Ocular Drug Delivery System

Prepared by:
Pranjal A Wagh
Roll no. 458
Final Year B. Pharm
2020-21
Contents
• Introduction
• Anatomy of Human eye
• Mechanism of Ocular Drug Absorption
• Barriers for Ocular Delivery
• Factors affecting Intraocular bioavailability
• Drawbacks of traditional ophthalmic formulations
• Classification of Ocular Drug Delivery System
• Formulations of Ocular Drug Delivery System
• Evaluation parameters of Ocular Drug Delivery System
Introduction to Ocular Drug Delivery System
• For many decades, treatment of an acute disease or a chronic illness has been mostly accomplished by delivery of
drugs using various pharmaceutical dosage forms, including tablets, capsules, pills, suppositories, creams, ointments,
liquids, aerosols and injectables as drug carriers.
• These conventional ophthalmic formulations have many disadvantages which result into poor bioavailability of drug
in the ocular cavity.
• Recently, several new techniques for drug delivery are made which are capable of controlling the rate of drug
delivery, sustaining the duration of therapeutic activity or targeting the delivery of the drug to a tissue.
• These advancements have led to the development of several novel drug delivery systems that could revolutionize
the method of medication and provide a number of therapeutic benefits.
• The various approaches that have been attempted to increase the bioavailability and the duration of the
therapeutic action of ocular drugs can be divided into two categories.
• The first one is based on the use of sustained drug delivery systems, which provide the controlled and continuous
delivery of ophthalmic drugs. The second involves maximizing corneal drug absorption and minimizing precorneal
drug loss.
Anatomy of Human eye
The eyeball measures about 2.5 cm in diameter, only a small portion (about 1/6th part) of the globular eye is
exposed in front, the rest is hidden in bony socket of the orbit on a cushion of fat and connective tissue. The wall of
the human eyeball consists essentially of three layers: Fibrous tunic, Vascular tunic and Retina.
Fibrous tunic
• Fibrous tunic, the outermost coat of the eyeball, consists of the anterior cornea and posterior
sclera.
• The cornea is a transparent coat that covers the colored iris.
✓ Cornea mainly consists of the following structures from the front to back, (I) Epithelium,
(II) Bowman’s membrane, (III) Stroma, (IV) Descemet’s Membrane, (V) Endothelium.
✓ The cornea is 0.5 to 1mm in thickness and normally it possesses no blood vessels except
at the corneoscleral junction.
• The sclera, the “white” of the eye, is a layer of dense connective tissue made up densely of
collagen fibers and fibroblasts.
✓ The sclera covers the entire eyeball except the cornea.
• At the junction of the sclera and cornea is an opening known as the scleral venous sinus (canal of
Schlemm).
Vascular tunic
• This middle layer is mainly vascular, consisting of the choroid, ciliary body and iris.
• Choroid lines the posterior five-sixths of the inner surface of the sclera. It is very rich in blood vessels.
• Ciliary body is the anterior continuation of the choroids consisting of ciliary muscle and secretary epithelial
cells. The major function of the ciliary body is the production of aqueous humor.
• Systemic drugs enter the anterior and posterior chambers largely by passing through the ciliary body
vasculature and then diffusing in to the iris where they can enter the aqueous humor.
• The ciliary body is one of the major ocular sources of drug-metabolizing enzymes, responsible for drug
detoxification and removal from the eye.
• Iris is the visible colored part of the eye and extends interiorly from the ciliary body lying behind the cornea
and in front of the lens.
• The pigment granules of the iris epithelium absorb light as well as lipophilic drugs. This type of binding is
characteristically reversible, allowing release of drug overtime.
• As a result, the iris can serve as a reservoir for some drugs, concentrating and then releasing them for longer
than otherwise expected.
Retina
• The innermost layer is the retina, consisting of the essential nervous system responsible for vision.
• Retina lines the posterior three quarters of the eyeball and is the beginning of the visual pathway.
• The retina is situated between the clear vitreous humor in its inner surface and the choroids on
its outer surface.
• Retina consists of two distinct chambers, anterior and posterior.
Lens
• Behind the pupil and iris, within the cavity of the eyeball, is the lens.
• Protein called crystallins, arranged like the layers of an onion, make up the lens.
• The lens is held in place by the zonules, which run from the ciliary body and fuse into the outer
layer of the lens capsule.
• The lens tends to develop cataract or opacities with age, interfering with vision.
Interior of the eyeball
• The lens divides the interior of the eyeball into two cavities; Anterior cavity and Vitrous chamber.
• The anterior cavity consists of two chambers.
• The anterior chamber that lies between the cornea and the iris.The posterior chamber that lies behind the
iris and in front of the lens.
• Aqueous humor is formed by ciliary bodies and occupies the posterior and anterior chambers, having a
volume of about 0.2mL.
• The fluid is constantly generated by pigmented and non- pigmented epithelium of ciliary body.
• The Vitreous chamber is filled with a viscous fluid, vitreous humor, which is a viscoelastic connective
tissue composed of small amounts of glycosaminoglycans, including of hyaluronic acid and proteins such as
collagen.
Conjunctiva
• The conjunctiva membrane covers the outer surface of the white portion of the eye and the inner surface of
the eyelids. In most places it is loosely attached and thereby permits free movement of the eyeball, this makes
possible subconjunctival injection.
• The conjunctiva forms an inferior and a superior sac except for the cornea, the conjunctiva is the most
exposed portion of the eye.
Mechanism of Ocular Drug Absorption
Drugs administered by instillation must penetrate the eye and do so primarily through the cornea
followed by the non-corneal routes. These non-corneal routes involve drug diffusion across the
conjunctiva and sclera and appear to be particularly important for drugs that are poorly absorbed
across the cornea.
Corneal permeation
• The permeation of drugs across the corneal membrane occurs from the precorneal space.
Transcellular transport occurs i.e. transport between corneal epithelium and stroma.
• The productive absorption of most ophthalmic drugs results from diffusional process across
corneal membrane.
• Maximum absorption thus takes place through cornea, which leads the drug into aqueous humor.
• Outer epithelium has only access to small ionic and lipophilic molecules.
Non-corneal permeation
• Primary mechanism of drug permeation is diffusion across sclera & conjunctiva into intra ocular tissues.
• Although like cornea, the conjunctiva is composed of an epithelial layer covering an underlying stroma, the
conjunctival epithelium offers substantially less resistance than does the corneal epithelium.
• This route is however, not productive as it restrains the entry of drug into aqueous humor.
• This route is important for hydrophilic and large molecules, such as insulin and p-aminoclonidine, which have
poor corneal permeability.
Barriers for Ocular Delivery
• Drug loss from the ocular surface
After instillation, the flow of lacrimal fluid removes instilled compounds from the surface of eye. Another source of
non-productive drug removal is its systemic absorption instead of ocular absorption. Systemic absorption may take
place either directly from the conjunctival sac via local blood capillaries or after the solution flow to the nasal cavity.
• Lacrimal fluid-eye barriers
Corneal epithelium limits drug absorption from the lacrimal fluid into the eye. The corneal epithelial cells form tight
junctions that limit the paracellular drug permeation. Therefore, lipophilic drugs have typically at least an order of
magnitude higher permeability in the cornea than the hydrophilic drugs.
• Blood-ocular barriers
The eye is protected from the xenobiotics in the blood stream by blood-ocular barriers. These barriers have two
parts: blood-aqueous barrier and blood-retina barrier. The anterior blood-eye barrier is composed of the
endothelial cells in the uvea (The middle layer of the eye beneath the sclera. It consists of the iris, ciliary body, and
choroid).
This barrier prevents the access of plasma albumin into the aqueous humor, and also limits the access of hydrophilic
drugs from plasma into the aqueous humor. The posterior barrier between blood stream and eye is comprised of
retinal pigment epithelium (RPE) and the tight walls of retinal capillaries. Unlike retinal capillaries the vasculature of
the choroid has extensive blood flow and leaky walls. Drugs easily gain access to the choroidal extravascular space,
but thereafter distribution into the retina is limited by the RPE and retinal endothelia.
Factors affecting Intraocular bioavailability
• Inflow & outflow of lacrimal fluids
• Efficient naso-lacrimal drainage
• Interaction of drug with proteins of lacrimal fluid
• Dilution with tears
• Corneal barriers
• Active ion transport at cornea
Drawbacks of traditional ophthalmic formulations
1. They have poor bioavailability because of
a) Rapid precorneal elimination
b) Conjunctival absorption
c) Solution drainage by gravity
d) Induced lacrimation
e) Normal tear turnover
2. Frequent instillation of concentrated medication is required to achieve a therapeutic effect.
3. Systemic absorption of the drug and additives drained through nasolacrimal duct may result in undesirable side
effects.
4. The amount of drug delivered during external application may vary. The drop size of ocular medication is not
uniform and those delivered is generally not correct.
5. Presence of viscous vehicles can cause blurred vision.
Classification of Ocular Drug Delivery System
Formulations of Ocular Drug Delivery System
o Mucoadhesives
• Mucoadhesives are retained in the eye by virtue of non-covalent bonds established with the corneal conjunctival mucin
for extending pre-ocular residence time.
• They are basically macromolecular hydrocolloids with hydrophilic functional groups, such as hydroxyl, carboxyl, amide
and sulphate having capability for establishing electrostatic interactions.

o Phase transition system

• These are liquid dosage forms which shift to the gel or solid phase when instilled in the cul-de-sac.
• After converting into gel it remains in contact with the cornea of the eye for prolonged period of time due to which
drug elimination also slow down.
o Niosomes
• Niosomes are the vesicles, containing non-ionic surfactants, that can entrap both hydrophilic and lipophilic drugs
either in aqueous layer or in vesicular membrane made of lipid materials.
• It helps in preventing the metabolism of the drug by enzymes present at the tear/corneal surface.
• Vesicles prepared from self-assembly of hydrated non-ionic surfactants molecules are called niosomes.
• Niosomes and liposomes are equiactive in drug delivery potential and both increase drug efficacy as compared
with that of free drug.
• Niosomes are preferred over liposomes because the former exhibit high chemical stability and economy. These
types of vesicles were first reported in the cosmetic industries.
• Nonionic surfactants used in formation of niosomes are polyglyceryl alkyl ether, glucosyl dialkyl ether, crown
ether, polyoxyethylene alkyl ether, ester-linked surfactants, and steroid-linked surfactants and spans, and tweens
series.
Advantages of niosomes
• The vesicle suspension being water based offers greater patient compliance over oil based systems.
• Since the structure of the niosome offers place to accommodate hydrophilic, lipophilic as well as amphiphilic drug
moieties, they can be used for a variety of drugs.
• The characteristics such as size, lamellarity etc. of the vesicle can be varied depending on the requirement.
• The vesicles can act as a depot to release the drug slowly and offer a controlled release.
Structure of Niosome:

Types of Niosomes:
1. Small unilamellar vesicles (SUV, size 0.025-0.05 μm)

2. Multilamellar vesicles (MLV, size >0.05 μm)

3. Large unilamellar vesicles (LUV, size >0.10 μm)

Applications of niosomes
• Drug targeting:
Niosomes can be used to target drugs to the reticulo-endothelial system.

• Anti-neoplastic treatment

• Delivery of peptide drugs:

Use of niosomes to successfully protect the peptides from gastrointestinal peptide breakdown is being
investigated.

• Use in studying immune response

Due to their immunological selectivity, low toxicity and greater stability; niosomes are being used to
study the nature of the immune response provoked by antigens.

• Niosomes as carriers for haemoglobin

o Liposomes
• Liposomes are usually within the size range of 10 nm to 1 μm or greater.
• These vesicular systems are composed of aqueous core enclosed by phospholipid bilayers of natural or
synthetic origin.
• They have the ability to entrap hydrophilic compound in the aqueous compartment and to incorporate
hydrophobic molecule in the lipid bilayers.
• Majority of liposomal formulations utilize phosphatidylcholine (PC) and other constituents such as
cholesterol and lipid-conjugated hydrophilic polymers as the main ingredients. Incorporation of cholesterol
enhances the stability by improving the rigidity of the membrane.
• Stability of liposomes depends upon the various properties such as surface charge, size, surface hydration,
and fluidity of lipid bilayers.
• Surface charge determines interaction of liposomes with ocular membrane.
• Positively charged liposomes display better corneal permeation than the neutral and negatively charged
liposomes.
• Neutral liposomes upon systemic administration evade the elimination by reticuloendothelial system (RES).
• Amphiphilic nature of phospholipids allows these molecules to form lipid bilayers. This unique feature is
utilized for the preparation of liposomes.
Types of Liposomes:
Liposomes are structurally classified on the basis of lipid bilayers such as small unilamellar vesicles (SUVs) or
multilamellar vesicles (MLVs).
Furthermore, on the basis of size, liposomes are classified into small unilamellar vesicles (SUVs), giant unilamellar
vesicles (GUVs), and large unilamellar vesicles (LUVs).
Unilamellar vesicles are composed of single layer of lipid such as lecithin or phosphatidylglycerol encapsulating
aqueous interior core.
Multilamellar vesicle is composed of various layers of lipid bilayers. MLVs are metastable energy configuration having
different facets depending upon the polydispersity of the liposomal formulation.
Application of Liposomes

• Liposomes have been investigated for ophthalmic drug delivery since it offers advantages as a carrier system.
• It is a biodegradable and biocompatible nanocarrier. It can enhance the permeation of poorly absorbed drug
molecules by binding to the corneal surface and improving residence time.
• It can encapsulate both the hydrophilic and hydrophobic drug molecules. In addition, liposomes can improve
pharmacokinetic profile, enhance therapeutic effect, and reduce toxicity associated with higher dose.
• Owing to their versatile nature, liposomes have been widely investigated for the treatment of both anterior and
posterior segment eye disorders. Current approaches for the anterior segment drug delivery are focused on
improving corneal adhesion and permeation by incorporating various bioadhesive and penetration enhancing
polymers. However, in the case of posterior segment disorders, improvement of intravitreal half-life and targeted
drug delivery to the retina is necessary.
• Currently verteporfin is being used clinically in photodynamic therapy for the treatment of sub foveal choroidal
neovascularization (CNV), ocular histoplasmosis, or pathological myopia effectively.Verteporfin is a light-activated
drug which is administered by intravenous infusion.
o Nanoparticles
• Nanoparticles are solid particles of polymeric nature ranging in size from 10-1000 nm.
• The drugs are bound to small particles, which are then dispersed into aqueous vehicle(20).
• Due to very small in size these are not washed away with tears quickly.

o Pharmacosomes
• They are the vesicles formed by the amphiphilic drugs.
• Any drug possessing a free carboxyl group can be esterified to the hydroxyl group of a lipid molecule thus
generating an amphiphilic prodrug.
• These are converted to pharmacosomes on dilution with tear.
o Ophthalmic inserts
• Inserts are defined as a thin disks or small cylinders made with appropriate polymeric material and fitting into the lower or
upper conjunctival sac.
• Their long persistence in preocular area can result in greater drug availability with respect to liquid and semisolid
formulation.
• Ophthalmic inserts are elliptical flexible wafer, multilayered system consisting of drug as core surrounded by rate controlling
membrane and designed to be placed in cul-de-sac between sclera and eyelid.

Advantages of ophthalmic inserts Disadvantages of ocular inserts

• Increasing the contact time and thus improve bioavailability. • Initial discomfort, their movement around the
eye.
• Providing prolonged drug release and thus a better efficacy.
• Occasional inadvertent loss during sleep or
• Reduction of systemic side effects and thus reduces adverse effects. while rubbing the eye.
• Reduction of the number of administration and thus better patient compliance. • Interference with vision and a difficult
• Administration of an accurate dose in the eye and thus a better therapy. placement.
• Increased shelf life with respect to aqueous solutions.
• Exclusion of preservative, thus reducing the risk of sensitivity reactions.
• A possibility of incorporating various novel chemical technological approaches
such as pro-drugs, micro particulates, salts acting as buffers.
Classification of Ophthalmic inserts:
1. Insoluble ophthalmic inserts
2. Soluble ophthalmic inserts
3. Bio erodible ophthalmic inserts

1. Insoluble ophthalmic inserts

They have been sub-classified into: Diffusion Inserts E.g. ‘Ocuserts’, Osmotic Inserts and Contact lenses presoaked in
drug solution.

Diffusion Inserts (Ocuserts)

• Ocusert system is a novel ocular drug delivery system based on porous membrane.
• A central reservoir of drug is surrounded by the polymeric membrane, which allows a constant movement of the
drug in to the tissue by diffusion.
• A controlled rate of delivery is provided by interaction between the membrane molecule and the drugs.
• The first major important factor in the rate of drug release is the driving force, which is maintained by saturated
concentration of the drug in the reservoir.
Osmotic Inserts
• The osmotic inserts are generally composed of a centre part surrounded by a peripheral part and are of two types.
• Type 1
✓ The central part is composed of a single reservoir of a drug with or without an additional osmotic solute dispersed
throughout polymeric matrix, so that the drug is surrounded by the polymer as discrete small deposits.
✓ The second peripheral part of this inserts compresses a covering film made up of insoluble semi permeable
polymer.
✓ The osmotic pressure against the polymer matrix causes its rupture in the form of apertures.
✓ Drug is then released through these apertures from the deposits near the surface of the devices.
• Type 2
✓ The central part is composed of two distinct compartments.
✓ The drug and the osmotic solutes are placed in two separate compartments, the drug reservoir being surrounded
by an elastic impermeable membrane and the osmotic solute reservoir by a semi permeable membrane.
✓ The second peripheral part is similar to that of type 1.
✓ The tears diffuse into the osmotic compartment inducing an osmotic pressure that stretches the elastic membrane
and contracts the compartment including drug, so that the active component is forced to the single drug release
aperture.
Contact lenses presoaked in drug solution
• Contact lenses are shaped structures comprising covalently cross linked hydrophilic or hydrophobic polymer that
forms a three dimensional network or matrix capable of retaining water, aqueous drug solution or solid
components.
• Contact lenses are classified into 5 groups.
a) Rigid
b) Semi-rigid
c) Elastomeric
d) Soft hydrophilic
e) Bio-polymeric
• When contact lens (including 35-80% water) is soaked in solution, it absorbs the drug. Drug release depends
markedly on the amount of drug, the soaking time of the contact lens and the drug concentration in the soaking
solution.
• Advantages
1. Concerning the high cost of contact lenses it should be mentioned that disposable contact lenses have been
commercially available and it should be possible to incorporate a drug into such devices at an acceptable cost.
2. The contact lenses are the only class of ophthalmic inserts that have the ability to correct any refractive error
that the patient may have and thereby provide improved visual acuity, while the medication is being administered.
• Disadvantages
1. Cleaning and rinsing procedures can induce deterioration of the devices.
2. The cost of medicated contact lens is high.
2. Soluble ophthalmic inserts
• They can be broadly divided in to two types, the first one being based on natural polymers and the other one
synthetic or semi synthetic polymers.
I. Natural polymers
Natural polymer used to produce soluble ophthalmic inserts is collagen. The therapeutic agent is preferably
absorbed by soaking the insert in a solution containing the drug, drying and rehydrating it before use on the eye.
The amount of drug loaded will depend on the amount of binding agent present, concentration of the drug
solution in to which the composite is soaked as well as duration of soaking. As the collagen dissolves, the drug is
gradually released from the interstices between the collagen molecules.
II. Synthetic or semi-synthetic polymer
The soluble ophthalmic inserts, containing synthetic and semi-synthetic polymers, offer the additional advantages
of being of a generally simple design, of being based on products well adapted for ophthalmic use and easily
processed by conventional methods. The main advantage is decreased release rate, but still controlled by
diffusion.
3. Bioerodible ophthalmic inserts
• These are composed of matrix of homogenous dispersion of a drug coated with hydrophobic material, which is
substantially impermeable to the drug.
• The main components used for the production of this type of inserts are the so-called Bioerodible polymers i.e.,
materials that undergo hydrolysis of chemical bonds and hence dissolution.
• Bioerosion is defined here as the property of a material to innocuously disintegrate or breakdown from a unit
structure or entity, over a prolonged period of time, in response to the environment in the eye.
• When bioerosion process takes place geometrically of inserts is modified and it is difficult to adequately control
the release.
• The bioerodible polymers used in erodible inserts are carboxymethylcellulose, hydrophobic polyacrylic acids,
hydroxypropyl methylcellulose, and silicone based pro-polymers.
• There are three devices, which are marketed to date namely
✓ Lacrisert
✓ Soluble Ocular Drug Inserts (SODI)
✓ Minidisc.
Evaluation parameters of Ocular Drug Delivery
System
o Thickness:
• Thickness of the film is an important factor while considering its drug release from ocular delivery systems. If
thickness varies from one film to another, the drug release from the film also varies.
• It is measured using digital micro meter and dial caliper at different points and mean value is calculated.
o Surface pH:
• It is measured by pH paper or pH meter.
• Inserts are allowed to swell in a closed petri dish at room temperature for 30 min in 0.1 mL of distilled water.
The swollen devices are then removed and placed on pH paper to determine the surface pH. After 1 min the
color developed is compared with the standard colour scale.
o Weight uniformity:
• Weight variation between the formulated films can lead to difference in drug content and in vitro behavior.
• Evaluation is carried out by weighing 10 films by an electronic balance. The average weight and standard
deviation are calculated.
o Drug content uniformity:
• The cast film is cut at different places and tested for drug as per monograph spectrophotometrically.
• Average drug content of three films is determined usually.

o Tensile strength and percentage elongation at break:

• The tensile strength of ocuserts refers to tension or force required to tear off the insert apart into two pieces.
• To determine elongation and tensile strength, the film is pulled by means of a pulley system.
• Weights are gradually added and the weight necessary to break the film is noted as break force.

o Moisture uptake:
• The percentage moisture uptake test is carried out to check the physical stability or integrity of the film.
• Ocular films are weighed individually and placed in a desiccator containing 100 mL of saturated solution of sodium
chloride (~ 75 % humidity).
• After three days, films are taken out and reweighed; the percentage moisture uptake is calculated by using following
formula.
𝐹𝑖𝑛𝑎𝑙 𝑤𝑒𝑖𝑔ℎ𝑡 − 𝐼𝑛𝑖𝑡𝑖𝑎𝑙 𝑤𝑒𝑖𝑔ℎ𝑡
𝑃𝑒𝑟𝑐𝑒𝑛𝑡𝑎𝑔𝑒 𝑚𝑜𝑖𝑠𝑡𝑢𝑟𝑒 𝑢𝑝𝑡𝑎𝑘𝑒 = × 100
𝐼𝑛𝑖𝑡𝑖𝑎𝑙 𝑤𝑒𝑖𝑔ℎ𝑡
o In - vitro evaluation methods:

• Bottle method:
✓ In this, dosage forms are placed in the containing dissolution medium maintained at specified temperature and
pH.
✓ The bottle is shaken.
✓ A sample of medium is taken out at appropriate intervals and analyzed for the drug content.

• Diffusion method:
✓ Drug solution is placed in the donor compartment and buffer medium is placed in between donor and receptor
compartment. Drug diffused in receptor compartment is measured at various time intervals.

• Modified rotating basket method:

✓ Dosage form is placed in a basket assembly connected to a stirrer. The assembly is lowered into a beaker
containing buffer medium and 37ºC.
✓ Samples are taken at appropriate time intervals and analyzed for drug content.

• Modified rotating paddle apparatus:

✓ Here, dosage form is placed into a diffusion cell which is placed in the flask of rotating paddle apparatus.
✓ The buffer medium is placed in the flask and paddle is rotated at 50 rpm. The entire unit is maintained at 37ºC.
✓ Aliquots of sample are removed at appropriate time intervals and analyzed for drug content.
o In vivo release study:
• Ocuserts are placed into the lower cul-de-sac of rabbits.
• The inserts are inserted into one eye of seven rabbits at same time and another eye served as control.
• After certain time intervals, the inserts are carefully removed and analyzed for remaining drug content by UV
analysis.
• The amount of drug remaining is subtracted from initial drug content of inserts to find the amount of drug
released in the rabbit eye.

o Corneal residence time evaluation:

• Precorneal residence time of ocular drugs has been assessed by certain invasive techniques and non-invasive
techniques.
• These approaches however, require isolation of ocular tissues or the use of radioisotopes.

o Accelerated stability studies:

• These are carried out to predict the breakdown that may occur over prolonged periods of storage at normal shelf
condition.
• Here, the dosage form is kept at elevated temperature or humidity or intensity of light, or oxygen.
• Then after regular intervals of time sample is taken and analyzed for drug content. From these results, graphical
data treatment is plotted and shelf life and expiry date are determined.
o Sterility Tests:
• Sterility is one of the most vital requirements for an ophthalmic preparation.
• The tests for sterility are intended for detecting the presence of viable forms of microorganisms in ophthalmic
preparations.
• The principle governing these tests is that if the microorganisms are placed in a medium which provides nutritive
material and water, kept at a favourable temperature, the organisms will grow and their presence can be indicated
by turbidity in the originally clear medium.
• Sterility tests are therefore performed by the:
1. Membrane filtration technique
2. Direct-inoculation technique

o Leakage test:
• This test is mandatory for opthalmic ointments, which evaluates the intactness of the ointment tube and its seal.
• Ten scaled containers are selected, and their exterior surfaces are cleaned. They are horizontally placed over
absorbent blotting paper. Maintained at 60 ± 3 0 C for 8 h.
• The test passes if leakage is not observed from any tube. If leakage is observed, the is repeated with an additional
20 tubes.
• The test passes if not more than 1 tube shows leakage out of 30 tubes.