PARAMETERS FOR EVALUATION OF PARENTERAL PRODUCTS

1. Meera Sharma
13HPH026

2. Evaluation Tests
A) Sterility test
B) Clarity test
C) Leakage test
D) Pyrogen test
E) Assay

3. Sterility Test
 It is done by detecting the presence of viable forms of bacteria, fungi and
yeast in parenteral products.
 The test for Sterility must be carried out under strict aseptic conditions in order
to avoid accidental contamination of the product during test.
 All glassware required for the test must be sterile.
 The test for Sterility may be carried out either by:
a) Membrane filtration method
b) Direct inoculation method

4. PRINCIPLE:
If bacteria or fungi are placed in a medium which provide nutrition & water, & kept at a
favourable temperature, organism will grow & their presence can be indicated by their
turbidity in the clear solution.
STEPS INVOLVED IN STERILITY TESTING :
1. Selection of the sample
2. Selection of the quantity of product to be used
3. Method of testing
4. Observation & results

5. Selection of the sample
 Sample must be representative of the whole of bulk material & lot of final
containers.
 Random sampling is taken from the final containers.
 As per IP 2007 the guideline for minimum number of items recommended to
be tested are:

6. No. of items in the batch Minimum no. of items recommended to
be tested
1. Injectable preparations
Not more than 100 containers 10% or 4 containers whichever is greater
More than 100 but not more than
500 containers
10 containers
More than 500 containers 2% or 20 containers whichever is less
2. Ophthalmic or other non-injectable
preparations
Not more than 200 containers 5% or 2 containers whichever is greater

7. More than 200 containers 10 containers
3. Surgical dressings
Not more than 100 packages 10% or 4 packages whichever is greater
More than 100 but not more than
500 packages
10 packages
More than 500 packages 2% or 20 packages whichever is less
4. Bulk solids
Less than 4 containers Each container
More than 4 containers but not more
than 50 containers
20% or 4 containers whichever is greater
More than 50 containers 2% or 10 containers whichever is greater

8. Selection of the quantity of products
to be used
 Depends mainly on the volume or weight of the container.
 Minimum samples to be used in each culture medium in the test for
sterility are:
Quantity of each container Minimum quantity to be used in each
culture medium
For liquid
Less than 1ml Whole contents of the container
1ml or more but less than 40ml Half the contents of a container
100ml or more 10% of the contents of a container but less
than 20ml
For solids
Less than 50mg The whole contents of a container
50mg or more but less than 300mg Half the contents of a container
300mg or more 100mg

9. Methods of testing
 Membrane filtration method
 Direct inoculation method

10. Membrane filtration method (METHOD 1):
 Membrane filtration Appropriate for
 Filterable aqueous preparations
 Alcoholic preparations
 Oily preparations
 Preparations miscible with or soluble in aqueous or oily (solvents with no
antimicrobial effect)

11. Media used for membrane filtration method:
FLUID THIOGLYCOLLATE MEDIUM :
 Specific role of some ingredients primarily intended for the culture of aerobic &
anaerobic bacteria.
 Incubation of the media: 14 days at 30 -35°C
SOYA-BEAN CASEIN DIGEST MEDIUM
 Primarily intended for the culture of fungi.
 Incubation of the media: 14 days at 20 -25°C

12. Membrane filter 0.45μ porosity
Filter the test solution
After filtration remove the filter
Cut the filter in to two halves
First halves (For Bacteria) Second halves (For Fungi)
Transfer in 100 ml culture media
(Fluid Thioglycollate medium)
Incubate at 30-350 C for not less then 7 days
Transfer in 100 ml culture media
(Soyabeans-Casein Digest medium)
Incubate at 20-250 C for not less then 7 days
Observe the growth in the media Observe the growth in the media
Steps:

13.  Suitable for samples with small volumes
 Volume of the product is not more than 10% of the volume
of the medium
 Suitable method for aqueous solutions, oily liquids,
ointments an creams
 Direct inoculation of the culture medium suitable quantity
of the preparation to be examined is transferred directly
into the appropriate culture medium & incubate for not
less than 14 days.
H.P.I
Direct inoculation method (METHOD 2):

14. Observation And Results
Culture media is examined during and after at the end of incubation. The following observations are
possible:
• No evidence of growth → passed the test for sterility
• There is evidence of growth → Re-testing is performed for same no. of sample, volume & media
as in original test → No evidence of growth → Passed the test for sterility.
• There is evidence of growth isolate → & identify the organism.
• Re-testing is performed with twice no. of sample
No evidence of growth → Pass the test for sterility.
There is evidence of growth → Failed the test for sterility

15. Clarity Test
 It is performed to ensure that the parenterals are free from visible foreign particles. Each parenteral
preparation in its final container is subjected individually to a visual inspection to exclude the
possibility of foreign particles.
 The unlabelled containers are held by the neck against strongly illuminated black ( for dark
particles)& white screen(for light colour particles).
 The contents of the container are slowly inverted & rotated ,then examined. . It may be dangerous
when the particle size is larger than R.B.C. & may block the blood vessel. This type of products are
immediately rejected from the batch.
 The limit test for particulate matter is prescribed in I.P. 1996 (A- 125)
 Applicable for: 100 ml or more volume containers of single dose IV given by IV infusion.
 Not applicable for: Multi-dose injections Single dose SVP Injectable solutions constituted from
sterile solids .

16. Sources of particulate matter:
Intrinsic contamination:
 Originally present in products e.g. Barium ions may react or leach with Sulphur ion which are already
present in formulation may produce barium sulphate crystals.
Extrinsic contamination
 Material comes from outside or environment e.g. coming off the material from body & cloths of
person Entry of particle from ceiling , walls & furniture May be in the form of cotton, glass rubber,
plastics, tissues, insect fragments, bacterial contamination, dust, papers etc…

17. Methods of monitoring particulate matter contamination :
 Methods of monitoring particulate matter contamination
 Visual method, Coulter counter method, Filtration method, Light blockage method
1. Visual method: Simple method Filled container are examined against strong illuminated screen by
holding neck & rotating it slowly or inverted it to keep out the foreign matter.
2. Coulter counter method: It is used for detection of particles less than 0.1 micrometer in diameter.
Based on electrode resistance. Sample is evaluated between two electrode & if particle found the
resistance of electrode is increased.

18. 3. Filtration method: It is used for counting the particles in hydraulic fluids. Sample is passed
through a filter & the material collected on the surface of the filter is evaluated under
microscope.
Disadvantage: Skilled & trained person is required
4. Light blockage method: Used for hydraulic oils Allows stream of fluid under test to pass
between a bright white light source & photoiodide sensor.

19. Seal packaging / Leaking test
 The sealed ampoules are subjected to small cracks which occur due to rapid temperature changes or due to
mechanical shocks.
 Vials & bottles are not suitable for this test because the sealing material used is not rigid.
Filled & sealed ampoules
Dipped in 1% Methylene blue solution
Under negative pressure in vacuum chamber
Vacuum released colored solution enter into the ampoule
Defective sealing

20. Pyrogen Testing
 Pyrogen = “Pyro” (Greek = Fire) + “gen” (Greek = beginning).
 Fever producing, metabolic by-products of microbial growth and death.
 Bacterial pyrogens are called “Endotoxins”. Gram negative bacteria produce more potent
endotoxins than gram + bacteria and fungi.
 Endotoxins are heat stable lipopolysaccharides (LPS) present in bacterial cell walls, not present in
cell-free bacterial filtrates
 Stable to at least 175oC; steam sterilization ineffective
 Water soluble; monomer unit of LPS can be 10,000 Daltons (1.8 nm) so endotoxins can easily pass
through 0.22μm filters
 Sources: Water (main), raw materials, equipment, process environment, people, and protein
expression systems if using gram negative bacteria.

21. Principle:
 Rabbits are used to perform this test because their body temp increases when pyrogen are introduced into their
bodies by parenteral route
 3 healthy adult rabbits of either sex, each weighing NLT 1.5 kg are selected
 Do not use any rabbit
 having a temp higher than 39.8 o C
 Showing temp variation >0.2 o C between two successive reading in the determination of initial temp
 Same test is performed within 7 days of actual test
 Animal showing temp increase over 0.6 o C should be removed from pyrogen testing

22. Method :
 Dissolve the substance being examined in, or dilute it with a pyrogen free saline solution
 Warm the liquid being examined to approx. 38.5o C temp before injection
 The volume of injection is NLT 0.5ml/kg & NMT 10ml/kg of body weight
 Withhold water during test
 Clinical thermometer is inserted into the rectum of rabbit to record body temp
 2 normal reading of rectal temp are should be taken prior to the test injection at an interval of half an hr
& its mean is calculated- initial temp
 The solution under test is injected through an ear vein
 Record the temp of each rabbit in an interval of 30 min for 3 hrs
 The difference between initial temp & maximum temp is recorded- taken as response

23. No. Of rabbits Individual
temperature rise(℃)
Temperature rise in
group(℃)
Test
3 rabbits 0.6 1.4 passed
If above test not
passed
3+5 rabbits taken
0.6 3.7 passed
If above test do not pass then test is performed again
If the above test do not pass then the sample is said to be pyrogenic
Interpretation of result

24. Bacterial endotoxin (LAL) test )
 To detect or quantify endotoxins of gram-ve bacterial origin
 Reagent: Amoebocyte lysate from horseshoe crab (Limulus polyphemus or Tachypleus
tridentatus).
 The name of the test is also Limulus amebocyte lysate (LAL) test
• Mechanism of LAL Test:
The test is based on the primitive blood-clotting mechanism of the horseshoe crab
enzymes located with the crab's amebocyte blood cells endotoxin
Initiation of an enzymatic coagulation cascade
Proteneous gel

25.  Test:
 Equal Volume of LAL reagent and test solution (usually 0.1 ml of each) are mixed in a depyrogenated test-
tube
 Incubation at 37°C, 1 hour
 Remove the tube - invert at (180°) observe the result
 Pass-fail test

26. LAL test
Three different techniques:
1. The gel-clot technique - gel formation
2. The turbidometric technique - the development of Turbidity after cleavage of an endogenous
substrate
3. The chromogenic technique - the development of color after cleavage of a synthetic peptide-
chromogen complex
Advantages of LAL test
Fast - 60 minutes vs. 180 minutes
Greater Sensitivity ,Less Variability
Much Less False, Positives ,Much Less Expensive
 Alternative to Animal Model, cheaper,
Particularly useful for:
Radiopharmaceuticals and cytotoxic agents
Blood products
Water for injection

27. Commercially derived LAL reagents :
Bleeding adult crabs into an anticlotting solution washing and centrifuging to
collect the amoebocyte lysate in 3% NaCl lysate is washed and lyophilized for
storage activity varies on a seasonal basis and standardization is necessary.

28. Assay
 Assay is performed according to method given In the monograph of that parental preparation
in the pharmacopoeia
 Assay is done to check the quantity of medicament present in the parenteral preparation