This document outlines the procedure for conducting an acute eye irritation/corrosion test of a substance using albino rabbits. The objective is to determine the degree of eye irritation or corrosion caused by a test substance. A single dose of the substance is applied to one eye of each rabbit, while the other eye serves as a control. Lesions to the conjunctiva, cornea, and iris are observed and graded at various time intervals over 72 hours. Based on the grading scores of the test eye compared to the control eye, the sensitization potential of the substance can be determined.

1. ExperimentNo. 11
Determination ofacute eye irritation/
corrosionof a test substance
Mr. Vishal Balakrushna Jadhav
Assistant Professor (Pharmacology)
GES’s Sir Dr. M. S. Gosavi COPER, Nashik-5
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2. Overview of Discussion
• Objective
• Principle
• Requirements
• Procedure
• Observation table
• Result and interpretation
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3. Objective
To perform acute eye irritation/ corrosion of a test substance.
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4. Principle
Albino rabbit is the preferred animal for testing ocular
preparations. The basic principle involved in the test is after
application of single dose of test drug to one of the eye of the
rabbit, the degree of eye irritation/corrosion determines the
effect of test substance.
Following pretreatment with a systemic analgesic and induction
of appropriate topical anesthesia, the substance to be tested
is applied in a single dose to one of the eyes of the
experimental animal; the untreated eye serves as the control.
The degree of eye irritation/corrosion is evaluated by scoring
lesions of conjunctiva, cornea, and iris, at specific intervals.
Other effects in the eye and adverse systemic effects are also
described to provide a complete evaluation of the effects. The
duration of the study should be sufficient to evaluate the
reversibility or irreversibility of the effects. 4

5. Animals showing signs of severe distress and/or pain at any
stage of the test or lesions should be humanely killed, and
the substance assessed accordingly.
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6. Requirements
 Animals: Albino rabbits
 Drugs: Buprenorphine (0.01 mg/kg), 0.5% proparacaine
hydrochloride or 0.5% tetracaine hydrochloride
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7. Procedure
Animals
 Select young adult albino rabbits of both sexes. Examine both the
eyes of animals carefully and if they have any ocular defects, corneal
injury and eye–irritation then exclude from the study.
 Select and house the animals individually. Maintain the room
temperature at 20±3°C with relative humidity of 30%-70% and provide
artificial light for maintaining 12 hours light and dark cycle. Provide
conventional laboratory diets with unrestricted supply of water ad
libitum to all animals. Initially test should be conducted by using one
animal. If adverse effect is not seen in the preliminary test, then
continue with two more rabbits.
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8. Doses of drug and application procedures
 For liquids: A dose of 0.1 ml is used. Instill the drug directly.
 For solids: 0.1 ml v/v or not more than 100 mg. Ground the test
substance to fine powder, compact the powder gently then measure
the volume.
 For aerosols: Dose estimate can be made by using weighing paper.
Spray the test substance on to the paper by placing directly in front of
the rabbit eye. The weight increase of the paper is an approximate
amounts prayed into eye. The dose of volatile material is calculated by
measuring weight of the spray bottle before and after removal of test
substance. Spray the aerosols in one burst of about one second from
10 cm distance directly in front of the eye. At the time of spray, rabbit
should be held open.
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9. Testing method
 Buprenorphine 0.01 mg/kg is given through subcutaneous route 60
minutes before application of test substance, to provide analgesic
effect in order to avoid/minimize pain caused by test substance.
Apply 1 or 2 drops of topical ocular anaesthetic (0.5% proparacaine
hydrochloride or 0.5% tetracaine hydrochloride) before 5 minutes of
test drug application to both eyes to normalize pain caused by the
test drug.
 Then place the test substance in the conjunctival sac of one of each
animal by gently pulling the lower lid away from the eye ball. The
eye lids are then smoothly held together for about one second to
prevent drug loss. The untreated eye will be considered as control.
 If a test animal shows any signs of pain during the test period,
additional analgesia called as rescue dose (0.03 mg/kg) of
buprenorphine should be given and the same would be repeated
for every 8 hours if the same condition persists. 9

10. If any clinical signs of pain and distress or if any ophthalmic lesions will
be shown after eight hours of drug treatment, then give
buprenorphine 0.01 mg/kg in every 12 hours with meloxicam 0.5
mg/kg SC in every 24 hours until that condition will be stabilized.
 Don’t wash the eyes of the animals for at least 24 hours following
instillation of test drug except in case of solids and immediate
corrosive substances. In such cases satellite groups may be used
(extra animals, preferably 2 rabbits) and conditions of washing
should be documented carefully.
 Observe the animals carefully for 24 hours. Terminate the
experiment if animal shows continuous signs of pain and distress.
Observe the reversibility of the test drug effect for 21 days (in case
of mild/moderate lesion). If the reversibility will be seen before 21
days, then terminate the experiment (if no lesions will be seen
even after 3 days).
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11. Clinical observations
Evaluate the presence or absence of ocular lesions after one hour of
application of test substance. Evaluate daily for first 3 days in a
specified time at least two times daily, with a minimum of 6 hours
between observations and note down the observations.
Animals with severe corneal perforation, ulceration, blood in the
anterior chamber, absence of light reflex, grade 4 corneal opacity,
ulceration of conjuctival membrane or necrosis should be killed
humanely.
The grading of ocular reaction is given in the below table and the
observations are noted accordingly for 1, 24, 48 and 72 hours of
post-instillation of test substance.
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12. 12
Table- Grading of ocular lesions

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19. Observation table
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Parts of eye
Grading of eye after test substance
instillation at different time intervals
Control eye
(in hours)
Testeye
(in hours)
1 24 48 72 1 24 48 72
Cornea
Iris
Conjunctivae
Lids/nictatingmembrane
(Chemosis- eye irritation)

20. Result and interpretation
Comparison of ocular lesion grading between control eye and
test eye estimates the sensitization potential of test drug.
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21. ANY QUESTION???
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