The efficacy of antimicrobial preservation of a pharmaceutical preparation on its own or, if necessary, with the addition of a suitable preservative has to be ascertained during the development of the product. The primary purpose of adding antimicrobial preservatives to dosage forms is to prevent adverse effects arising from contamination by micro-organisms that may be introduced inadvertently during or subsequent to the manufacturing process. However, antimicrobial agents should not be used solely to reduce the viable microbial count as a substitute for good manufacturing procedures. There may be situations where a preservative system may have to be used to minimise proliferation of micro-organisms in preparations that are not required to be sterile.

1. EVALUATION OF EFFICACY OF
ANTIMICOBIAL PRESERVATIVE
SAURAV BHANDARI
ASSISTANT PROFESSOR
DEPT. OF QUALITY ASSURANCE
ISF COLLEGE OF PHARMACY
WEBSITE: - WWW.ISFCP.ORG
EMAIL: BHANDARISAURAV89@GMAIL.COM
ISF College of Pharmacy, Moga
Ghal Kalan, GT Road, Moga- 142001, Punjab, INDIA
Internal Quality Assurance Cell - (IQAC)
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2. The efficacy of antimicrobial preservation of a pharmaceutical preparation on
its own or, if necessary, with the addition of a suitable preservative has to be
ascertained during the development of the product.
The primary purpose of adding antimicrobial preservatives to dosage forms is
to prevent adverse effects arising from contamination by micro-organisms that
may be introduced inadvertently during or subsequent
to the manufacturing process.
However, antimicrobial agents should not be used solely to reduce the viable
microbial count as a substitute for good manufacturing procedures.
There may be situations where a preservative system may have to be used to
minimise proliferation of micro-organisms in preparations that are not required
to be sterile.
GENERAL CONSIDERATIONS 2

3. The following tests are provided to demonstrate, the effectiveness of any
added preservatives, during the shelf lives of the preparations to ensure that
the antimicrobial activity has not been impaired by storage in multiple dose
products made with aqueous bases or vehicles,.
parenteral,
otic,
nasal,
ophthalmic,
oral and
Topical
The tests apply only to the product in the original, unopened container in
which it was supplied by the manufacturer.
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GENERAL CONSIDERATIONS

4. The test consists of
1.Challenging the preparation in its final container with a prescribed inoculum
of suitable micro-organisms,
2.Storing the inoculated product at a prescribed temperature,
3.Withdrawing samples from the container at specified intervals of time and
counting the organisms in the samples removed.
The preservative properties of the product are considered adequate if,
1.In the conditions of the test, there is a significant fall or
2.No increase in the number of micro-organisms in the inoculated preparation
after storage for the times and at the temperatures prescribed.
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GENERAL CONSIDERATIONS

5. The organisms specified for use in the tests are intended to be representative of
those that might be expected to be found in the environment in which the
preparation is manufactured, stored and used.
Precautions
Challenge tests should be conducted under conditions that prevent accidental
contamination of the product during the test but the precautions taken to
prevent contamination should not affect the survival of organisms in the
product being examined.
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GENERAL CONSIDERATIONS

6. Test organisms
The following test organisms are used in the test
Candida albicans ATCC 10231
Aspergillus niger ATCC 16404
Escherichia coli ATCC 8739
Pseudomonas aeruginosa ATCC 9027
Staphylococcus aureus ATCC 6538
Media
For the initial cultivation of the test organism, use Soyabean Casein Digest Agar
Medium for bacterial cultures and Sabouraud-dextrose agar for C albicans and
A. niger, or any other media not less nutritive than the said media.
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REQUIREMENTS FOR TEST

7. From a recently grown stock culture of each of the test organisms, subculture
on the surface of a suitable volume of the above stated media.
Incubate the bacterial cultures at 30° to 35° for 18 to 24 hours and incubate the
cultures of C. albicans and A. niger at 20° to 25°C for 48 hours and 7 days
respectively.
Using sterile saline solution, harvest the bacterial and C. albicans cultures and
dilute suitably with the sterile saline solution to bring the count to about 1 x
108
per ml.
Similarly harvest A. niger culture with sterile saline solution containing
0.05 per cent w/v of polysorbate 80 and adjust the spore count to about 1 x 108
per ml with sterile saline solution.
7PREPARATION OF INOCULUM

8. Alternatively, the stock culture organisms may be grown in a suitable liquid
medium, and the cells may be harvested by centrifugation, washed and
resuspended in sterile saline solution to give the required microbial or spore
count.
Determine the number of colony-forming units (CFU) per ml in each
suspension. This value serves to determine the size of inoculum to be used in
the test.
If the standardised suspensions are not used within 2 hours, it should be stored
in a refrigerator.
Periodically monitor the stored suspensions by the plate-count method to
determine any loss of viability.
8PROCEDURE

9. Inoculate each original product container or product tube (when original container is
not suitable for inoculation with a sterile syringe fitted with needle, transfer 20 ml per
capped bacterial tube) with one of the standard microbial suspensions using a ratio
equivalent to 0.1 ml of inoculum suspension to 20 ml of product and mix.
The final concentration should be between 1 x 105
and 1 x 106
micro-organisms per ml
of the product.
Determine the number of viable micro-organisms by the plate count method in each
inoculum suspension and from there calculate the initial concentration of micro-
organisms per ml of product being examined.
Incubate the inoculated containers or tubes at 20° to 25°.
Determine the viable count (by the plate count method) at 7, 14, 21 and 28 days
subsequent to inoculation. Record also any change observed in the appearance.
9PROCEDURE

10. The preservative is effective in the product examined if
(a)the concentration of viable bacteria are not more than 0.1 per cent of the
initial concentrations by the 14th
day,
(b)the concentrations of viable yeasts and moulds remain at or below the initial
concentration during the first 14 days and,
(c)the concentration of each test micro-organism remains at or below these
designated levels during the remainder of the 28-day test period.
10INTERPRETATION

11. For testing purposes, the USP has divided test articles into four separate
categories:
Category 1 – Injections, other parenterals including emulsions, otic,
sterile nasal products made with aqueous bases or vehicles.
Category 2 – Topically used products made with aqueous bases or vehicles,
non-sterile nasal products, and emulsions, including those applied to mucous
membranes.
Category 3 – Oral products other than antacids made with aqueous bases or
vehicles.
Category 4 – Antacids made with an aqueous base.
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PRODUCT CATEGORIES

12. 12
IP 2007

13. 13
IP 2007

14. 14
IP 2007

15. 15
IP 2007

16. USP EP JP
Type of Product
Organism Time Viable Count
Reduced by
Time Viable Count
Reduced by
Time Viable Count
Reduced by
Parenterals (multidose
and ophthalmics) Bacteria 7 d 101
6 h 102
14 d 103
14 d 103
24 h 103
28 d NI
28 d NI 28 d NR
Fungi 7 d NI 7 d 102
14 d NI
14 d NI 28 d NI 28 d NI
28 d NI
Oral (liquid products
only)
Bacteria 14 d 101
14 d 103
14 d 101
28 d NI 28 d NI 28 d NI
Fungi 14 d NI 14 d 101
14 d NI
28 d NI 28 d NI 28 d NI
Topical Bacteria 14 d 102
2 d 102
14 d 102
preparations 28 d NI 7 d 103 28 d NI
28 d NI
Fungi 14 d NI 14 d 102
14 d NI
28 d NI 28 d NI 28 d NI
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