BACKGROUND
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.1
REQUIREMENTS
Animals: Albino rabbits
Drugs: Buprenorphine (0.01 mg/kg),
0.5% proparacaine hydrochloride or 0.5% tetracaine hydrochloride
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.
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 amount sprayed 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.
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 subject 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. 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).
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.
Table 1: Grading of ocular lesions.
| Parts of the eye with observation | Grading |
| Cornea (Opacity: degree of density readings should be taken from most dense area)* | |
| No ulceration or opacity | 0 |
| Scattered or diffuse areas of opacity (other than slight dulling of normal lustre); details of iris clearly visible | 1 |
| Easily discernible translucent area; details of iris slightly obscured | 2 |
| Nacrous area; no details of iris visible; size of pupil barely discernible | 3 |
| Opaque cornea; iris not discernible through the opacity | 4 |
| Iris | |
| Normal | 0 |
| Markedly deepened rugae, congestion, swelling, moderate circumcorneal hyperaemia; or injection; iris reactive to light (a sluggish reaction is considered to be an effect | 1 |
| Hemorrhage, gross destruction, or no reaction to light | 2 |
| Conjunctivae (Redness refers to palpebral and bulbar conjunctivae; excluding cornea and iris) | |
| Normal | 0 |
| Some blood vessels hyperaemic (injected) | 1 |
| Diffuse,crimson colour; individual vessels not easily discernible | 2 |
| Diffuse beefy red | 3 |
| Chemosis (Swelling refers to lids and/or nictating membranes) | |
| Normal | 0 |
| Some swelling above normal | 1 |
| Obvious swelling, with partial eversion of lids | 2 |
| Swelling, with lids about half closed | 3 |
| Swelling, with lids more than half closed | 4 |
Observation table.
| Parts of eye | Grading of eye after test substance instillation at different time intervals | |||||||
| Control eye (in hours) | Test eye (in hours) | |||||||
| 1 | 24 | 48 | 72 | 1 | 24 | 48 | 72 | |
| Cornea | ||||||||
| Iris | ||||||||
| Conjunctivae | ||||||||
| Lids/nictating membrane (Chemosis) | ||||||||
CONCLUSION
Comparison of ocular lesion grading between control eye and test eye estimates the sensitization potential of test drug.
REFERENCES
1. OECD Guideline For The Testing Of Chemicals. Available at: http://www.oecd.org/chemicalsafety/testing/48108995.pdf. Accessed on 10 February 2016.
Also read:
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- Analgesic activity study of drugs by tail-flick method
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