SOP for Preservative Efficacy Testing

OBJECTIVES

To lay down a procedure for testing the Efficacy of Preservatives.

SCOPE

This SOP is applicable for testing the Efficacy of Preservatives added to products manufactured at PharmaInfo Limited

RESPONSIBILITY

Microbiologist.

QC Manager

MATERIALS AND EQUIPMENT

1. Micropipette
2. Micropipette tips: 1ml, 10ml, 100ml
3. Sterile normal saline (0.9 %w/v) with 0.05% polysorbate-80
4. Sterile normal saline with 0.1% peptone
5. 70% IPA
6. Sample for Testing
7. Media: SDA and SCDA
8. Test culture suspension 1 X 108 cells of each organism: Escherichia coli (ATCC No. 8739), Pseudomonas aeruginosa (ATCC No. 9027), Staphylococcus aureus (ATCC No. 6538), Candida albicans (ATCC No. 10231), Aspergillus niger (ATCC No. 16404))

PROCEDURE

Ensure that all the media employed should be tested for their growth-promoting properties

INOCULUM PREPARATION

• Inoculate the surface of the Soybean Casein Digest Agar and Sabouraud Dextrose Agar media plates with 1 ml of the microorganisms from the recent culture suspension as given in Table-1
• Incubate the plates at suitable conditions as given in the Table-1.
• Harvest the cells except for Aspergillus niger after the incubation by washing the growth on the surface of the respective solid agar media using Sterile Normal Saline(0.9%w/v), collect it in a sterile container and treat this as Stock solution.
• For harvesting of Aspergillus Niger, Use Normal saline with 0.05% Polysorbate 80.
• From the Stock solution, do tenfold serial dilutions using sterile normal Saline as diluent to get a count of 1 x 108 CFU / ml
• Determine the number of CFU per ml of these harvested suspensions by plate count Method using suitable Agar media and incubate at appropriate temperatures.
• Use the initial count of suspensions to calibrate the size of the inoculum to be used in the Test.
• Use the Bacterial and Candida albicans suspensions within 24 hours of harvest.
• Fungal suspensions shall be refrigerated and used for up to 7 Days.
• For microorganisms and media to be used, incubation conditions, and microbial recovery time, refer to Table-1 given below

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Table-1

Organism	Suitable Medium	Incubation Temperature	Inoculum Incubation Time	Microbial Recovery Incubation Time
Escherichia coli (ATCC No. 8739)	Soybean–Casein Digest Broth;	32.5 ± 2.5	18 to 24 hours	3 to 5 days
Pseudomonas aeruginosa (ATCC No. 9027)	Soybean–Casein Digest Agar	32.5 ± 2.5	18 to 24 hours	3 to 5 days
Staphylococcus aureus (ATCC No. 6538)	Soybean–Casein Digest Broth;	32.5 ± 2.5	18 to 24 hours	3 to 5 days
Candida albicans (ATCC No. 10231)	Soybean–Casein Digest Agar	22.5 ± 2.5	44 to 52 hours	3 to 5 days
Aspergillus niger (ATCC No. 16404))	Soybean–Casein Digest Broth;	22.5 ± 2.5	6 to 10 days	3 to 7 days

TEST PROCEDURE

• Use five Product containers if the volume per container is sufficient.
• If the volume is not sufficient, transfer the product into five sterile screw-capped containers.
• Reconstitute the product container if it is in dry powder form, as per the instruction on the label.
• Standardize the volume of the inoculum to be between 0.5% and 1.0% of the volume of the product and the concentration to be between 1 × 105 and 1 × 106 cfu per mL of the product.
• Add the inoculum into the product containers using a Sterile Syringe or Pipette.
• Determine the initial count of the inoculated containers by Plate Count Method.
• Incubate the inoculated containers at 22.5 ± 2.5
• Withdraw samples from containers at the intervals of 14 and 28 Days and determine the number of CFUs present in Sample containers by the Plate Count Method.
• Observe and record any changes in appearance of the sample during the sampling as per report.
• Incorporate an Inactivator in the diluents such as sterile normal saline (0.9 % w/v) with polysorbate-80 (0.05 % w/v) and 0.05 % Soya lecithin or with peptone (0.1 % w/v) and 0.05 % Soya lecithin or sterile normal saline (0.9 % w/v) with 0.05 % polysorbate-80.
• Using the Initial count of the test preparation, calculate the change in log10 values of the concentration of cfu per mL for each microorganism at the respective intervals.
• Express this in terms of Log Reduction as per Annexure-I and report as Complies if results comply to the criteria as given in Table-2.

ACCEPTANCE CRITERIA

Sr. No.	Microorganism	Acceptance Criteria
1	Bacteria	Not less than 1.0 log reduction from the initial count at 14 days, and no increase from the 14 days’ count at 28 days.
2	Yeast and Moulds	No increase from the initial calculated count at 14 and 28 days.

Note: No increase is defined as not more than 0.5 log10 unit higher than the previous value Measured.

ATTACHMENTS

Preservative Efficacy Test Report for Oral Preparations

ABBREVIATIONS

SOP: Standard Operating Procedure

QC: Quality Control

REVISION HISTORY

Nil

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