Leakage tests are used to test package integrity and detect leaks that could allow contamination. There are four main types of leakage tests: visual inspection, bubble test, dye test, and vacuum ionization test. Visual inspection is the simplest but least sensitive method, while dye test is widely used in industry. Clarity and particulate contamination tests are also important to ensure parenteral products are free of visible particles and meet sub-visible particle limits. The light obscuration and microscopic particle count tests are commonly used to detect sub-visible particles.

1. Leaker tests:
Leakage occurswhenadiscontinuityexistsinthe wall of apackage thatcanallow the passage
of gas under the action of a pressure or concentration differential existing across the wall. Presence of
capillarypores ortinycrackscan cause microbesorotherdangerouscontaminantstoenterthe ampoules
or package or mayleadto the leakage of contentstooutside.Thismaycause contaminationof the sterile
contents and also spoilage of appearance of the package. Changes in temperature during storage can
cause expansion and contraction of the ampoule or package and thereby causing interchange of its
contents if an opening exists.
Purpose:
Leakage test is employedto test the package integrity. It is employedto detect incompletely
sealedampoulesothattheymaybe discarded.Package integrityreflectsitsabilitytokeepthe productin
and to keep potential contamination out.
Types of leaker tests:
Leaker tests are 4 types
a) visual inspection
b) bubble test
c) dye test
d) vacuum ionization
a) Visual inspection
Visual inspectionisthe easiestleakertestmethodto perform.This methodis used
for the evaluation of large volume parenteral. To increase the sensitivity of the method the visual
inspectionof the sample containermaybe coupledwiththe applicationof vacuumtomake leakagemore
readilyobservable.Thismethodissimpleandinexpensive. Thisis lesssensitivemethodbutitssensitivity
is increased by applying pressure/vacuum.
b) Bubble test:
The test package is submerged in liquids. A differential pressure is applied on the
container. The container is observed for bubbles. Sometimes, surfactant added liquid is used for
immersion of test package. Any leakage is evident after the application of differential pressure as the
generation of foaming in immersion liquid. The method is simple and inexpensive. The location of the
leakscanbe observedinthismethod. Generationof adifferentialpositive pressure of 3psi inside the vial
and observation of any leakage using magnifying glass within a maximum test time of 15 minutes.
However, it is relatively insensitive and the findings are operator dependent and are qualitative. The
optimizedconditionscanbe achievedusingasurfactantimmersionfluidalongwiththe darkbackground
and High intensity lighting.
C) Dye test:

2. The testcontainerisimmersedinadye bath.Vacuumandpressureisappliedforsometime.The container
isremovedfromthe dye bathandwashed.The containeristheninspectedforthe presenceof dye either
visuallyorbymeansof UV spectroscopy.The dye usedmaybe of blue,green,yellowish-greencolor.The
dye testcan be optimizedbyuse of asurfactantandor a low viscosityfluidinthe dyesolutiontoincrease
the capillarymigrationthroughthe pores.The dye test is widelyacceptedinindustryandis approvedin
drug use.The test is inexpensive andisrequiresno special equipmentrequiredforvisual dye detection.
However, the test is qualitative, destructive and slow. The test is used for ampoules and vials.
D) Vacuum ionization test:
Vacuumionizationtestisusefulfortestingleakage inthe vialsorbottled
sealedundervacuum.Thistestisusedfortestingof the lyophilizedproducts.Highvoltage,highfrequency
field is applied to vials which to cause residual gas, if present to glow. Glow intensity is the function of
headspace vacuumlevel.The blue glowisthe indicative of vacuumwhile the purple glow indicativeof no
vacuum. The sensitivity of the method is not documented. This test is rapid and is nondestructive test.
However, the proteins present in the test sample may be decomposed. This method is used for the
lyophilized vials of biopharmaceuticals.
2) CLARITY TEST (PARTICLE CONTAINMENT TEST):
Clarity is a relative term, itsmean a clear solution having
a highpolishconveysto the observerthatthe product is of exceptional qualityandpurity.Claritytestis
carriedout to checkthe particulate matterinthe sample.Itispracticallyimpossiblethateveryunitof lot
is perfectly free from visible particulate matter,that is, from particles that are 30 to 40 micrometer and
large in size.
PRINCIPLE:
Thistestisperformedtocheckthe particulate contaminationof injectionsandinfusionsconsists
of extraneous, mobile and undissolved particles, other than gas bubbles, unintentionally present in the
solution.
USP limits for large volume infusion
if particle size is 10 um (or) larger/ml then Particle limit is 50.
if Particle size is 25 um (or) larger/ml then particle limit is 5.
TYPES OF TEST:
Particulate matter can be detected in parenteral product by two methods,
1. Test for visible particles
2. Test for sub visible particles
1) Test for Visible particles:
Visual inspection by naked eye:

3. The testis intendedtoprovideasimple procedureforthe visual assessmentof the qualityof parenteral
solutions as regards visible particles. In visual inspection, each injectable is inspected visually against
white andblackbackgrounds. The white backgroundaidsindetectionof darkcoloredparticles. The light
or reflective particles will appear against the black background.
Some visual-enhancingaidscanincrease the efficiency. A magnifyinglensat2.5 × magnificationsetatthe
eye level facilitatesthe inspection. Microscopicexaminationenhancesdetectionof particulate matterin
injectables. Visual inspection gives the qualitative estimation of the particulate matter. Acceptance
Standards is that each container checked must not contain any visible particulate matter.
2. Test for Sub visible particle:
This test isperformedto checkparticulate contaminationof injectionsandinfusions
consistsof extraneous,mobile un-dissolvedparticles,otherthangas bubbles,unintentionallypresentin
the solutions.
This is further divided into two methods:
1. Method 1 ((Light Obscuration Particle Count Test)
2. Method 2 (Microscopic Particle Count Test)
Whenexamininginjectionsandinfusionsforsub visible particles,Method1 is preferably applied. Butin
case of preparations having reduced clarity or increased viscosity,the test is carried out according to
Method 2. e. g. Emulsions, colloids, and liposomal preparations.
METHOD 1 (Light obscuration particle count test):
Principle:
This test is based on the principle of light blockage which allows an automatic determinationof the size
of particles and the number of particles according to size.
Apparatus:
An electronicparticle countingsystemthatusesa lightobstructionsensorwitha suitable feedingdevice
is used.
General precautions:
The test is carried out under conditions limiting particulate contamination, preferably in a laminar flow
cabinet.Verycarefullywashthe glassware andfiltrationequipmentused,exceptforthe membranefilters,
with a warm detergent solution and rinse with abundant amounts of water to remove all traces of
detergent.
Immediatelybefore use,rinse the equipmentfromtoptobottom, outside andtheninside,withparticle-
free waterR. Take care nottointroduce airbubblesintothe preparationtobe examined,especiallywhen
fractions of the preparation are being transferred to the container in which the determination is to be
carried out.
PROCEDURE:

4. Mix the contents of the sample by slowly inverting the container 20 times successively.
Clean the outer surfaces of the container opening using a jet of particle-free water R, avoiding any
contaminationof the contents. Eliminategasbubblesbyappropriate measuressuchasallowingtostand
for 2 min or sonicating. For large-volume parenterals, single units are tested. For small-volume
parenterals less than 25mL in volume, the contents of 10 or more units are combined in a cleaned
containertoobtaina volume of notlessthan25 ml. Small-volume parenteralshavingavolume of 25 mL
or more may be tested individually.
For large-volume parenteralsorfor small-volume parenteralshavingavolume of 25 mL or more, fewer
than10 unitsmaybe tested,basedonanappropriate samplingplan. Remove 4portions,eachof notless
than 5 mL, and count the number of particles equal to or greater than 10 μm and 25 μm. Calculate the
mean number of particles for the preparation to be examined.
Evaluation:
Test 1.A – Solutionsforinfusionorsolutions forinjectionsuppliedincontainerswithanominal content
of more than 100 mL: The preparationcomplieswiththe testif the average numberof particlespresent
in the unitstesteddoesnot exceed25 per millilitre equal toor greaterthan 10 μm and doesnot exceed
3 per millilitre equal to or greater than 25 μm.
Test1.B – Solutionsforinfusionorsolutionsforinjectionsuppliedincontainerswithanominal contentof
lessthan100 mL The preparationcomplieswiththe testif the averagenumberof particlespresentinthe
unitstesteddoesnotexceed6000 percontainerequal toorgreaterthan10 μm and doesnotexceed600
per container equal to or greater than 25 μm. For preparations supplied in containers with a nominal
volume of 100 mL,applythe criteriaof test1.B. If the average numberof particlesexceedsthelimits,test
the preparation by the microscopic particle count test.
METHOD 2 (Microscopic Particle Count Test):
Use a suitable binocular microscope, filter assembly for retaining particulate contamination and
membrane filterforexamination. The microscopeisequippedwithanocularmicrometercalibratedwith
an objective micrometer, a mechanical stage and, 2 suitable illuminators.
ocular micrometer
is a circular diameter graticule and consists of a large circle divided by crosshairs into quadrants,
transparent and black reference circles 10μm and 25μm in diameter at 100 magnifications, and a linear
scale graduated in 10 μm increments. The large circle is designated the graticule field of view (GFOV).
Mechanical stage
capable of holding and traversing the entire filtration area of the membrane filter.
Illuminators:
two illuminators are required.
an episcopic brightfield illuminator internal to the microscope,

5. the other is an external, focusable auxiliary illuminator.
Filter assembly:
The filterassemblyforretainingparticulatecontaminationconsistsof afilterholdermadeof glassorother
suitable material, and is equipped with a vacuum source and a suitable membrane filter.
Membrane filter: The membrane filter is of suitable size, black or dark grey in color, non-gridded or
gridded, and 1.0 μm or finer in nominal pore size.
Precautions:The testis carriedout in laminar-flow cabinet. Verycarefullywashthe glassware andfilter
assemblyused,exceptforthe membrane filter,withawarmdetergentsolutionandrinse withabundant
amounts of water to remove all traces of detergent. Air bubbles should not be present.