, 90 and 324 mg/kg bw per day in the main study, and in the recovery phase a control and 324 mg/kg bw/day group were included. One mortality was reported during the study, a high dose female, and test substance-related adverse clinical signs were reported in the high dose group. Other test substance-related changes noted in the high dose group included changes in body weights, activated partial thromboplastin time (APTT) values, and in absolute and relative organ weights. Based on the results of the study, the no observed adverse effect level (NOAEL) for VOHO Hemp Oil was determined to be 90 mg/kg bw/day in both male and female Wistar rats.
... As the extract was of a comparably high purity of CBD, the authors decided to still include the study for comparative reasons. Similarly, Dziwenka et al. [9,15] recently provided 2 studies of hemp extracts; while the 2020 study [15] did not provide raw data necessary for BMD modelling, the 2021 study [9] was included for comparative reasons as well. ...
... As the extract was of a comparably high purity of CBD, the authors decided to still include the study for comparative reasons. Similarly, Dziwenka et al. [9,15] recently provided 2 studies of hemp extracts; while the 2020 study [15] did not provide raw data necessary for BMD modelling, the 2021 study [9] was included for comparative reasons as well. ...
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... In the study by Marx et al. (2018), dose-dependent increases of alanine aminotransferase (ALT), alkaline phosphatase (ALP), gamma-glutamyl transferase (GGT) and bilirubin were also found. In the EMA summary data, increases in ALT and ALP were detected, while no changes in transaminases were detected in the study by Dziwenka et al. (2020) with the extract with low CBD content. The LOAELs ranged from 12 to 90 mg/kg bw equivalent of CBD in the different studies. ...
... Different preparations containing CBD have been evaluated for potential genotoxicity in several in vitro and in vivo tests. Mutagenicity of different CBD preparations was negative in bacterial reverse mutation assays (Marx et al., 2018;Dziwenka et al., 2020). In vitro micronucleus assay using highly purified CBD and assessing structural and numerical chromosomal aberrations has been reported to be positive (Russo et al., 2019) in human HepG2 cells. ...
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Background The genotoxic and cancerogenic impacts of population-wide cannabinoid exposure remains an open but highly salient question. The present report examines these issues from a continuous bivariate perspective with subsequent reports continuing categorical and detailed analyses. Methods Age-standardized state census incidence of 28 cancer types (including “All (non-skin) Cancer”) was sourced using SEER*Stat software from Centres for Disease Control and National Cancer Institute across US states 2001–2017. It was joined with drug exposure data from the nationally representative National Survey of Drug Use and Health conducted annually by the Substance Abuse and Mental Health Services Administration 2003–2017, response rate 74.1%. Cannabinoid data was from Federal seizure data. Income and ethnicity data sourced from the US Census Bureau. Data was processed in R. Results Nineteen thousand eight hundred seventy-seven age-standardized cancer rates were returned. Based on these rates and state populations this equated to 51,623,922 cancer cases over an aggregated population 2003–2017 of 124,896,418,350. Regression lines were charted for cancer-substance exposures for cigarettes, alcohol use disorder (AUD), cannabis, THC, cannabidiol, cannabichromene, cannabinol and cannabigerol. In this substance series positive trends were found for 14, 9, 6, 9, 12, 6, 9 and 7 cancers; with largest minimum E-Values (mEV) of 1.76 × 10 ⁹ , 4.67 × 10 ⁸ , 2.74 × 10 ⁴ , 4.72, 2.34 × 10 ¹⁸ , 2.74 × 10 ¹⁷ , 1.90 × 10 ⁷ , 5.05 × 10 ⁹ ; and total sum of exponents of mEV of 34, 32, 13, 0, 103, 58, 25, 31 indicating that cannabidiol followed by cannabichromene are the most strongly implicated in environmental carcinogenesis. Breast cancer was associated with tobacco and all cannabinoids (from mEV = 3.53 × 10 ⁹ ); “All Cancer” (non-skin) linked with cannabidiol (mEV = 1.43 × 10 ¹¹ ); pediatric AML linked with cannabis (mEV = 19.61); testicular cancer linked with THC (mEV = 1.33). Cancers demonstrating elevated mEV in association with THC were: thyroid, liver, pancreas, AML, breast, oropharynx, CML, testis and kidney. Cancers demonstrating elevated mEV in relation to cannabidiol: prostate, bladder, ovary, all cancers, colorectum, Hodgkins, brain, Non-Hodgkins lymphoma, esophagus, breast and stomach. Conclusion Data suggest that cannabinoids including THC and cannabidiol are important community carcinogens exceeding the effects of tobacco or alcohol. Testicular, (prostatic) and ovarian tumours indicate mutagenic corruption of the germline in both sexes; pediatric tumourigenesis confirms transgenerational oncogenesis; quantitative criteria implying causality are fulfilled.
... The toxicity of the extracts observed in the present study was therefore attributed to the presence of cytoxic secondary plant metabolites in the solvent extracts. In the Ames assay with extracts of C. sativa diluted with olive oil as well as the extracts produced with an isopropanol and supercritical CO 2 extraction method, toxicity was evident for strains TA 98, TA 1535, TA 1537 and E. coli WP2 uvrA at≥50 μg/plate, with and without S9, in the plate incorporation and/or pre-incubation tests (Dziwenka et al. 2020). These results are similar to results of other researches that demonstrated cytotoxicity of plant extracts including betel and tobacco leaf extracts and some Nigerian folk medicines to root-tip cells of A. cepa (Sopova et al. 1983;Abraham and Cherian 1978). ...
... In another assessment of extracts of hemp (C. sativa) using the Ames reverse mutation assay, the extracts produced with an isopropanol and supercritical CO 2 extraction methods were diluted with olive oil and the undiluted extract formulated as a solution in DMSO; no mutagenic effect was observed in the four strains of Salmonella typhimurium (TA98, TA100, TA1535 and TA1537) and one strain of E. coli (WP2 uvrA) that were used (Dziwenka et al. 2020). In the present study, methanol and ethyl acetate extracts of the areal parts of C. sativa dissolved in 2.5% acetone as solvent, induced genotoxiciy in the A. cepa root meristem cells. ...
... Body weight and food consumption are used to evaluate the adverse effects caused by the tested drug. And body weight and food consumption are usually correlated and need comprehensive analysis (Dziwenka et al., 2020). Compared with the control group, the female body weight and food consumption of the medium and high dose group was reduced, which had a good dose correlation. ...
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... 5 This was the case despite a relatively high dose and an efficient route of administration, which could be associated with toxicity in chronic settings. [17][18][19][20] Given this context, it appears unlikely that there is a direct physiological role for CBD in anabolic and inflammatory signaling within skeletal muscle tissue. ...
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... Although, the gold standard for measuring of functional recovery following rat sciatic nerve injury is the SFI. However, the application of additional tools such as automated recording of motor activity of rats using Photobeams Activity System is recommended (Dziwenka et al. 2020;Wang et al. 2018). ...
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Implantation of a nerve guidance conduit (NGC) carrying neuroprotective factors is promising for repairing peripheral nerve injury. Here, we developed a novel strategy for repairing peripheral nerve injury by gold nanoparticles (AuNPs) and brain-derived neurotrophic factor (BDNF)-encapsulated chitosan in laminin-coated nanofiber of Poly(l-lactide-co-glycolide) (PLGA) conduit and transplantation of rat adipose-derived stem cells (r-ADSCs) suspended in alginate. Then, the beneficial effect of AuNPs, BDNF, and r-ADSCs on nerve regeneration was evaluated in rat sciatic nerve transection model. In vivo experiments showed that the combination of AuNPs- and BDNF-encapsulated chitosan nanoparticles in laminin-coated nanofiber of PLGA conduit with r-ADSCs could synergistically facilitate nerve regeneration. Furthermore, the in vivo histology, immunohistochemistry, and behavioral results demonstrated that the AuNPs- and BDNF-encapsulated chitosan nanoparticles in NGC could significantly reinforce the repair performance of r-ADSCs, which may also contribute to the therapeutic outcome of the AuNPs, BDNF, and r-ADSCs strategies. In this study, we found that the combination of AuNPs and BDNF releases in NGC with r-ADSCs may represent a new potential strategy for peripheral nerve regeneration.
... The polyphenolic extract of clove buds was reported to be non-mutagenic and it exhibited antimutagenic potential against known mutagens such as tobacco and sodium azide [17]. Hemp extract was shown to be non-mutagenic in an Ames test conducted in accordance with U.S. FDA Redbook and ICH guidelines [18]. The seed oil of Helianthus annuus Linné (sunflower) was reported to be non-mutagenic in an Ames test performed according to the OECD guideline for testing of chemicals [19]. ...
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