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Oxymetazoline Ototoxicity in a Chinchilla Animal Model
Sam J. Daniel, Olubunmi V. Akinpelu, Sofia Sahmkow, W. Robert J. Funnell and Fadi Akache
Otolaryngology -- Head and Neck Surgery 2012 146: 114 originally published online 16 August 2011
DOI: 10.1177/0194599811419082
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Original Research—Otology and Neurotology
Oxymetazoline Ototoxicity in a Chinchilla
Animal Model
Sam J. Daniel, MD, MSc, FRCSC1,2,
Olubunmi V. Akinpelu, MD, MSc2, Sofia Sahmkow, MD1,
W. Robert J. Funnell, PhD1, and Fadi Akache, MSc1
Sponsorships or competing interests that may be relevant to content are disclosed at the end of this article.
Abstract
Objective. To investigate possible ototoxic effects of a onetime application of oxymetazoline drops in a chinchilla
animal model with tympanostomy tubes.
Study Design. A prospective, controlled animal study.
Setting. The Research Institute of the Montreal’s Children
Hospital, McGill University Health Centre.
Subjects and Methods. Ventilation tubes were inserted in
both ears of 12 animals. One ear was randomly assigned to
receive oxymetazoline drops (0.5 mL). The contralateral ear
did not receive any drops, serving as a control ear.
Outcome Measures. Distortion product otoacoustic emissions were measured bilaterally for a wide range of frequencies (between 1 and 16 kHz) before and 1 day after the
application of oxymetazoline in the experimental ears. Two
months later, the animals were sacrificed and all cochleae
were dissected out and processed for scanning electron
microscopy.
Results. In this established chinchilla animal model, the measured distortion product otoacoustic emission amplitudes and
the morphological appearance on scanning electron microscopy were similar for both control and experimental ears.
Conclusion. Oxymetazoline did not cause ototoxicity in a
chinchilla animal model 2 months after a single application
via a tympanostomy tube.
Otolaryngology–
Head and Neck Surgery
146(1) 114–118
Ó American Academy of
Otolaryngology—Head and Neck
Surgery Foundation 2012
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DOI: 10.1177/0194599811419082
http://otojournal.org
million operations performed yearly in North America.1-3 It
is common practice in many pediatric centers to use oxymetazoline drops (off-label) to control bleeding after a myringotomy and tube insertion or to unblock clots in ventilation
tubes.4 Oxymetazoline has also been used off-label to prevent otorrhea after PE tube insertion in some centers.5,6 To
date, only one study has assessed the ototoxicity of oxymetazoline, using a model with an osmotic pump implantation
delivering the product slowly over a 2-week period. The
aim of this study is to evaluate the safety of a one-time
application of oxymetazoline drops during tympanostomy
tube insertion in our established chinchilla animal model
simulating the clinical procedure.
Materials and Methods
The study was performed on 14 female chinchillas
(Chinchilla laniger) under the supervision of the McGill
University Animal Care committee that approved and monitored the protocol. These animals had commercial food
and water ad libitum and had a mean body weight of 500
6 75 g. Ventilation tubes were inserted in both ears of all
animals. In 12 animals, 1 ear was randomly assigned to
receive 0.5 mL of oxymetazoline (Drixoral, Schering
Plough Canada) at the time of ventilation tube insertion;
the other ear received no drops and served as control for
that particular animal. Two animals were used as a positive
control group. These received 0.5 mL of gentamicin sulfate
(Garasone, Schering Plough Canada), in 1 randomly
selected ear, to demonstrate the validity of the model.
Ototoxicity was expected in the ear under test in this group
to confirm that the drops indeed reached the cochlea
through the method described here.
Keywords
oxymetazoline, ototoxicity, tympanostomy tubes, scanning
electron microscopy
Received September 27, 2010; revised July 8, 2011; accepted July 14,
2011.
M
yringotomy with ventilation tube placement has
become one of the most common surgical procedures performed on children, with more than 1
1
2
Department of Otolaryngology, Head and Neck Surgery, McGill University
Montreal Children’s Hospital, Montreal, Quebec, Canada
Corresponding Author:
Sam J. Daniel, MD, MSc, FRCSC, Department of Otolaryngology, Head and
Neck Surgery, McGill University, Montreal Children’s Hospital, 2300 Tupper
Avenue, Montreal, Quebec, Canada H3H 1P3
Email: [email protected]
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Daniel et al
115
Sample Size Estimation
Based on an initial pilot study, setting a at 0.05, a minimum
absolute difference of 15, and a power of 0.80, a sample
size of 8 animals was deemed to be sufficient. We used 12
animals, considering the possibility of unexpected events
leading to animal loss during the study.
Surgical Procedure
The animals were anaesthetized using intraperitoneal
injections of ketamine (35 mg/kg) and xylazine (5 mg/kg)
and then positioned on their sides to allow for examination of the external auditory canal and tympanic membrane. Any debris or wax seen was cleared off using a
curette. With a speculum in the external auditory canal
and under an operating microscope (Zeiss, Germany), an
incision was made in the anterior, inferior quadrant of the
tympanic membrane. Afterward, a Reuter Bobbin tube
(1.14 mm wide, Medtronic Xomed Inc, Jacksonville,
Florida) was inserted in the eardrum. This procedure was
carried out on both ears of each of the animals.
Oxymetazoline Application
The head of the animal was positioned such that the external auditory canal of the randomly selected experimental
ear faced upward. Then 0.5 mL of oxymetazoline drops was
instilled directly over the opening of the ventilation tube.
The head was maintained in that position for 5 minutes.
ethanol solutions, 30% (15 minutes), 50% (15 minutes),
70% (15 minutes), 80% (15 minutes), 95% (15 minutes),
and 100% (15 minutes). Finally, they were critical-point
dried, mounted on stubs, and coated with gold in a sputter
coater. The samples were analyzed under a field-emission
scanning electron microscope (Hitachi S4700). The control
and experimental micrographs were compared visually with
regard to the shape of the stereocilia, outer hair cell loss,
and inner hair cell loss using the method described by
Korver.7
Statistical Analysis
The difference in DPOAE amplitudes between the control
ear and the treated ear was calculated for each animal, at
each frequency, before and after oxymetazoline application. We also calculated the 95% confidence intervals for
each of the differences reported both at baseline and post
oxymetazoline.
Results
Gentamicin Group
The 2 ears that were given Garasone (gentamicin) as a positive control group had severe damage to the inner and outer
hair cells and loss of architecture of the stereocilia, indicating that in this model drops applied through the tympanostomy tubes reached the inner ear in sufficient quantity to
cause damage. Both gentamycin and oxymetazoline are
marketed in aqueous forms.
Assessment of Ototoxicity Using the DPOAE Test
DPOAEs
We used the Smart distortion product otoacoustic emission (DPOAE) high-frequency software/hardware package
(Intelligent Hearing Systems, Miami, Florida). The tests
were performed in a quiet environment at all times.
Thermal stability of the animal was maintained at 36°C
with an infrared lamp at 2 feet above the operating table.
All tests were done using inhalational anesthesia induction with 5% isofluorane and maintenance with 2% isofluorane. The otoacoustic emissions were recorded for
both ears between 1 and 16 kHz. Two-tone stimuli at 55
and 65 dB SPL were emitted with a frequency ratio (F1/
F2) of 1.22 and averaged 32 times. The 2F1-F2 DPOAE
amplitude was used in assessing hearing function.
Otoacoustic emission measurements were used to compare the response of the control and tested ears before
(baseline) and 1 day after oxymetazoline application.
Baseline DPOAEs were similar in both control and experimental ears, showing that the experimental and control ears
were similar at the onset of the experiment. Table 1 shows
the amplitudes obtained for control and experimental ears at
low, middle, and high frequencies at baseline and post treatment, while Figure 1 shows the apparent similarities in the
values obtained for both groups. The 95% confidence intervals for the means of the differences between the control
and experimental ears at baseline and post oxymetazoline
treatment for each frequency are shown in Table 2. These
do not reveal any significant difference between the treated
ears and untreated ears in each animal.
Assessment of Ototoxicity Using Scanning Electron
Microscopy
Two months after the instillation of oxymetazoline, all animals were sacrificed by an overdose of anesthetic using 40
mg/kg of pentobarbital. After decapitation, the cochleae
were removed quickly and washed in a saline solution.
They were then fixed in 1% glutaraldehyde for 1 hour and
left in a 3% glutaraldehyde solution for 3 days at 4°C. The
bone covering the scala vestibuli and the cochlear duct was
removed. The specimens were then dehydrated in a series of
Scanning Electron Microscopy
Twenty four cochleae were processed (12 control, 12
experimental). Two cochleae (1 experimental and 1 control
from 2 different animals) were rejected for poor processing.
The scanning electron microscopy analysis of all remaining
cochleae did not show any damage in the structure of the
inner ear. Both control and experimental ears had no discernible hair cell loss in the basal turns. The outer and inner
hair cell layers were intact, there was no loss of hair cells,
and the integrity of the stereocilia was preserved, indicating
that oxymetazoline did not cause any hair cell damage. The
basal turns of the cochleae were studied, as this is where
most ototoxicity would manifest itself if present. Figures 1
and 2 show representative electron microscopy sections of
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116
Otolaryngology–Head and Neck Surgery 146(1)
Table 1. Mean Distortion Product Otoacoustic Emission Changes in the Control and Experimental Ears
1
Control ears
F2, kHz
1.1
Baseline, dB
20.2
Post treatment
0.8
SD
2.5
Change
1
Experimental ears
F2, kHz
1.1
Baseline, dB
21.2
Post treatment
20.2
SD
3
Change
1
2
3
4
5
6
7
8
9
10
11
12
1.3
21.5
20.5
2
1
1.55
0.7
2.7
1.5
2
1.85
4.8
7.8
2
3
4.42
12.3
14.3
2
2
5.24
20.8
23.8
0.5
3
6.24
24.2
27.2
0.2
3
7.42
26.3
30.3
1
4
8.84
22.1
24.1
1
2
10.5
19.7
22.7
1.8
3
12.5
17.1
19.1
1
2
14.8
18.2
20.2
0.8
2
1.3
20.9
2.3
2
3.2
1.55
4.6
6.6
1
2
1.85
7.2
11.2
0.4
4
4.42
14.1
17.1
2
3
5.24
21.6
23.1
1
1.5
6.24
25.3
25.9
0.1
0.6
7.42
29.5
34.5
0.5
5
8.84
23.6
24.6
3
1
10.5
22.1
24.1
0.5
2
12.5
19.2
22.2
2
3
14.8
20.6
23.6
1
3
Abbreviations: SD, standard deviation.
Figure 1. Representative electron microscopy section of outer
hair cells from a cochlea exposed to oxymetazoline revealing
normal structures and architecture.
outer hair cells from oxymetazoline and gentamicin exposed
cochleas, respectively.
Discussion
Oxymetazoline HCl in aqueous solution is an antihistamine
decongestant that has been described by some authors as
having antibiotic properties with no observed ototoxic
effects.5,6 Drixoral contains oxymetazoline HCl 0.05% in
aqueous solution with nonmedicinal ingredients including
benzalkonium chloride, edetate disodium, propylene glycol,
and water. Being an adrenergic a-agonist and a directly
acting sympathomimetic, Drixoral is used as a vasoconstrictor to relieve nasal congestion.8 The sympathomimetic action
of oxymetazoline constricts the smaller arterioles of the nasal
passages, producing a prolonged decongestant effect for up to
12 hours.8,9 This property is most likely responsible for its
hemostatic effect at the myringotomy incision site. It is not
uncommon to have some bleeding at the time of myringotomy incision. Although this is rarely significant, controlling
it can prolong the time of surgery, and its occurrence can
later lead to blockage of the PE tube.10 The prophylactic use
of oxymetazoline or the related xylometazoline to prevent the
occurrence of blocked tubes was the practice of 13% of otolaryngologists in a recent survey looking at trends in methods
used to prevent and treat blocked PE tubes.11 Oxymetazoline
has also been used in human subjects to reduce the occurrence of posttympanostomy otorrhea, which is a common
complication of PE tube insertion.5 In a recent animal study,
oxymetazoline was shown to possess antimicrobial properties,
inhibiting middle-ear pathogens; no associated ototoxicity
was reported.6
Off-label use of oxymetazoline raises some concerns
regarding its safety within the inner ear.12 With a molecular
weight of 296.84 g/mol, oxymetazoline will easily cross the
round-window membrane (RWM), as it is known that substances whose molecular weights are less than 500 g/mol will
rapidly pass through the RWM.13 Miller et al14 demonstrated
a reduction of up to 60% in cochlear blood flow when the
cochlea was exposed to epinephrine either topically or systemically. Similarly, a 25% reduction in cochlear blood flow
was found when phenylephrine was applied to the RWM of
gerbils.15 However, the use of phenylephrine in human subjects as a middle-ear decongestant to prevent PE tube
obstruction did not produce hearing loss clinically.16
Our chinchilla animal model has been used previously to
demonstrate the severe ototoxic and vestibulotoxic effects
of gentamicin and the safety of dexamethasone eardrops.17
Our current results clearly indicate that a one-time application of oxymetazoline at the time of ventilation tube insertion does not lead to demonstrable ototoxicity either
functionally or morphologically. This corroborates the earlier claim made by Isaacson et al.6 Although extrapolations
from animal studies to humans should be done carefully, the
chinchilla animal model is a very sensitive model for toxicity due to ototopical drugs. The thickness of its round
window is only 10 to 14 mm, compared with 40 to 70 mm
in humans, thereby allowing a much easier penetration of
the toxic agents into the cochlea.18 It can be deduced, therefore, that oxymetazoline, being safe through the thinner
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Daniel et al
117
Table 2. Differences between DPOAE Amplitude Recorded in Control versus Experimental Ears at Baseline and Post Oxymetazoline
Treatment for Each Animal across All Tested Frequencies
F2, kHz
1.1
1.3
1.55
1.85
4.42
5.24
6.24
7.42
8.84
1.5
12.5
14.8
Mean of Difference at Baseline
SD
95% CI
Mean of Difference Post Oxymetazoline
SD
95% CI
1.06
0.7
1.2
20.7
21.4
20.8
20.6
2.2
1.8
1.5
2.6
1.9
5.6
4.8
6.3
6.4
5.1
6.5
7.1
6.0
5.7
5.5
6.2
4.9
63.17
62.72
63.57
63.63
62.89
63.68
64.02
63.40
63.23
63.12
62.27
62.76
2.33
1.89
2.3
21.8
20.9
21.7
21.2
3.1
2.2
1.9
1.8
1.7
6.9
7.6
8.1
7.9
9.5
8.2
7.7
6.8
8.5
9.1
6.5
7.3
63.91
64.31
64.59
64.48
65.38
64.65
64.65
63.85
64.82
64.53
63.68
64.14
Abbreviations: CI, confidence interval; SD, standard deviation.
Conclusion
Oxymetazoline (Drixoral) ear drops are sometimes used off
label to stop bleeding after tympanostomy tube insertion or
to unblock a clotted tube. When tested in a chinchilla
animal model, the oxymetazoline drops did not produce any
damage to the outer hair cells demonstrable by either
DPOAE measurements or SEM findings. Further study of
the effects of oxymetazoline on cochlear blood flow and on
the electrophysiological activities of the cochlear hair cells
would help in making conclusive statements on safety.
Author Contributions
Figure 2. Representative electron microscopy section of outer
hair cells from a cochlea exposed to Garasone, revealing altered
architecture and damaged stereocilia.
RWM of the chinchilla, may be also safe in the much
thicker human RWM. In our study, oxymetazoline was
applied once after placement of the ventilation tube to
mimic the clinical procedure whereby patients would
receive drops of oxymetazoline as a hemostatic agent or to
unblock the tympanostomy tube. It is expected that the
drops will be present for several minutes only, but their
proximity to the RWM allows them to diffuse into the inner
ear. This was shown in our positive-control experiment in
which severe damage was noted in the outer hair cells,
demonstrating that the route of administration was adequate
for the drug to get to the RWM. The oxymetazoline used in
the animals described in this study was procured from sterile bottles sold over the counter (Drixoral). Since oxymetazoline is not commercially available as eardrops, the risk of
contamination remains an issue. Although the pH of oxymetazoline is slightly acidic, we did not observe any symptoms
in the animals to suggest pain or discomfort following oxymetazoline application.
Sam J. Daniel, conception and design of the study, analysis and
interpretation of data, helping write and revise the article critically
for important intellectual content, final approval of the version to be
published; Olubunmi V. Vakinpelu, acquisition and interpretation of
the data, drafting the article, final approval of the version to be published; Sofia Sahmkow, acquisition and interpretation of the data,
drafting the article, final approval of the version to be published;
W. Robert J. Funnell, interpretation and analysis of the data, participated in the drafting of the article, final approval of the version to
be published; Fadi Akache, acquisition of data, participated in the
drafting the article, final approval of the version to be published.
Disclosures
Competing interests: None.
Sponsorships: None.
Funding source: Canadian Institute of Health Research (CIHR)
and the Canadian Foundation for Innovation (CFI) (only role is
funding research grant).
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