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Humanwell Pharmaceutical Ethiopia PLC 1

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SOP/SL/011 Title: Page No.

Preservative Efficacy Test Page 1 of 8

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Preservative Efficacy Test

Prepared by： X X
Department Name Date

Approve by： Y Y
Department Name Date

Authorized by： Z Z
Department Name Date

1. Purpose
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The purpose of this SOP is to provide standard procedure for performing the preservative
efficacy test for nonsterile and sterile dosage forms.
2. Scope

This procedure is applicable to - nonsterile and sterile dosage forms in Humanwell

Pharmaceutical Ethiopia PLC

3. Validity

This SOP is valid only until next revision date and if it bears control seal.

4. Responsibility

The microbiology division & the quality assurance manager are responsible for SOP
compliance.
5. Material and Equipment
5.1. Equipment

 Laminar air flow

 Autoclave
 Water bath
 Incubator at 376°C
 Dry heat sterilizer
 Refrigerator
 Gas burner
 Petri dishes (20 × 100 mm)
 Micropipette
 pH Meter
 Spectrophotometer
 Vortex mixer
 Glassware
 Colony counter
5.2. Reagent
5.2.1. Harvesting fluid-1 (JUSP sterile saline TS)
 Sodium chloride: 9.0 g
 Distilled water: q.s. 1000 ml
 Sterilize in the autoclave at 121°C for 15 minutes
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5.2.2. Harvesting fluid 2

 Sodium chloride: 9.0 g
 Polysorbate 80 (w/v): 0.5 g
 Distilled water q.s.: 1000 ml
 Sterilize in the autoclave at 121°C for 15 minutes

5.2.3. Diluent

 Sodium chloride: 9.0 g

 Polysorbate 80 (w/v): 0.5 g
 Peptone 49: 1.0 g
 Distilled water q.s.: 1000 ml
 Sterilize in the autoclave at 121°C for 15 minutes

5.3. Media: perform growth promotion test for all media used in preservative efficacy
test
5.3.1. Tryptone soya agar (TSA)
 Pancreatic digest of casein: 15.0 g
 Papaic digest of soya bean: 5.0 g
 Sodium chloride: 5.0 g
 Agar: 15.0 g
 Distilled water q.s.: up to 1000 ml
 Final pH: 7.3 to 0.2 (after sterilization) at 25°C
 Suspend 40 g in 1 l distilled water or deionized water
 Mix and boil to create solution
 Sterilize in the autoclave at 121°C for 15 minutes

5.3.2. Sabouraud dextrose agar (SDA)

 Peptone: 10.0 g
 Dextrose: 40.0 g
 Agar: 15.0 g
 Distilled water q.s.: up to 1000 ml
 Final pH 5.6 to 0.2 (after sterilization)
 Suspend 65 g in 1 l distilled water or deionized water
 Mix and boil to create solution
 Sterilize in the autoclave at 121°C for 15 minutes
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5.4. Test organisms

 Aspergillus niger (ATCC no. 16404)

 Candida albicans (ATCC no. 10231)
 Escherichia coli (ATCC no. 8739)
 Pseudomonas aeruginosa (ATCC no. 9027)
 Staphylococcus aureus (ATCC no. 6538)

Note: Other microorganisms may be included in the test on an optional basis, if they represent
likely contaminants to the preparation. Zygosaccharomyces rouxii may be used for oral
preparations containing a high concentration of sugar.

 The viable microorganism used in the test must not be more than five passages
removed from the original ATCC culture.
 One passage is defined as the transfer of organisms from an established culture to fresh
medium and count all transfers.
 Resuscitate the cultures received from the ATCC according to the ATCC directions.

6. Procedure
6.1. Method
6.1.1. Preparation of inoculum

 Inoculate the surface of tryptone soya agar slant for bacteria and Sabouraud
dextrose agar slant for fungi from recently revived stock culture of each of the test
microorganisms.
 Incubate the bacterial cultures at 32.5 to 2.5°C for 18 to 24 hours.
 Incubate the culture of C. albicans at 22.5 to 2.5°C for 48 hours and the culture of
A. niger at 22.5 to 2.5°C for 6 to 10 days or until good sporulation is obtained.
 Harvest the growth of bacteria and C. albicans with 10 ml of sterile harvesting fluid
1 and the growth of A. niger with 10 ml of harvesting fluid 2.
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 Dilute the suspension with respective sterile fluid to reduce the microbial count to
about 1 × 108 colony-forming units (CFUs) per liter.
 Determine the number of colony-forming units per liter in each suspension by plate
count method, using the conditions of media and microbial recovery incubation
times listed in Table 3 to confirm the initial CFUs per ml estimate.
 Use the bacterial and yeast suspensions within 24 hours of harvesting.
 Use the fungal suspension up to 7 days of harvest if stored under refrigeration.

6.2. Challenging formulated product

 Conduct the test in original product container or in sterile, capped containers of
suitable size into which a sufficient volume of product, at least 20 ml, has been
transferred.
 Inoculate each product container with one of the prepared and standardized
inoculum suspensions and mix well.
 Use the volume of suspension between 0.5 and 1.0% of the volume of the product
so that each liter of product after inoculation contains between 1 × 105 to 1 × 106
cells of the test microorganisms.

Table 2
Sampling Intervals per Product Category

Sampling Intervals
Product 6 4 7 14 28
Category hours hours days days days
Category la + + + + +
Category Ib + + + + +
Category Ic + + + + +
Category II + + + + +
Note: “+” indicates sampling intervals.

 Estimate the initial concentration of viable microorganisms in each test

preparation based on the concentration of microorganisms in each of the
standardized inoculum suspensions.
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 Incubate the inoculated product containers at 22.5 to 2.5°C.

 Sample each container at the appropriate intervals specified in Table 2.
6.3. Standard plate count method-
Preparation of sample
 Transfer aseptically 1 ml, accurately measured, of sample from each inoculated
product container to a dilution bottle containing 9 ml of sterile diluent and mix
well. This is 10:1 dilution.
 Transfer aseptically 1 ml, accurately measured, from 10:1 dilution to a second
dilution blank containing 9 ml of sterile diluent and mix well. This is 10:2
dilution.
 Continue tenfold dilution up to 10-’s or suitable as required.

Total aerobic bacterial count

 Pipette 1 ml aliquot from each dilution to duplicate sets of sterile petri dishes.
 Promptly pour into each petri dish about 15 to 20 ml of sterile melted tryptone
soya agar medium previously melted and cooled to approximately 45°C.
 Cover the dishes and mix the sample well with agar by tilting and rotating the
petri dishes.

Table 3 Culture Conditions for Inoculum Preparation and Microbial

Recovery
Suitabl Incubatio
e n Incubatio Microbial
Mediu Temperat n Recovery
Organism m ure Time Time
Escherichia coli TSA 32.5– 18–24 h 3–5 days
(ATCC no. 8739) 2.5°C
Pseudomonas TSA 32.5– 19–24 h 3–5 days
aeruginosa 2.5°C
(ATCC no. 9027)
Staphylococcus aureus TSA 32.5– 18–24 h 3–5 days
(ATCC no. 6538) 2.S°C
Candida albicans SDA 22.5– 44–52 h 3–5 days
(ATCC no. 10231) 2.S°C
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Aspergillus niger SDA 22.5– 6–10 day 3–5 days

(ATCC no. 16404) 2.5°C

Note: TSA = tryptone soya agar; SDA = Sabouraud dextrose agar.

 Pour one plate with uninoculated agar medium and uninoculated diluent as negative
control.
 After the agar has solidified, invert the dishes and incubate them at 32.5 to 2.5°C for
3 to 5 days.

Total molds and yeasts count

 Pipette 1 ml aliquot from each dilution to duplicate sets of sterile petri dishes.
 Promptly pour into the petri dishes about 15 to 20 ml of sterile melted Sabouraud
dextrose agar medium previously melted and cooled to approximately 45°C.
 Cover the dishes and mix the sample with agar carefully by tilting and rotating the
petri dishes.
 Pour one plate with uninoculated agar medium and uninoculated diluent as negative
control.
 After the agar has solidified, invert the dishes and incubate them at the conditions
listed in Table 3.

6.4. Acceptance Criteria

The criteria for evaluation of antimicrobial activity are given in Table 4 in terms of the
log reduction in the number of viable microorganisms using as baseline the value
obtained for the inoculum. “No increase” is defined as not more than 0.5 log unit higher
than the previous value obtained.

Table 4 Acceptance Criteria for Preservative Efficacy

Required Log Reduction from the Initial Calculated Count

at Interval
Product 7 14 28
Category Organism days days days
Category Ia Bacteria 1 3 NI
Fungi NI NI NI
Category Ib Bacteria — 2 NI
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Fungi — NI NI
Category Ic Bacteria — 1 NI
Fungi — NI NI
Category II Bacteria and — NI NI
fungi
Note: NI = no increase.

6.5 RECORDS

Use Preservative Efficacy test CoA

7. Abbreviations
 SOP - Standard Operating Procedure

8. Related Documents /Applicable document/

None

9. Revision History

Version
Revision Description
No.

1 New