Safety and Tolerability of Overdosed Artiﬁcial
Tears by Abraded Rabbit Corneas
and Jean-Se´ bastien Garrigue
Purpose: Preservative-free cationic emulsion-based artiﬁcial tear (AT) is an innovative eye drop based on the
technology with cetalkonium chloride (CKC) as the cationic agent. The cationic emulsion Catio-
norm is designed for the management of mild-to-moderate dry eye disease (DED) patients that present cornea
epithelium alterations. The aim of the present study was to evaluate the safety and tolerability of overdosed ATs
by altered corneal epithelium in vivo and assess the usefulness of the ex vivo eye irritation test (EVEIT) as a
predictive alternate toxicity test method.
Methods: The experimental procedure, treatment duration, and instillation frequency closely mimic in vivo the
ex vivo protocol described by Pinheiro et al. and discussed in the Discussion and Conclusion section of this
article. Two to 3-month-old female New Zealand white rabbits, n=6 per group, were treated with ATs (21
instillations/day over 3 days) following corneal abrasion. Corneal ﬂuorescein staining, in vivo confocal mi-
croscopy (IVCM), and slit lamp examinations were performed to assess corneal epithelium recovery and the
ocular tolerability of the overdosed ATs.
Results: All abraded eyes experienced almost complete epithelium recovery within 3 days following treatments
with Cationorm, Optive, Vismed, and Saline. Benzalkonium chloride (BAK, 0.02%) treatment resulted in
82.4% reepithelialization. IVCM data illustrated corneal epithelium normal recovery. Acute local tolerability of
the overdosed ATs was conﬁrmed using Draize and McDonald–Shadduck’s test scales.
Conclusions: The different ATs were demonstrated to be well tolerated by abraded corneas in vivo, and the
extreme overdosing regimen did not hamper the wound healing process of the rabbit eye in comparison to
saline. These data did not conﬁrm the ones obtained with the nonvalidated ex vivo eye irritation test.
Keywords: cationic emulsions, cornea abrasion, in vivo model, wound healing, safety evaluation
Dry eye disease (DED) is a complex multifactorial
disease of the ocular surface that manifests by struc-
tural alterations of the cornea (keratitis) and the presence of
symptoms of discomfort of variable severity (scratchy and
dryness sensation, stinging or burning, pain, and eye red-
Patients with DED may also experience a blurred
vision, eye fatigue, and heavy eyelid sensations. The tear
ﬁlm (TF), whose normal function is to protect, hydrate, and
nurture the corneal cells and thus preserve vision’s quality,
is often altered and instable in DED patients, thus not able to
play its roles.
Artiﬁcial tears (ATs) represent the ﬁrst line
therapy for restoring the TF and for the management of the
symptoms and signs of DED patients.
ATs exist with different formulation strategies used to re-
store a healthy TF.
Aqueous-based AT formulations, with
or without gel-forming polymers (eg, carboxymethyl cellu-
lose, hydroxypropyl guar, and hyaluronic acid), are classi-
cally used in aqueous-deﬁcient DED,
ATs are used for evaporative DED.
For the temporary
relief of the less severe (ie, mild to moderate) symptoms,
patients are advised to instill one drop in the affected eye
when needed and up to 4 drops per day.
tion of the efﬁcacy and safety of most of the ATs was
performed in 1–3 month clinical trials in mild-to-moderate
DED patients treated with up to 4 instillations per eye and
SANTEN SAS, Novagali Innovation Center, Evry Cedex, France.
Iris Pharma, La Gaude, France.
ªPhilippe Daull et al. 2018; Published by Mary Ann Liebert, Inc. This Open Access article is distributed under the terms of the Creative
Commons License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any
medium, provided the original work is properly cited.
JOURNAL OF OCULAR PHARMACOLOGY AND THERAPEUTICS
Volume 34, Number 10, 2018
Mary Ann Liebert, Inc.
The management of the more severe symptoms generally
requests the need of prescription eye drop containing potent
anti-inﬂammatoryactives like cyclosporine,
However, depending on the severity of the
discomfort some patients might feel the need to instill ATs in
their eyes above the recommended dosing regimen of 4 eye
drops per day, with the risk of developing acute toxicity events.
The safety of the ATs is generally demonstrated during the
preclinical development stage of the ATs in in vitro and in vivo
relevant animal models with dosing regimens exceeding the
expected human posology. This approach usually allows for the
deﬁnition of a margin of safety (fordrugs under development),
which can be deﬁned as the range between the minimal thera-
peutic dose and the minimal toxic dose of a drug. However, it is
often not possible to precisely determine a margin of safety for
ATs, as for the latter the maximal dose assayed in the animal
ocular tolerability/toxicology tests is not high/extreme enough
for the development of adverse events to appear (and thus de-
ﬁne the minimal toxic dose) due to the inherent verygood safety
of unpreserved ATs.
With the idea of reducing the use of animal models for
toxicology and safety evaluations, alternative in vitro and ex
vivo models are being developed. Unfortunately, most of
them do not generate sufﬁciently reliable data to be validated
and used as good alternative test methods.
Recently, an ex
vivo eye irritation test (EVEIT) was developed and used as an
alternate assay model to evaluate the toxic effects of ATs on a
wounded corneal epithelium subjected to hourly (24 instil-
lations per day) instillations over a 3–6 day period. This
highly overdosed dosage regimen in the nonphysiological
experimental setting of the EVEIT assay suggests dramatic
ocular toxicity of the preservative-free cationic emulsion
Cationorm and the Purite
preserved Optive eye drops. The
goal of the present study was to evaluate the effects of these 2
eye drops in a rabbit model with similarly wounded corneas
and receiving 21 instillations (one eye drop every 45 min) per
day over a 3-day experimental period and, thus, assess the
reliability of the EVEIT assay data.
Thirty female New Zealand white rabbits (Hypham,
Roussay, France) of approximately 2–3 months of age were
used in this study. Iris Pharma Internal Ethics Committee
approved the protocol. All animals were treated according
to the Directive 2010/63/UE European Convention for the
Protection of Vertebrate Animals used for Experimental and
Other Scientiﬁc Purposes and to the Association for Re-
search in Vision and Ophthalmology (ARVO) statement for
the Use of Animals in Ophthalmic and Vision Research. All
animals were housed individually in standard cages under
identical temperature (18C–3C), humidity (45%–80%
relative humidity), and controlled enrichment conditions
and exposed to a 12-h light/12-h dark cycle in continuously
ventilated rooms (15 air volumes per hour). They received a
standard dry pellet diet and water ad libitum.
Experimental procedure and treatment groups
The animals were randomly assigned to 5 groups (n=6per
group) deﬁned as follows: a negative control group receiving
saline (0.9% NaCl), a toxic control group treated with a 0.02%
benzalkonium chloride (BAK) solution (in saline), and 3
treatment groups: Cationorm (Santen SAS, Evry, France),
Vismed (Horus Pharma, Saint-Laurent du Var, France), and
Optive (Allergan, Inc., Dublin, Ireland). Twenty microliters of
each eye drop was applied for up to 21 instillations (except on
the day of induction with 16instillations) per day (45 min apart)
in the right eye and started 1h after the completion of the
corneal abrasion procedure. The treatment period lasted 3 days.
The study design used in this experiment is presented in Fig. 1.
Corneal abrasion procedure
Rabbits were deeply anesthetized by an intramuscular in-
jection of a mixture of ketamine (35 mg/kg)/xylazine (5 mg/
kg), and local cornea anesthesia was performed by instillation
of a drop of 0.4% oxybuprocaine (Cebesine
Chauvin, Montpellier, France) in the study eye. Standardized
superﬁcial corneal abrasions (4 per right eye) were performed
with a classic biopsy punch (2mm in diameter) to a depth of
approximately 35 mm (Fig. 2). An analgesic (buprenorphine,
20 mg/kg, SC) was administered 30min before corneal abra-
sion and once a day during the following 2 days.
Corneal ﬂuorescein staining
and healing measurement
One drop of a 0.5% ﬂuorescein sodium solution (Fluor-
esceine Faure, 0.4-mL unit-dose vials, Novartis Pharma SAS,
FIG. 1. Schematic repre-
sentation of the experimental
design of the in vivo study.
2 DAULL ET AL.
France) was instilled into the inferior conjunctival sac using a
micropipette. The cornea was examined through a biomi-
croscope by light passing through a cobalt blue ﬁlter. Pictures
of cornea lesions (area stained by ﬂuorescein) were taken
using a CCD camera and analyzed using ImageJ software to
measure the size of the 4 abrasions on each cornea.
Local tolerability evaluation with Draize,
McDonald–Shadduck, and IVCM scales
Ocular examinations for clinical evaluations. Both eyes
were examined under a light source for cornea, conjunctiva,
and iris adverse reaction scoring according to Draize’s scale.
Slit lamp examinations of both eyes were performed to as-
sess cornea, conjunctiva, iris, and the inner parts of the
eye adverse reactions according to McDonald–Shadduck’s
scale. Figure 1 presents the schedule of the different ob-
servations performed during the experiment; slit lamp ob-
servations were performed at baseline and at the end of the
experiment, while ocular observations were also performed
each day before the ﬁrst and after the 10th and 21st instil-
lation of the day.
In vivo confocal microscopy
A laser scanning in vivo confocal microscope Heidelberg
Retina Tomograph (HRT II)/Rostock Cornea Module
(RCM, Heidelberg Engineering GmbH, Heidelberg, Ger-
many) was used to examine the cellular structure of the
cornea. Pictures (400 ·400 mm) of the superﬁcial and basal
epithelium, of the anterior and posterior stroma, and the
endothelium were taken in the center of the cornea with at
least one wounded area in the ﬁeld of the microscope.
Statistical analysis was performed with GraphPad Prism
software, and intergroup comparisons were performed by 1-
way or 2-way ANOVA followed by Tukey’s multiple com-
parison test. The signiﬁcance threshold was set at P<0.05.
Effects of overdosed ATs on the healing process
Four standardized lesions were performed in the central
part of the cornea (Fig. 2). The total abrasion area at day 1
(immediately after the debridement procedure) was approxi-
mately 15%, ranging from 14.8% –1.4% to 16.2% –0.5% for
the different groups (Fig. 3A). Twenty one eye drops per day
of various ATs were instilled for 3 days. At the end of the
treatment period (day 3), the size of the remaining lesion was
measured by ﬂuorescein staining (Figs. 2 and 3B). The re-
maining lesion size varied from 0.3% –0.5% to 0.9% –2.1%
for the 3 commercially available ATs and saline, with no
statistically signiﬁcant difference between them. Only the
0.02% BAK solution had a lesion at day 3 that remains vis-
ible with the naked eye (Fig. 2) following ﬂuorescein stain-
ing. The area of the remaining lesion represents 2.8%–1.3%
of the total cornea area. Corneal reepithelialization at day 3
was almost complete (from 94.4% –12.3% to 97.9% –3.5%)
FIG. 2. Illustration of the cornea defect during the ex-
periment. Corneal ﬂuorescein staining of abraded rabbit
eyes at the beginning of the treatment period (day 1) and
after 3 days of treatment (day 3). Color images available
online at www.liebertpub.com/jop
FIG. 3. (A) Percentage of cornea abrasion (–SD) at day 1
and day 3 for the different treatment groups. There is no
statistically signiﬁcant difference between the groups (2-
way ANOVA, Tukey’s multiple comparison test). (B) Per-
centage of reepithelialization (–SD) at day 3. *P<0.05 (1-
way ANOVA, Tukey’s multiple comparison test) versus
TOLERABILITY OF OVERDOSED ARTIFICIAL TEARS 3
following the treatment with saline and the 3 commercially
available ATs (Fig. 3B). Surprisingly, the 21 instillations per
day of a 0.02% BAK solution were not accompanied by a
worsening of the lesion, that is, an increase in the size of the
abraded area; the healing process remained effective, even
though it appeared to be slower.
Ocular tolerability of overdosed ATs
by diseased corneas
Examination of the ocular surface (cornea and conjunc-
tiva), as well as the inner parts of the eye (iris) and the
aqueous ﬂare, was performed, with a slit lamp and by IVCM
at baseline, during the treatment period, and at the end of the
experiment to characterize the potential side effects that may
result from the high number of daily instillations. Ocular ex-
amination of the cornea, conjunctiva, and iris before the ﬁrst
and after the 10th and 21st instillations of each day did not
reveal any major ﬁndings throughout the experiment. The
resulting mean score per group on the conjunctiva and cornea
using the Draize’s scale (Fig. 4) remains extremely low for all
treatments, including the 0.02% BAK solution. The only
ﬁndings (minor in intensity and duration) that affected the
conjunctiva: slight conjunctival discharge and redness present
in most of the eyes of each group. They were seen only on the
ﬁrst day of instillation just after the corneal abrasion. No
ﬁndings were observed on days 2 and 3 (Fig. 4). Slit lamp
biomicroscopic observations on day 3 revealed a slight con-
junctiva congestion and a slight iris hyperemia for one eye in
the Vismed group. The other ﬁndings affected the cornea.
Some opacities were observed for one eye in the saline group,
2 eyes in the Vismed and Optive groups, 4 eyes in the Ca-
tionorm group, and for all the eyes in the 0.02% BAK group
(Fig. 5). However, the overall mean score per group on the
McDonald–Shadduck’s scale remains low, even for the 0.02%
BAK group. There was no statistically signiﬁcant difference
between saline and the 3 commercially available ATs (Ca-
tionorm, Vismed, and Optive); however, saline, Vismed, and
Optive were signiﬁcantly different from the 0.02% BAK.
IVCM pictures of the superﬁcial and basal epithelium, the
anterior and posterior stroma, and the endothelium (Fig. 6) did
not reveal signiﬁcant differences between the AT groups and
the saline group. Superﬁcial epithelial cells appeared to be
relatively large with an irregular size, with some smaller cells
visible in the 0.02% BAK and saline groups. In a basal epi-
thelium that looked overall homogeneous within the different
groups, areas with irregular intercellular spaces were visible,
with hyper-reﬂective cells being more present in the 0.02%
BAK and Optivegroups compared to the other treatments. The
anterior stroma in the 0.02% BAK group presented numerous
hyper-reﬂective elongated spindle-shaped cells with a linear
cell body suggesting apoptosis. Fewer presumably apoptotic
cells can also be seen in the anterior stroma from the other
treatment groups. The posterior stroma and endothelium ap-
peared to be normal for all treatment groups.
Discussion and Conclusion
DED is a very common eye disease that affects millions
of people worldwide.
It manifests by symptoms of dis-
comfort associated with clinical signs of corneal and con-
junctival alterations. The vast majority of DED patients
experience mild-to-moderate signs and symptoms that are
generally treated by repeated instillations of ATs.
the patients are advised to instill ATs when necessary, that
is, upon feeling sensations of discomfort, the dosage regi-
men of ATs for the alleviation of DED symptoms rarely
exceeds 4 instillations per eye and per day.
for the management of very severe symptoms, more fre-
quent instillations of ATs might be needed on an acute basis
(eg, one drop per hour) before switching to prescription (Rx)
eye drops designed for the treatment of severe DED con-
An hourly dosing regimen in a 12-h awaken
FIG. 4. Mean Draize test score (–SEM) for the different
treatment groups before the ﬁrst and after the 10th and 21st
instillations at days 1, 2, and 3.
FIG. 5. Mean McDonald–Shadduck’s test score (–SEM) for
the different treatment groups at day 3 for corneal opacity degree
(A) and corneal opacity area (B).*P<0.05 (1-way ANOVA,
Tukey’s multiple comparison test) versus 0.02% BAK.
4 DAULL ET AL.
period per day would represent 12 eye drops per day, quite a
high burden for the patient. Indeed, an hourly dosing regi-
men is not sustainable for the patients in the long term,
especially when Rx medicine containing potent (and safe)
anti-inﬂammatory compounds, such as cyclosporine, de-
signed for the treatment of severe DED conditions in dry eye
patients that did not improve with ATs, are available and
were demonstrated to be effective following the instillation
of just one drop per day and per eye.
Recently, an ex vivo eye irritation assay was used to
compare commercially available ATs with the goal of ex-
ploring the toxicity of ATs.
To make sure that some toxic
events arise following the instillations of the ATs an hourly
dosage regimen (ie, 24 eye drops a day) was applied over
the 3-day experiment. The cultured half eye balls received
72 eye drops over a 3-day period. With such overdosing
toxicity side effects were indeed observed in this EVEIT
assay for Cationorm and Optive.
The aim of the present work was to explore in vivo the
potential side effects that may be induced on abraded rabbit
cornea epithelium by Cationorm and other commercially
available ATs following a high dosing regimen. Four small
and standardized abrasions were performed on the corneas of
rabbits’ right eye before receiving one eye drop every 45min,
that is, 21 instillations per day over a 3-day period (except on
the day of induction with 16 instillations). This design was
chosen to mimic in vivo the extreme instillation protocol (24
eye drops per day) used in the ex vivo irritation test, EVEIT,
and conﬁrm, or not, the relevance of the EVEIT assay as an
alternate toxicity test method for the evaluation of eye drops.
The data presented on Figs. 2 and 3 demonstrate that the
healing process in the rabbit is normal and that even one in-
stillation every 45 min (ie, 58 instillations over 3 days) of Ca-
tionorm for 3 days has no negative impact on abraded rabbit
eyes compared to saline instillations. Indeed, BID instillations
of cationic emulsions
were demonstrated to contribute to
the promotion of the healing process of abraded cornea in a rat
scrapping assay. The IVCM pictures (Fig. 6) conﬁrmed struc-
turally that the corneal epithelium did heal properly. In addi-
tion, in an in vivo rabbit acute toxicity assay, the animals
received 15 instillations over a 90-min period, that is, one in-
stillationevery 5 min for 90 min;no toxic reactions were found
following the applications of cetalkonium chloride (CKC)-
containing cationic emulsions.
During the development pro-
cess of the cationic emulsions (Cationorm, Ikervis), the 28-day
local tolerability studies (6 daily instillations over 28 days)
conﬁrmed that the cationic emulsions, with CKC as the cationic
agent, were very well tolerated by rabbit eyes.
safety proﬁle in animal models was also observed in patients
during the different clinical trials evaluating the safety and efﬁ-
cacy of the cationic emulsions.
Nosika trial, Cationorm vs. Vismed, 4 instillations per day over 3
months in patients with mild-to-moderate DED signs and
symptoms; 22.7% of the patients treated with Cationorm have a
complete cornea clearing (ie, a completely healed cornea) as soon
as 7 days after treatment initiation.
By comparison, Vismed
treatment reached this effectiveness (22.0%) only after 3 months
In addition, the annual update safety reports of
Cationorm also conﬁrmed that Cationorm is very well tolerated
by the altered and inﬂamed ocular surface of DED patients.
The improved wound healing and anti-inﬂammatory prop-
erties of the cationic emulsions demonstrated during clinical
trials (Nosika and Sansika trials) in patients were suggested
previously by in vitro studies in human corneal epithelial cells
and in vivo.
Indeed, in these early experiments, it was
noticed that the cationic emulsions were able to improve the
wound healing process (both in pace and ‘‘quality’’). The
reason for the wound healing clinical efﬁcacy of the cationic
emulsions was furtherexplored at the mechanistic level. Recent
studies demonstrated that CKC was a speciﬁc inhibitor of
protein kinase C (PKC) alpha
(Daull, 2018, in press). Re-
cently, Pﬂugfelder and collaborators (2010) demonstrated that
FIG. 6. IVCM representa-
tive pictures of the superﬁcial
and basal epithelium, the an-
terior and posterior stroma,
and the endothelium of the
cornea for the different treat-
ment groups. IVCM, in vivo
TOLERABILITY OF OVERDOSED ARTIFICIAL TEARS 5
PKCa‘‘inhibition’’ improved wound healing by reducing the
level of inﬂammatory cell inﬁltration in the wounded area with
PKCaknockout mice which have a ‘‘more rapid corneal epi-
thelial wound healing, perhaps due to a decreased neutrophil
Hence, the inhibition of PKCaby CKC might
explain why cationic emulsions like Cationorm and the Ikervis
vehicle did perform so well in the improvement of corneal
epithelium healing in the clinical trials.
Following the rationale for reducing the use of live animals
in toxicology studies, alternate toxicity test methods are being
developed. These new in vitro (and ex vivo) test methods are
particularly valuable for the scientiﬁc community and the so-
ciety in general. However, these alternate test methods used for
toxicity evaluation need an extended validation phase to make
sure that they produce reliable data that can conﬁdently be used
to adequately assess the toxicity of the tested eye drops or
Unfortunately, most of these alternate irritation
assays failed in their validation phase. Indeed, the interagency
coordinating committee on the validation of alternative meth-
ods (ICCVAM) and ICCVAM Ocular Toxicology Working
Group did not recommend the use of the bovine corneal opacity
and permeability, Cytosensor
Microphysiometer (CM), hen’s
egg test-chorioallantoic membrane, isolated chicken eye, and
isolated rabbit eye test methods to distinguish substances not
labeled as irritants.
In most of the cases the false positive rates
were high. Recently, Frentz et al. developed an EVEIT as an
alternative to the in vivo evaluation of toxicity or ocular toler-
So far, the EVEIT assay has not yet undergone
irritation test method by Abdelkader et al. in 2015
et al. in 2016
in their reviews.
In a direct comparison of the EVEIT assay and an in vivo
assay in rabbits (this study) based on the Draize test, the most
reliable eye irritation test, used for more than 70 years
drug development to address regulatory requirements for
safety testing, it appears that the EVEIT assay failed to ad-
equately predict the toxicity of compounds not classiﬁed as
irritant. In conclusion, the EVEIT as it is presently designed
and used appears not to be an adequate alternate test method
for safety testing of eye drops. Modifying its design so that
the EVEIT test method more closely mimics the eye’s normal
lubrication and physiology would probably improve its pre-
dictability and decrease its overprediction rate and position
this test method, once it has passed formal validation, as a
potential alternate toxicity assay for eye drops.
Author Disclosure Statement
No competing ﬁnancial interests exist.
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Received: March 22, 2018
Accepted: August 15, 2018
Address correspondence to:
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1, rue Pierre Fontaine
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TOLERABILITY OF OVERDOSED ARTIFICIAL TEARS 7