The document summarizes key tests conducted on parenteral products, including sterility, pyrogen, and bacterial endotoxin tests. The sterility test detects any microorganisms in the product using culture media and incubation. Samples are selected randomly and inoculated directly or via membrane filtration. Two weeks of incubation allows any growth to be observed. The pyrogen test detects fever-causing endotoxins which may be present. The rabbit pyrogen test injects samples into rabbits and monitors their temperature increase. The bacterial endotoxin (LAL) test uses an extract of horseshoe crab blood cells that clots in response to endotoxins. These tests help ensure parenteral products are free of

1. Evaluation of Parenterals
Presented by
D.Harshali
PE/2017/307
NIPER Hyderabad
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2. Parenteral product requirements
 Must be sterile
 Free from pyrogens
 Should be isotonic
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3. Tests for parenterals
 Finished product quality control tests
 Mainly seven tests for paenterals
1. Sterility test
2. Pyrogen test
3. Leaker test
4. Particulate test
5. Clarity test
6. Closure integrity test
7. Content uniformity or weight variation test
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4. Sterility test
 Sterility is defined as freedom from the presence of viable
micro-organisms
 Sterility test is defined as the microbiological test applied to sterile product to
show that the product manufactured and processed under specification guided by
cGMP
 It is destructive test so it is impossible to test every item for sterility
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5.  If bacteria or fungi placed in the medium which provide nutrition &
Water and kept at a favourable temperature, organism will grow & their
Presence can be detected by their turbidity in the clear solution
 Steps involved in sterility testing
1. Selection of the sample
2. Selection of the quantity of the product to be used
3. Method of testing
4. Observation & Results
Principle of the test
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6. Mechanisms of sterilization
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Sterilization
Physical
methods
Chemical
methods
Heat Filtration Radiation
Dry heat Moist heat Ionizing Nonionizing

7. Mechanisms of sterilization
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D-Value( Decimal Reduction time) – Time in minutes at any defined temp to
destroy 90% of viable organisms
 Z- Value (Thermal destruction time) – Number of degrees of temperature change
to produce tenfold change in D-value
 F- value – Lethality value
1. Dry heat sterilization
 For thermostable substances ( above 140°c)
 Act by oxidizing the proteins
a) Tyndallization
 Applicable for drugs unstable at 150°c
 Heating 80-100°c for 1 hr in 3 successive days
b) Pasteurization
1. Holder pasteurization- Milk heated at 145°F (62.8°c) held for 3o min & quickly
cooled

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c) Hot air oven
Used for glasswares, metalware, anhydrous oils
Temperature(°c) Time(min)
170 60
160 120
150 150
140 180
2. Moist heat sterilization
 More rapid & efficacious
 Applicable for rubbers closures, ampoules, syringes, surgical dressings
 Denaturation & coagulation of proteins
 Autoclave conditions

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Temperature(°c) Stream pressure Ib/sq
.inch
Holding
time(min)
115-118 10 30
1121-124 15 15
126-129 20 10
135-138 30 3
3. Radiation sterilization
 For thermolabile materials
 Act by damaging nucleic acids
a) Ionizing radiation
 X-rays & Gamma rays cause mutations & destroy microbes by stopping
reproduction
 Creates free H+, OH, OOH radicals
b) Nonionizing radiation
UV –light (253.7nm) cause damage to DNA by dimerization of adjacent thymine
molecules

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4. Fitration sterilization
 For thermolabile materials
a) Depth filters- For sterilization of gases
b) Membrane filters-
Pore size= 0.45 Um( Millipore grade HA)
0.22Um ( Millipore grade GS)
 Integrity & performance is evaluated by Bubble point pressure method
Sterilization methods Biological indicators D-value
Dry heat Bacillus atrophaeus, B.subtilis
var niger
NLT-2.5
Moist heat B.Stearothermophillus NLT-1.5
Ethylene oxide B.Atrophaeas NLT-2.5
Ionizing radiation B.Pumilus 0.15-0.20
Filtration Pseudomonas diminuta -

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12.  Sample must be representative of the whole of the bulk materials & lot of final
containers
 Random sampling is taken from the final container
 Minimum sample size related to batch size ( BP 2001)
Selection of the sample
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13. Selection of the sample
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14. Selection of quantity of the product to be used
 Depends mainly on the volume or the weight of the container
 Minimum samples to be used in each culture medium in the test for sterility
are: (BP 2001)
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15. Methods used for sterility testing
 Methods used
1. Membrane filtration 2. Direct inoculation
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16. Media used for sterility testing
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1. Fluid thioglycolate medium
For aerobic and anaerobic bacteria
Incubation of the media: 14 days at 30-35°c
2. Soyabean casein digest medium
For culture of fungi
Incubtion media- 14 days at 20-25°c
 Nutritive properties of media
1. Aerobic : Staphylococcus aureus, Bacillus subtilis, Pseudomonas
aeruginosa
2. Anaerobes : Clostridium sporogenus
3. Fungi : Candida albicans, Aspergillus niger

17. Steps involved in membrane filtration
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18. Direct inoculation method
 Suitable for samples with small volumes
 Volume of the product is not more than 10% of the
volume of the culture media
 Suitable for aqueous solutions, oily liquids.
Creams, ointments
 Appropriate quantity of the preparation to be
examined is transferred directly to the culture medium
and incubate for not less than 14 days
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19. Observation and Result
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 No evidence of growth- Passed the test for sterility
 Evidence of growth- Re-testing is performed foe the same number
of sample, media and volume – if no evidence of growth then passed
then test for sterility
 If evidence of growth then isolate and identify the organism
Retesting is performed with twice the no of sample
1. No growth-passed the test
2. If growth occurs-Failed the test for sterility

20. Pyrogen testing
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 Introduction
 Pyro-(Greek-Fire), gen (beginning)
 Fever producing metabolic by-products of microbial growth and death
 Bacterial pyrogens called endotoxins gram negative bacteria produce more potent
than gram positive
 Characteristics of endotoxins
1.Thermostable
2. Water soluble
3. Unaffected by bactericides
4. Non volatile

21. Pyrogen testing
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 Physiological effects of pyrogen
1. Elevate circulating levels of inflammatory cytokines
2. produces fever, blood coagulation, hypotension. Lymphopenia
3. shock
4. characterized by cardiovascular dysfunction
5. vasodilation
6. vasoconstriction
7. endothelium dysfunction
8. Multiple organ dysfunction or failure

22. Pyrogen testing
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1. Rabbit test 2. LAL test

23. Rabbit test
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Why the Rabbit ?
1. Reproducible pyrogenic responce
2. Other species not predictable responce
3. Similar threshold pyrogenic response to the humans
4. Economic
 Requirements
 Healthy rabbits of weight 1.5 kg
 Newziland Albino or Belzium white strain

24. Rabbit test
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 Preliminary test (Sham test)
 Performed for conditioning and training the rabbits
 IV injection 10mL/Kg body weight of sterile pyrogen free saline solution
 Rectal temp is recorded
 Any animal showing temp variation of 0.6°c is nat used in main test
 Main test
 Carry out the test in a group of 3 rabbits
Sample preparation- Dissolve substance to be tested in a pyrogen free saline
solution warm it to 38.5°c before administration

25. Rabbit test
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Procedure
Inject the solution under examination into the marginal ear veins of each rabbit within
10 min ( Inj volume NLT-0.5mL/Kg & NMT-10mL/Kg body wt)
Record the rectal temp of each animal at ½ hr intervals for 3hrs after injection
The difference between the initial temp & the highest temp recorded for the rabbit is
taken as its responce

26. Rabbit test
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Result

27. Bacterial Endotoxin test (LAL test)
 Limulus - genus of horseshoe crab
Amoebocyte - blood cells
 Lysate - blood extract
 Developed by F. Bang and J. Levin in 1960's
 The method is based on the F. Bang's studies who observed in 1956 that gram-
negative bacteria, even if killed, cause the blood of the American horseshoe crab
(Limulus polyphemus) to turn into a semi-solid mass
 American or japanese horseshor crab (Limulus polyphemus) are used
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28. Bacterial Endotoxin test (LAL test)
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 Principle of the test
Addition of solution containing endotoxin to the solution of lysate produce
turbidity
 Rate of reaction depends on the conc of endotoxins, PH, temp
 Reference standard is the freeze dried
 The test is based on the primitive blood clotting mechanism of horse shoe
crab

29. Bacterial Endotoxin test (LAL test)
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The endotoxin limits for pharmaceutical product is calculated by
formula K/M:
Where, where K is the threshold pyrogenic dose of 5 EU/kg, 0.2 EU/kg
for intrathecal injection, and M is the maximum dose in units/kg
 The accepted limits of endotoxins in pharmaceutical products

30. Bacterial Endotoxin test (LAL test)
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 Three techniques
There are three basic methods commercially available and currently approved by FDA
for end-product release testing:
1.The gel-clot, based on gel formation:
 The formation of a gel-clot indicates the presence of endotoxin in a sample .
 The maximum sensitivity is 0.015 EU/mL
2. The turbidimetric test:
Based on the development of turbidity after cleavage of an endogenous substrate.
Tubidimetric systems depend upon an increasing conc of insoluble coagulin released as
the test progresses. The maximum sensitivity is 0.001 EU/mL
3. The chromogenic assay:
 Based on the development of color after cleavage of a synthetic chromogen
complex. The maximum sensitivity is 0.005 EU/mL

31. Bacterial Endotoxin test (LAL test)
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Advantages
1. Invitro test
2. More sensitive
3. Quantitative result
4. Less time consuming
5. Less expensive
 Limitations
1. Specific for gram negative pyrogens only
2. Clotting enzymes are heat labile. PH sensitive
 Applications
1. Pharmaceuticals - Parenteral dosage forms,LVPs, SVPs
2. Biologicals - Blood products, plasma fractions, vaccines
3. Medical device- Nebulisers
4. Validation of dry heat sterilization
5. Diagnosis of disease caused by gram negative bacteria

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