Original Article

Toxicologic Pathology
2018, Vol. 46(6) 693-705

Toxicity and Toxicokinetic Study of ª The Author(s) 2018

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Subcutaneously Administered RPh201 DOI: 10.1177/0192623318786428
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in Minipigs

Yuval Ramot1, Zadik Hazan2, Andre Lucassen2, Konstantin Adamsky2,

Vanessa Ross3, Nigel Young3, Matt Saunders3, Helmut Ehall3,
and Abraham Nyska4,5

Abstract
Mastic gum extracts are widely used as herbal remedies and are being tested for several clinical indications. Nevertheless, infor-
mation on their safety is limited. RPh201 is an extract of the mastic gum, formulated and stabilized in a proprietary method, which is
being developed as a novel drug candidate for neurological indications. The aim of this study was to assess the systemic toxic
potential of RPh201, administered twice weekly by subcutaneous injections to minipigs, after 39 weeks of administration followed
by a recovery period of 6 weeks. No clinical or dose-related signs were observed, but treatment-related findings were seen at the
injection sites of the high-dose animals, composed of abscesses, chronic inflammation, and subcutaneous fibrosis. Abscesses >30
mm in size, graded as marked severity, were confined to the high-dose group and were considered as adverse. Minimal-slight
subcutaneous and lymph nodes abscesses seen in control, low, and intermediate doses, related to the vehicle (cottonseed oil), were
not considered as adverse. Additionally, minimal-to-slight cystic spaces or vacuolation related to the vehicle were observed in the
skin, lymph nodes, kidney, and lungs. These findings were considered not to be adverse. The no-observed-adverse-effect level was
considered to be 12.5 mg/kg/occasion.

Keywords
Pistacia lentiscus, mastic gum, RPh201, toxicity, safety, minipigs, toxicokinetics

Mastic gum is extracted from the Pistacia lentiscus plant,
which grows in the Greek island of Chios (Suzuki et al.
2017). It has been traditionally used as a dietary additive and
as a flavoring agent (Spyridopoulou et al. 2017), but it has also
been widely used as a herbal remedy, mostly in the Mediterra-
nean area (Jin et al. 2017). In recent years, there is a growing
interest in the potential beneficial properties of mastic gum
extracts, and a large number of scientific studies reported on
their biological activities (Jin et al. 2017; Spyridopoulou et al.
2017; Suzuki et al. 2017). These range from antibacterial activ-
ities (Karygianni et al. 2014; Koychev et al. 2017; Miyamoto,
Okimoto, and Kuwano 2014) via antiviral activities (Ezz Eldin
and Badawy 2015; Loizzo et al. 2008; Ozcelik et al. 2005;
Sakagami et al. 2009) to antioxidant activities (Ahmad et al.
2010; Gholami et al. 2016; Hatamnia et al. 2016; Tolooei and
Mirzaei 2015; Toul et al. 2017). In addition, many studies have
reported on the anticancer properties of the mastic gum
extracts, both for solid and hematologic cancers (Balan et al.
2005; Balan et al. 2007; Dimas et al. 2009; Giaginis and Theo-
charis 2011; He et al. 2006; Loutrari et al. 2006; Magkouta
et al. 2009; Moulos et al. 2009; Sakagami et al. 2009).
Taking into consideration this long list of potential positive
properties, mastic gum extracts are being incorporated into
health and care products and are a major component in the food
industry (Vlastos et al. 2015). They are also being tested for
many clinical indications such as Crohn’s disease (Kaliora
et al. 2007a, 2007b), healing of peptic ulcers (Al-Habbal, Al-
Habbal, and Huwez 1984; Huwez and Al-Habbal 1986; Huwez
et al. 1998), and dental ulcers (Saez, Lopez, and Romero 2005;
Takahashi et al. 2003). In spite of their increasing popularity,
data on their safety are very limited.
1
Hadassah-Hebrew University Medical Center, Jerusalem, Israel
2
Regenera Pharma, Nes-Ziona, Israel
3
Envigo Ltd, Alconbury, Cambridgeshire, United Kingdom.
4
Consultant in Toxicologic Pathology, Timrat, Israel
5
Tel Aviv University, Tel Aviv, Israel
Corresponding Author:
Abraham Nyska, Toxicologic Pathologist, Haharuv 18, P.O. Box 184, Timrat
36576, Israel.
Email: anyska@bezeqint.net
694
Table 1. Experimental Design, Group Allocation, and Number of Animals.
Group Treatment Dose (mg/kg/occasion)a
1 Control 0 4
2 RPh201 3.1
3 RPh201 12.5
4 RPh201 50.0
a
Expressed in terms of the free base.
RPh201 is an extract of the mastic gum, formulated and
stabilized in a proprietary method, which is being developed
as a novel drug candidate for neurological indications. It has
already been shown to be effective for patients with previous
nonarteritic ischemic optic neuropathy in a phase 2 clinical trial
(Hazan et al. 2016). However, information on its long-term
safety profile is limited. We have recently reported that
RPh201 was found to have no adverse effects in Sprague-
Dawley rats when administered subcutaneously (Ramot et al.
2017). In this study, injection site reactions were observed to be
of greater severity in the high-dose group, but they were of mild
severity and reversible.
In order to gain more information on the safety and toxico-
kinetic profile of RPh201, we have conducted a study accom-
plished in compliance with the Good Laboratory Practice
(GLP) standards. The study included 39-week subcutaneous
(twice weekly) administration in minipigs, with a 26-week
interim phase and a 6-week recovery period.
Materials and Method
Animal Husbandry and Maintenance
A total of 32 male and 32 female Göttingen minipigs, approx-
imately 3 to 4 months old, weighing 8.3 to 12.3 kg, were
supplied by Ellegaard Göttingen Minipigs (Dalmose, Den-
mark). The animals were housed in a limited access animal
facility. Room controls were set to maintain temperature and
relative humidity at the range of 15 to 24 C and 40% to 70%,
respectively. The rooms were artificially lit for 12 hr each day.
The study animals were acclimatized for at least 4 weeks
before treatment initiation.
Potable drinking water from the public supply was available
without restrictions via automatic valves that were checked
daily. All animals were supplied Special Diets Services SMP2
811571 Minipig Diet on arrival, and after a short period, ani-
mals were transitioned onto Teklad Minipig Diet 8753C;
8753C diet was supplied from no later than the start of pre-
treatment recordings until study completion.
The Göttingen minipig was chosen for this study based on
physiologically similar cardiovascular, gastrointestinal, and,
notably, integumentary systems to humans (Manno et al.
2016; Ramot et al. 2015; Vezzali et al. 2011).
Toxicologic Pathology 46(6)
Number of Animals
Main Study Interim Phase Recovery Phase
Male Female Male Female Male Female
4 3 3 2 2
4 4 3 3 0 0
4 4 3 3 0 0
4 4 3 3 2 2
The study was conducted in accordance with the require-
ments of the European Commission Directive 2004/10/EC, and
in compliance with 21 CFR part 58, Good Laboratory Practice
for Nonclinical Laboratory Studies, the UK Good Laboratory
Practice Regulations (Statutory Instrument 1999 No. 3106, as
amended by Statutory Instrument 2004 No. 994), and the Orga-
nization for Economic Cooperation and Development Princi-
ples of Good Laboratory Practice (as revised in 1997), ENV/
MC/CHEM(98)17. The experiment was conducted at Envigo,
Cambridgeshire, UK.
Test Material and Treatment Protocol
The test material, RPh201, was supplied in a BHT-stabilized
cottonseed oil by Regenera Pharma, Nes-Ziona, Israel. BHT-
stabilized cottonseed oil was also used as vehicle. An oil for-
mulation has been used because the botanical drug substance is
essentially insoluble in aqueous carriers. Cottonseed oil has
been specifically used because it has relatively low irritability
compared to other oils (2001). The formulation used in the
safety study is the same as the one that has been used in the
clinical studies in humans (phase 1–3). The study design is
summarized in Table 1 and consisted of a control group and
three RPh201 treatment groups. Main study animals were
treated for 39 weeks with an interim sacrifice point at 26
weeks. Recovery animals were treated for 39 weeks followed
by a 6-week off-dose period. Treatment was administered
twice weekly as subcutaneous injections on the left and right
sides of the flanks. Two sites on each side were marked and
used in rotation.
Clinical Observations and Mortality and
Physical Examination
Animals were inspected visually at least twice daily, and pens
were inspected daily for evidence of ill-health. A detailed
weekly physical examination was performed on each animal.
Detailed clinical observations were performed on both dosing
days during weeks 1 to 4 and thereafter on a dosing day once
weekly during weeks 5 to 13 and once every two weeks there-
after. These observations were done before injection, 1- to 2-hr
postdosing and at the end of the same working day. The injec-
tion sites were observed 1- and 3-hr postdosing on dosing days.
Ramot et al.
During the recovery phase, observations of the animals and
their pens were recorded at least once per day.
The weight of each animal was recorded (prior to feeding)
weekly during the acclimatization period, on the day that treat-
ment commenced (week 0), weekly throughout the study, and
before necropsy. Food consumption was calculated by weigh-
ing the amount of food supplied to each animal and subtracting
any remaining food together with an additional estimate of any
that had been spilt. This was recorded daily during the accli-
matization period and throughout the study. The eyes of the
animals were examined by means of a binocular indirect
ophthalmoscope pretreatment, on weeks 25 to 27 and 37 to
39 on all main and recovery animals, and at recovery week 6
for all recovery animals.
Electroretinogram (ERG) and Tonometry
Under general anesthesia, an ERG was conducted on both eyes
of main and recovery animals. Both scotopic and photopic
ERG evaluations were carried out on the same day. The ani-
mals were habituated to darkness for a minimum of 2 hr prior to
the scotopic evaluation. Under the same general anesthetic as
for ERG, measurement of intraocular pressure was carried out
via tonometry on both eyes of main and recovery animals.
These investigations were performed pretreatment, on weeks
25 to 27 and 37 to 39 on all main and recovery animals, and at
recovery week 6 for all recovery animals.
Electrocardiography
Electrocardiograph tracings were recorded predose and 0.5, 1,
2, 4, 8, and 24 hr after completion of dosing from all animals
pretreatment and on weeks 13, 26, 37, and 39. Recovery ani-
mals were tested on recovery week 6.
Clinical Pathology and Toxicokinetics and Urinalysis
Blood samples were obtained via the jugular vein/cranial vena
cava from all animals pretreatment, on weeks 13 and 26, at
week 39 from both the main and recovery-phase animals, and
at recovery week 6 from the recovery-phase animals. The blood
was analyzed for hematological, coagulation, and biochemical
parameters. Urine samples were collected at the same
time points.
Blood samples for toxicokinetics and immunogenicity were
obtained from the main-phase animals on day 1 and week 13,
26, and 39 at 0.5, 1, 2, 4, 8, and 24 hr after dosing. Samples
from all treated animals and two samples from each control
animal per occasion were analyzed. One sample around Tmax
(2-hr time point) and one sample in the elimination phase (8-hr
time point) were selected for analysis.
Euthanasia and Necropsy
Animals of the main study were sacrificed following 39 weeks
of treatment with an interim sacrifice at 26 weeks. Recovery
animals were sacrificed following 39 weeks of treatment and 6
695
weeks of recovery. Animals were euthanized with an overdose
of sodium pentobarbitone solution (200 mg/ml) by intravenous
injection followed by exsanguination. All animals were subject
to a detailed necropsy.
Organ Weights
Organ weights of the following organs were recorded: adrenal
glands, brain, epididymides, heart, kidneys, liver, lungs, ovar-
ies, pituitary gland, prostate gland, spleen, testes, thymus, thyr-
oid gland, and uterus with cervix.
Microscopic Observations
Tissues were preserved in 10% neutral-buffered formalin
except for the testes that were preserved in modified David-
son’s fluid and the eyes that were preserved initially in David-
son’s fluid and subsequently transferred to 70% industrial
methylated spirit (IMS).
Histopathological evaluation of hematoxylin and eosin–
stained tissues was performed on all animals. The following
tissues were evaluated: adrenals, thoracic aorta, brain, cecum,
colon, dosing sites, duodenum, epididymides, esophagus, eyes,
femur, gallbladder, Harderian gland, heart, ileum, jejunum,
kidneys, lacrimal gland, liver, lungs, lymph nodes (mandibular,
mesenteric, and left inguinal), optic nerves, ovaries, pancreas,
parathyroids, Peyer’s patches, pituitary, prostate, salivary
glands (submandibular, parotid, and sublingual), sciatic nerves,
skeletal muscle, skin with mammary glands, spinal cord,
spleen, sternum, stomach, testes, thymus, thyroid, trachea,
urinary bladder, uterus with cervix, and vagina. In addition,
Oil Red O staining was performed on the lungs from a few
animals to confirm presence of oil, and Gram’s stain was per-
formed on the injection sites from a few animals with abscesses
to check for the presence of bacteria. Findings were either
reported as “present” or assigned a severity grade. In the latter
case, one of the following five grades was used—minimal,
slight, moderate, marked, or severe.
Abscesses seen in the injection sites and lymph nodes were
graded according to the following criteria:
Minimal: between one and five abscesses up to 2 mm in
size; slight: more than five abscesses up to 2 mm and/or
abscesses up to 10 mm in size. Usually, only one abscess
larger than 2 mm was present at a single-injection site;
moderate: abscesses 10 to 30 mm in size; marked:
abscesses larger than 30 mm in size.
Abscesses graded as moderate or marked were usually a
single abscess that was detected macroscopically, although
smaller abscesses (generally smaller than 2 mm) were some-
times present at the same injection site. The distinction
between moderate and marked was made from the size of the
macroscopic description, as abscesses larger than 30 mm were
too large to fit on the slide and may also have ruptured during
harvesting and preparation of the slide. Recovering abscesses
usually occurred singularly at an injection site and were graded
696
Table 2. Liver Weights at Week 39.
Males
1M 2M
Group N¼4 N¼4
Absolute weight 494.295 (33.799) 401.979 (38.997)
Adjusted weight 493.186 402.519*
Females
Group 1F 2F
N¼4 N¼4
Absolute weight 542.674 (42.711) 447.243 (27.839)
Adjusted weight 544.619 445.872
*p < .05.
**p < .01.
in the same way based on their size. The same grading system
was used for abscesses in lymph nodes that were all graded as
minimal or slight.
Statistical Analysis
All statistical analyses were carried out separately for males
and females (except for electrocardiography, for which sexes
were combined) using the individual animal as the basic
experimental unit. A parametric analysis was performed for
body weight, food consumption, electrocardiography, organ
weight, and clinical pathology data if Bartlett’s test for var-
iance homogeneity was not significant at the 1% level. The F1
approximate test was applied. If the F1 approximate test for
monotonicity of dose-response was not significant at the 1%
level, Williams’ test for a monotonic trend was applied. If the
F1 approximate test was significant, suggesting that the dose-
response was not monotone, Dunnett’s test was performed
instead. A nonparametric analysis was performed if Bartlett’s
test was still significant at the 1% level following both loga-
rithmic and square-root transformations. The H1 approximate
test, the nonparametric equivalent of the F1 test described
above, was applied. If the H1 approximate test for monotonicity
of dose-response was not significant at the 1% level, Shirley’s
test for a monotonic trend was applied. If the H1 approximate
test was significant, suggesting that the dose-response was not
monotone, Steel’s test was performed instead.
For clinical pathology data, if 75% of the data (across all
groups) were the same value, Fisher’s exact tests were per-
formed. Treatment groups were compared using pairwise com-
parisons of each dose group against the control. For organ
weight data, analysis of covariance was performed using ter-
minal body weight as covariate, unless nonparametric methods
were applied. The treatment comparisons were made on
adjusted group means in order to allow for differences in body
weight, which might influence the organ weights. Significant
differences between the groups compared were expressed at the
5% (p < .05) or 1% (p < .01) level.
Toxicologic Pathology 46(6)
3M 4M
N¼4 N¼4
414.216 (26.632) 439.725 (17.048)
415.225* 439.285**
3F 4F
N¼4 N¼4
501.49 (40.638) 457.774 (46.865)
501.892* 456.797*
Results
Clinical Observations (Including Ocular ERG and
Tonometry, and Cardiac Electrocardiography) and
Organ Weights
The behavior of the animals has been unaffected following 39
weeks of treatment, and there were no test article–related clin-
ical signs. One female animal from group 1 had to be eutha-
nized early on day 88 for welfare reasons after sustaining an
injury during routine blood sampling.
There were no treatment-related effects on body weight or
food consumption during the 39 weeks of treatment or 6-
week recovery phase. The terminal investigations conducted
following 39 weeks of treatment revealed lower absolute and
body weight–adjusted liver weights in all treated groups
when compared to controls (Table 2). This observation did
not show a dose relationship, and the liver weights of indi-
vidual animals showed considerable overlap with the concur-
rent control range of values. Considering also that there were
no significant histopathological findings in the liver and the
absence of any differences in liver weights after a 6-week
off-dose period compared to controls, it was considered that
any variation in liver weights was fortuitous and unrelated to
treatment.
The terminal investigations, conducted following 26 weeks
of treatment, did not reveal any organ weight differences from
the control group, which were related to treatment.
All other differences from controls were minor, showed
considerable overlap with the concurrent control range of val-
ues or lacked dose dependency, are not considered to be test
item related, and are therefore attributed to normal biological
variation.
Posterior suture line opacity was observed in the lens of
the eye of four animals: one male from group 2 at week 39;
one male from group 4 at week 39; one female from group
4 at week 26, 39, and recovery week 6; and one female
from group 4 at recovery week 6. There was no effect on
Ramot et al. 697

Table 3. Hematology Results.

Males

Week 26

1M 2M 3M 4M

Group N¼9 N¼7 N¼7 N¼9

9
Neutrophils 10 /L 3.3 (0.698) 3.37 (1.239) 4.84 (2.881) 5.54 (2.682)*
Week 39
Group 1M 2M 3M 4M
N¼6 N¼4 N¼4 N¼6
Neutrophils 109/L 4.34 (0.909) 3.24 (1.268) 5.15 (1.534) 4.65 (1.213)

Females

Week 26

Group 1F 2F 3F 4F

N¼8 N¼7 N¼7 N¼9

Neutrophils 109/L 2.32 (0.432) 2.92 (0.59) 4.03 (1.623)* 3.62 (1.301)*
Week 39
Group 1F 2F 3F 4F
N¼6 N¼4 N¼4 N¼6
Neutrophils 109/L 3.32 (1.203) 2.58 (0.875) 3.74 (1.821) 3.07 (0.714)
RBC 1012/L 8.7 (0.86) 7.89 (0.409) 8.13 (0.393) 8.13 (1.39)
Hb g/dl 16.5 (0.88) 15.9 (1.92) 15.1 (1.02) 15.5 (1.94)
Week 6 recovery
Group 1F 2F 3F 4F
N¼2 N¼0 N¼0 N¼2
Neutrophils 109/L 2.44 (0.396) — — 2.92 (0.049)
RBC 1012/L 10.61 (0.849) — — 7.17 (0.417)
Hb g/dl 18.7 (0.92) — — 15.3 (2.62)
*p < .05.

the retina at ERG examination, and no ocular hypo or
hypertension was evident. There were no effects on electro-
cardiography parameters.
Clinical Pathology
Hematology investigations conducted in week 26 of treatment
revealed slightly higher mean neutrophil counts observed in
both sexes receiving 12.5 or 50 mg/kg/occasion when com-
pared to concurrent controls (Table 3). This slightly higher
neutrophil count was still evident in the week 39 hematology
investigations but only in males given 12.5 mg/kg/day when
compared to concurrent controls. There was full recovery
during the 6-week off-dose period, although this was assessed
in females only due to clotted samples in the males.
In week 39, a lower mean erythrocyte count and hemoglobin
concentration were observed in females receiving 50 mg/kg/
occasion in comparison with the controls (Table 3). The lower
mean values were mainly due to particularly low values
observed for one animal. There was partial recovery in both
parameters following 6 weeks off-dose, as one female still
showed lower erythrocyte count and hemoglobin concentration.
There were no other differences from the controls in clinical
pathology or urinalysis parameters observed at the week 13,
26, or 39 investigations that were considered to be related to
treatment.
Toxicokinetics
The rate of systemic exposure of minipigs to RPh201, measured
as the two components masticadienonic acid (MDA) and iso-
masticadienonic acid (IMDA), appeared to be characterized by
nonlinear, subproportional (dose dependent) kinetics over the
dose range from 3.1 to 50.0 mg/kg/day on day 1 and during
weeks 13, 26, and 39 of the 39-week subcutaneous toxicity study
(data not shown). However, the extent of systemic exposure of
minipigs to MDA and IMDA generally appeared to be charac-
terized by dose-independent (linear) kinetics over the dose range
3.1 to 50.0 mg/kg/day on day 1 and during weeks 13, 26, and 39
of the 39-week subcutaneous toxicity study. Systemic exposure
of minipigs to MDA was generally comparable to that of IMDA.
The data also provided evidence that the systemic exposure
of female minipigs to MDA and IMDA was generally slightly
lower than that of males after repeated administration and that
698 Toxicologic Pathology 46(6)

Table 4. Treatment-related Histopathological Findings in the Injection Sites.

Group/sex 1M 2M 3M 4M 1F 2F 3F 4F

Dose (mg/kg/occasion) 0 3.1 12.5 50.0 0 3.1 12.5 50.0

Abscess(es)
26 weeks
Minimal 0 1 1 1 0 0 1 0
Slight 0 0 0 1 0 0 0 0
Marked 0 0 0 0 0 0 0 1
Total 0 1 1 2 0 0 1 1
39 weeks
Minimal 1 1 2 2 0 0 1 1
Marked 0 0 0 1 0 0 0 1
Total 1 1 2 3 0 0 1 2
6-week recovery
Minimal 1 — — 1 1 — — 1
Slight 0 — — 1 0 — — 0
Total 1 — — 2 1 — — 1
Subcutis–resolving abscess(es)
39 weeks
Moderate 0 0 0 0 0 0 0 2
Marked 0 0 0 0 0 0 0 1
Total 0 0 0 0 0 0 0 3
Number of tissues examined 4 4 4 4 4 4 4 4
6-week recovery
Minimal 0 — — 0 0 — — 2
Total 0 — — 0 0 — — 2
Subcutis–fibrosis
39 weeks
Minimal 1 0 1 3 0 0 1 0
Slight 0 0 0 0 1 0 0 1
Moderate 0 0 0 0 0 0 0 2
Total 1 0 1 3 1 0 1 3
6-week recovery
Minimal 1 — — 0 0 — — 1
Slight 0 — — 2 0 — — 1
Total 1 — — 2 0 — — 2

there was some accumulation of MDA and IMDA at the 3.1
and 12.5 mg/kg/day dose levels in both sexes after repeated
subcutaneous administration of RPh201.
Macroscopic Observations
Injection sites. Clear oily liquid was found at the injection sites
of all groups at the 26- and 39-week sacrifice points. In the
6-week recovery group, clear oily liquid was also observed
in controls and animals previously treated with 50.0 mg/kg/
occasion with a similar incidence. Thickening was seen at
the 39-week sacrifice point in both sexes treated with 50 mg/
kg/occasion, control animals of both sexes, and in a single
female treated with 12.5 mg/kg/occasion. Pale white areas
were seen in all treated groups at the 26- and 39-week sacri-
fice points and in a male and female control animal. At the
6-week recovery group, pale areas were seen in controls and
animals previously treated with 50.0 mg/kg/occasion with a
higher combined incidence in previously treated animals.
Raised areas were seen at the week 26 time point in females
treated with 50 mg/kg/occasion and at the 39-week sacrifice
necropsy in both sexes treated with 50 mg/kg/occasion, in
female animals treated with 12.5 mg/kg/occasion, and in one
female control animal. At the 6-week recovery necropsy,
raised areas were seen in females treated with 50.0 mg/kg/
occasion.
Lymph nodes. At the 26-week sacrifice necropsy, enlargement
of the right and left inguinal lymph nodes was found in one
female receiving 50.0 mg/kg/occasion, with pale areas also
noted in the left inguinal node of a different female from the
same dose group. At the 39-week and 6-week recovery sacri-
fice points, clear oily liquid, enlargement, and pale areas were
seen in various lymph nodes (including axillary, inguinal, man-
dibular, and mediastinal lymph nodes) in animals treated with
50 mg/kg/occasion and in the control group. Enlargement was
also seen in one female animal treated with 12.5 mg/kg/occa-
sion, and pale areas were also seen in a male and female animal
in this group.
Ramot et al. 699

Figure 1. (A) Histopathological section of the subcutaneous injection site in minipig from the high-dose group (50 mg/kg), main phase of the study
(i.e., sacrifice at the end of 6 months of treatment). The tissue reaction consists of chronic inflammation (asterisks). These findings were
comparable to the control group and were considered as related to the vehicle. In addition, abscesses (arrow) in the subcutis (minimal grade)
700 Toxicologic Pathology 46(6)

Figure 1. (Continued). were seen, characterized by a cystic space containing aggregates of neutrophils with a variable chronic inflammatory
component and a fibrous capsule of varying thickness. H&E. (B) Histopathological section of the subcutaneous injection site in minipig from the
high-dose group (50 mg/kg), main phase of the study. Higher magnification of the previous photo. Note the abscess (arrows) in the subcutis
(minimal grade), characterized by a cystic space containing aggregates of neutrophils with a variable chronic inflammatory component and a fibrous
capsule of varying thickness. The surrounding tissue reaction consists of chronic inflammation (asterisk). H&E. (C) Histopathological section of
the subcutaneous injection site in minipig from the high-dose group (50 mg/kg), main phase of the study (i.e., sacrifice at the end of 6 months of
treatment). The tissue reaction consists of chronic inflammation (asterisks), and intermixed with empty spaces reflecting the washed-out cotton-
seed oil vehicle. These findings were comparable to the control group and were considered as related to the vehicle. In addition, abscesses
(arrows) in the subcutis (slight grade) were seen. H&E. (D) Histopathological section of the subcutaneous injection site in minipig from the high-
dose group (50 mg/kg), main phase of the study. Higher magnification of the previous photo. Note the abscess (arrows) in the subcutis (slight
grade). The surrounding tissue reaction consists of chronic inflammation (asterisk). H&E. (E) Histopathological section of the subcutaneous
injection site in minipig from the high-dose group (50 mg/kg), main phase of the study. The tissue reaction consists of chronic inflammation
(asterisks), and intermixed with empty spaces reflecting the washed-out cotton-seed oil vehicle. Note the abscess (arrows) in the subcutis
(marked grade). This grade of lesion is considered treatment related. H&E. (F) Histopathological section of the inguinal lymph node (regional to
the injection site) in minipigs from the intermediate-dose group (12.5 mg/kg), main phase of the study (i.e., sacrifice at the end of 6 months of
treatment). The tissue reaction consists of chronic inflammation (asterisks), and intermixed with empty spaces reflecting the washed-out cotton-
seed oil vehicle. These findings were comparable to the control group and were considered as related to the vehicle. In addition, minimal-grade
abscesses (arrows) were seen. H&E. (G) Histopathological section of the inguinal lymph node (regional to the injection site) in a minipig from the
intermediate-dose group (12.5 mg/kg), main phase of the study. Higher magnification of the previous photo. Note the minimal-grade abscess
(arrows). The surrounding tissue reaction consists of chronic inflammation (asterisk). H&E. (H) Histopathological section of the subcutaneous
injection site in a minipig from the high-dose group (50 mg/kg), recovery phase of the study. A resolving abscess. The tissue reaction is
characterized by fibrous tissue, mineralization (arrows), and with only few neutrophils present (asterisk). H&E. (I) Histopathological section
Ramot et al.
Microscopic Observations
Treatment-related findings. Treatment-related findings were
observed at injection sites of animals from all sacrifice points
and are summarized in Table 4. Since the findings were com-
parable for all four injection sites evaluated, only injection site
3 is presented in the table. The histopathological findings com-
prised of abscesses at the 26-week and 39-week sacrifice and
recovery phase and recovering abscesses and an increase in
fibrous tissue in the 39-week sacrifice and recovery phases.
Abscesses were characterized by a circular space containing
aggregates of neutrophils with a variable chronic inflammatory
component and a fibrous capsule of varying thickness.
Minimal-graded abscesses were also seen in the control group,
and this grade is considered to be related to the vehicle.
Abscesses graded as moderate or marked were confined to
animals receiving 50 mg/kg/occasion. Gram stain was per-
formed on the injection sites from a few animals with abscesses
to check for the presence of bacteria, and no bacterial colonies
were present. Resolving abscesses were seen at the 39-week
sacrifice point in females receiving 50.0 mg/kg/occasion.
These were characterized by fibrous tissue, mineralization, and
with few neutrophils present. At the 6-week recovery sacrifice,
the severity of the abscesses, resolving abscesses, and fibrosis
was confined to minimal or slight, which indicated partial
recovery from the findings seen at 26 and 39 weeks. Represen-
tative photos of the abscesses from the main phase are shown in
Figure 1A–E. Representative photos of the resolving abscesses
from the recovery phase are shown in Figure 1H–J.
Vehicle-related findings
Lungs: Vacuolation in the alveolar septae and intraluminal
vacuoles in blood vessels were seen in control animals and
those receiving 12.5 and 50 mg/kg/occasion (Table 5). Oil
Red O staining was performed on the lungs with vacuola-
tion from a few animals to confirm the presence of oil, and
all the tissues stained were positive confirming the pres-
ence of oil vacuolation in the alveolar septae and intra-
luminal vacuoles in blood vessels. Partial recovery was
evident for all findings at the 6-week recovery point.
Kidneys: Vacuolation in the glomeruli was seen in
control animals and those receiving 50.0 mg/kg/occasion
(Table 6). Partial recovery was evident for all findings at
the 6-week recovery point.
Injection sites: Cystic spaces and chronic inflammation
were seen in all treated groups and controls with the
severity generally correlating with the volume of vehicle
administered (Table 7). The chronic inflammation was
characterized by mononuclear cells (lymphocytes and
macrophages), foreign body giant cells, fibrosis, and
Figure 1. (Continued). of the subcutaneous injection site in minipig from the high-dose group (50 mg/kg), recovery phase of the study. Another
example of a resolving abscess. The tissue reaction is characterized by fibrous tissue (arrow) and with only few neutrophils present (asterisk).
H&E. (J) Histopathological section of the subcutaneous injection site in minipig from the high-dose group (50 mg/kg), recovery phase of the study.
Area of fibrosis (asterisks). The increase in subcutaneous fibrosis was also seen in the 39-week and 6-week recovery sacrifice, mainly in the high-
dose animals. H&E.
701
occasional neutrophils. Incidence and severity of the
findings did not show signs of recovery after 6 weeks.
Findings similar to those at the subcutaneous injection sites
were seen in the routine skin section of one control male
and one male receiving 50.0 mg/kg/occasion at the 26- and
39-week sacrifice point. Such findings were not observed at
the 6-week recovery group.
Lymph nodes: Cystic spaces and chronic inflammation were
seen in the left inguinal lymph node of all treated groups
and controls at 26 week, 39 week, and recovery phase
with the severity generally correlating with the volume of
vehicle administered (Table 8). Chronic inflammation
was characterized by mononuclear cells (lymphocytes and
macrophages), foreign body giant cells, fibrosis, and occa-
sional neutrophils. Abscesses, graded as minimal, were
seen in the left inguinal lymph node of a few animals killed
at 26 weeks in all treated groups but also in a female control
killed after 39 weeks of treatment and in the recovery
group. They were also seen in other lymph nodes in all
three phases of this study, which were examined histo-
pathologically due to being macroscopically abnormal.
These consisted of aggregates of neutrophils contained
within a small circular space surrounded by a minimal
fibrous capsule. Representative photos of the abscesses in
the inguinal lymph nodes from the main phase are shown in
Figure 1F–G. Considering the overall incidence of
abscesses in the left inguinal lymph node and other lymph
nodes in the study, this finding was considered to be related
to the vehicle. The incidence and severity of the findings at
the 6-week sacrifice point were similar to the 39-week
sacrifice point, therefore not showing signs of recovery.
Similar findings were also seen in the right inguinal lymph
node and in the axillary, mediastinal, medial iliac, aortic, and
superficial inguinal lymph nodes when a macroscopic abnorm-
ality was seen. Such abnormalities were present most com-
monly in controls and animals receiving 50.0 mg/kg/occasion.
Discussion
The aim of this study was to assess the systemic toxic potential
of RPh201, administered twice weekly by subcutaneous injec-
tions to minipigs, after 39 weeks of administration followed by
a recovery period of 6 weeks. This study shows that RPh201
was well tolerated for 39 weeks with no clinical or dose-related
signs observed and with no changes in body weight, food con-
sumption, ERG, intraocular pressure, electrocardiography or
blood chemistry, and urinalysis parameters.
702 Toxicologic Pathology 46(6)

Table 5. Vehicle-related Histopathological Findings in the Lungs. Table 7. Vehicle-related Histopathology Findings at Injection Sites.

Group/sex 1M 2M 3M 4M 1F 2F 3F 4F Group/Sex 1M 2M 3M 4M 1F 2F 3F 4F

Dose (mg/kg/occasion) 0 3.1 12.5 50.0 0 3.1 12.5 50.0 Dose (mg/kg/occasion) 0 3.1 12.5 50.0 0 3.1 12.5 50.0

26 weeks Cystic spaces

Vacuolation, alveolar septae 26 weeks
Minimal 2 0 1 3 1 0 0 2 Minimal 0 3 0 0 0 1 3 1
Slight 1 0 0 0 0 0 0 0 Slight 0 0 2 0 2 1 0 1
Moderate 0 0 0 0 0 0 0 1 Moderate 3 0 1 2 0 0 0 1
Total 3 0 1 3 1 0 0 3 Total 3 3 3 2 2 2 3 3
Blood Vessels–intraluminal vacuoles 39 weeks
Minimal 2 0 0 3 1 0 0 2 Minimal 0 2 0 0 1 3 0 0
Total 2 0 0 3 1 0 0 2 Slight 0 2 2 1 1 1 2 1
39 weeks Moderate 4 0 2 3 2 0 2 3
Vacuolation, alveolar septae Total 4 4 4 4 4 4 4 4
Minimal 3 0 1 0 3 0 1 3 6-week sacrifice
Slight 0 0 0 3 0 0 0 1 Slight 0 — — 0 0 — — 1
Total 3 0 1 3 3 0 1 4 Moderate 2 — — 2 2 — — 1
Blood vessels–intraluminal vacuoles Total 2 — — 2 2 — — 2
Minimal 2 0 0 2 3 0 3 2 Chronic inflammation
Total 2 0 0 2 3 0 3 2 26 weeks
6-week sacrifice Minimal 0 1 1 1 1 0 3 1
Vacuolation, alveolar septae Slight 3 1 2 2 0 0 0 2
Minimal 0 — — 0 0 — — 1 Total 3 2 3 3 1 0 3 3
Total 0 — — 0 0 — — 1 39 weeks
Minimal 1 1 1 0 2 2 2 2
Slight 3 1 3 4 1 1 2 2
Total 4 2 4 4 3 3 4 4
Table 6. Vehicle-related Histopathological Findings in the Kidneys. 6-week sacrifice
Minimal 0 — — 0 1 — — 0
Group/sex 1M 2M 3M 4M 1F 2F 3F 4F Slight 2 — — 1 1 — — 2
Moderate 0 — — 1 0 — — 0
Dose (mg/kg/occasion) 0 3.1 12.5 50.0 0 3.1 12.5 50.0
Total 2 — — 2 2 — — 2
26 weeks
Vacuolation, glomeruli
Minimal 1 0 0 2 1 0 0 2
areas (which contained either dark fluid or green purulent mate-
Total 1 0 0 2 1 0 0 2
39 weeks rial). The subcutaneous fibrosis seen at the injection sites was
Vacuolation, glomeruli considered secondary to chronic inflammation and was again
Minimal 1 0 0 2 1 0 0 1 more pronounced in animals given 50 mg/kg/occasion and to a
Slight 0 0 0 1 0 0 0 2 lesser extent in animals given 12.5 mg/kg/occasion. There was
Total 1 0 0 3 1 0 0 3 partial recovery from both the abscess formation and fibrosis.
6-week sacrifice
Necrosis was not seen in any of the injection sites, and
Vacuolation, glomeruli
Minimal 0 — — 1 0 — — 1 bacterial colonies were not seen in the abscesses (i.e., con-
Total 0 — — 1 0 — — 1 firmed negative by Gram staining). Therefore, these abscesses
were concluded to be sterile. Formation of sterile abscesses in
the skin and muscle was observed previously in humans, fol-
Treatment-related findings were seen at the injection sites. lowing repeated injections of oil-based formulations (Cordes
Abscesses, which were scored as minimal or slight, and subcu- and Arnold 1949; Jones 1996; Starmark, Forsman, and Wahl-
taneous fibrosis that was associated with pale areas were strom 1980). Chronic inflammation can result due to irritation
observed at the injection sites at the 12.5 or 50 mg/kg/occasion from the presence of the drug in a location with a relatively
dose levels. These findings correlated with a small increase in slow rate of absorption. The acute inflammation seen in a few
peripheral neutrophil count. At the 12.5 mg/kg/occasion dose injection sites may represent a milder reaction or an early stage
level, these abscesses were scored as minimal to slight and of the process, which might progress to abscess formation.
therefore were considered to be nonadverse and at this dose level Even though necrosis was not seen at any injection site and
or below there were no findings considered to be of toxicological no treated animals showed any clinical signs relating to the
importance. However, in animals given 50 mg/kg/occasion, injection sites or any effect on food consumption or body-
abscesses graded above slight corresponded with macroscopi- weight, the abscesses seen at the injection sites of some animals
cally thickened areas (with cystic pockets of dark fluid) or raised receiving 50 mg/kg/occasion were considered adverse due to
Ramot et al. 703

Table 8. Findings Related to the Vehicle in the Left Inguinal Lymph iliac, aortic, and mandibular lymph nodes), injection sites, and
Node. routine skin and subcutis sections. Generally, the incidence and
Group/Sex 1M 2M 3M 4M 1F 2F 3F 4F severity reflected the volume of material injected, with the
controls showing a similar incidence of findings to the animals
Dose (mg/kg/occasion) 0 3.1 12.5 50.0 0 3.1 12.5 50.0 receiving 50 mg/kg/occasion. The chronic inflammation in the
subcutis was considered to be related to the cystic spaces
26 weeks
Cystic spaces caused by the vehicle. The findings in the routine sections of
Minimal 0 0 0 0 0 1 1 0 the skin and subcutis were considered to be due to ventral
Slight 0 1 0 0 0 1 1 0 movement of injected material from the dorsally located injec-
Moderate 0 2 3 0 2 0 0 2 tion sites. After a 6-week recovery period, the lungs and kidney
Marked 3 0 0 3 0 0 0 1 showed partial recovery, while the findings in the subcutaneous
Total 3 3 3 3 2 2 2 3
injection sites and lymph nodes were similar to that seen at the
Chronic inflammation
Minimal 0 3 1 1 2 1 0 0 end of the treatment period. These findings were considered as
Slight 3 0 2 2 0 0 1 3 not adverse since they were of minimal-to-slight severity.
Total 3 3 3 3 2 1 1 3 The trafficking of foreign material, such as oil-based for-
Abscess(es) mulations, via lymphatic vessels has been known for many
Minimal 0 1 2 2 0 0 1 1 years (Clark and Clark 1917). Such trafficking can be mediated
Total 0 1 2 2 0 0 1 1
by antigen presenting cells or as passive absorption (Ramot
39 weeks
Cystic spaces et al. 2009). Alternatively, movement of oil-based compounds
Slight 0 0 0 0 1 0 0 0 can occur via the circulatory system (Peyre et al. 2004). This
Marked 0 0 0 1 0 0 0 0 can result in the formation of lipogranuloma at distant organs
Total 0 0 0 1 1 0 0 0 such as the peritoneum (Ramot et al. 2009).
Chronic inflammation Vegetable oils, and among them cottonseed oil, are capable
Slight 0 0 0 1 1 0 0 0
of solubilizing highly lipophilic materials and are in commer-
Total 0 0 0 1 1 0 0 0
Fibrosis cial use for many parenteral formulations (Strickley 2004).
Minimal 0 0 0 1 1 0 0 0 Nevertheless, information on the potential adverse effects
Total 0 0 0 1 1 0 0 0 related to the use of these vehicles, and specifically during
6-week sacrifice preclinical studies, is limited. Therefore, the information on
Cystic spaces the lack of adverse effects associated with the use of the cotton-
Slight — — — — 0 1 1 0
seed oil vehicle will be of importance for future drug safety
Moderate — — — — 0 0 0 2
Marked — — — — 1 0 1 0 assessments utilizing vegetable oils as a vehicle.
Total — — — — 1 1 2 2 Lens opacities, including suture line abnormalities, are
Chronic inflammation — — — — described as a normal background lesion in the Göttingen mini-
Minimal — — — — 1 1 2 2 pig (Loget 1995; Loget and Saint-Macary 2000). According to
Total — — — — 1 1 2 2 Loget and Saint-Macary (2000), the background incidence of
Fibrosis — — — — lens abnormalities in the Göttingen minipig is reported to be
Minimal — — — — 1 1 0 2
Slight — — —– — 0 0 1 0 2.8% for suture line abnormality, 8.3% for focal nuclear opa-
Total — — — — 1 1 1 2 city, 19.4% for posterior cortical pinpoint opacity, and 8.3% for
Abscess(es) — — — — posterior capsular opacity (Loget and Saint-Macary 2000).
Minimal — — — — 0 0 1 0 Lens focal nuclear opacities were reported in other animal
Total — — — — 0 0 1 0 species (e.g., dogs, rats, and Yucatan minipigs) and are non-
progressive and do not interfere with vision. It is probable that
this abnormality appears later in life and should not be consid-
their relatively large size, that is the size of the abscess was the ered adverse (Loget 1995).
“critical effect size,” which defines the breaking point between The nature and the appearance of the findings, the age of
adverse and nonadverse changes (Kerlin et al. 2016). occurrence, and the pattern of progression are in accordance
Cystic spaces were recorded in the subcutaneous injection with the background incidences of posterior suture line opaci-
sites, and these correlated with an abnormal oily clear liquid ties, which are seen in this species, strain, and age. Further-
material recorded macroscopically, which is considered to be more, no effects were observed following the ERG or tonicity
due to the presence of the vehicle (BHT-stabilized cottonseed assessments of the eyes or clinical signs that would suggest
oil). Thickened areas recorded macroscopically in the subcu- impaired eye function. It is therefore highly unlikely to be
taneous injection sites generally correlated with chronic associated with the administration of the test compound, espe-
inflammation in the subcutis seen microscopically. Findings cially if similar findings were not seen in any other species that
related to treatment with the vehicle were seen in the lungs, received the compound. In conclusion, under the conditions of
kidneys, lymph nodes (inguinal, axillary, mediastinal, medial, this study, the no-observed-adverse-effect level was considered
704
to be 12.5 mg/kg/occasion, after 39 weeks of administration
followed by a 6-week recovery period.
Authors’ Note
Drs. Zadik Hazan, Andre Lucassen, and Konstantin Adamsky are
employees of Regenera Pharma; Abraham Nyska is a consultant to
Regenera Pharma.
Authors Contribution
Authors contributed to conception or design (ZH, AL, KA, VR, NY,
MS, HE, AN); data acquisition, analysis, or interpretation (YR, ZH,
AL, KA, VR, NY, MS, HE, AN); drafting the manuscript (YR, ZH,
AL, KA, VR, NY, MS, HE, AN); and critically revising the manu-
script (YR, ZH, AL, KA, VR, NY, MS, HE, AN). All authors gave
final approval and agreed to be accountable for all aspects of work in
ensuring that questions relating to the accuracy or integrity of any part
of the work are appropriately investigated and resolved.
Declaration of Conflicting Interests
The author(s) declared no potential, real, or perceived conflicts of
interest with respect to the research, authorship, and/or publication
of this article.
Funding
The author(s) disclosed receipt of the following financial support for
the research, authorship, and/or publication of this article: The study
was funded by Regenera Pharma, Nes-Ziona, Israel.
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