A Standard Operating Procedure (SOP) for a Preservative Efficacy Test outlines the step-by-step process for conducting tests to determine the effectiveness of preservatives in cosmetic, pharmaceutical, and other products. This test is crucial to ensure that products remain safe and free from microbial contamination throughout their shelf life. Here’s a sample SOP for a Preservative Efficacy Test:

PURPOSE

To provide guidelines for preservative efficacy test and determine the effectiveness of preservatives in products by assessing their ability to inhibit the growth of microorganisms.

SCOPE

This SOP for Preservative Efficacy Test is applicable for preservatives used for aseptic filling to inhibit the growth of microorganisms in product. This SOP applies to all personnel involved in conducting Preservative Efficacy Tests for cosmetic, pharmaceutical, and related products.

RESPONSIBILITIES & AUTHORITIES

The Quality Control Manager is responsible to ensure that procedure & formats are followed entirely as approved.
The Microbiologist is responsible for preparation, review and to follow the SOP.

REFERENCE & REQUIREMENTS

USP

DEFINITIONS & ABBREVIATIONS

Preservative: A chemical that inhabits vegetative form microorganisms (such as bacteria and fungi).

PROCEDURE

Materials & Equipment’s

1. LAFH
2. Measuring cylinder
3. Sterile petri plates
4. Preservative
5. Sterile saline solution with 0.01% peptone
6. Sterile saline solution with 0.05% polysorbate 80
7. 70% IPA solution
8. Sterile and molten cooled soybean casein digest agar
9. Test organisms ( E.Coli , Salmonella , .C.albicans )

Precaution

1. Keep the hands clean and used strictly 70 % IPA to rinsed hand gloves throughout the operation. Dilution of each microbial inoculum should be made properly.
2. Plating of final dilution and counting of cfu for each microbial culture should be recorded properly which forms the baseline for comparison with the test.
3. Use separate tube with the product or separate container for each culture.
4. All the procedures of Preservative Efficacy test should be Carried out under the laminar air flow unit chamber

Procedure

From a recently grown stock culture of each of the test organism subculture on the surfaces of respective media as given below.

Candida albicans	ATCC-10231	Sabourad Dextrose agar	20-25ᵒC
Aspergillus niger	ATCC- 16404	Sabourad Dextrose agar	20-25ᵒC
E.coli	ATCC-8739	Soybean casein digest agar	30-35ᵒC
Salmonella	ATCC-14028	Soybean casein digest agar	30-35ᵒC
S.aureous	ATCC-6538	Soybean casein digest agar	30-35ᵒC

1. Using sterile solution about 10 ml of sterile normal saline with peptone (0.1 % w/v), harvest the bacteria and C. albicans culture and diluted suitably with the sterile saline solution to bring the count to about 1 X 108 per ml.
2. Similarly harvest A. niger culture with the sterile saline solution containing 0.05% w/v of Tween 80 & adjust the spore count to about 1 X 108 per ml with the sterile saline solution.
3. Determine the number of cfu per mL in each suspension by serial dilution method. Prepare ten-fold serial dilutions of each of the bacterial cultures and Candida albicans using sterile normal saline (0.9 % w/v) with peptone (0.1 % w/v); and Aspergillus niger using sterile normal saline, with polysorbate 80.
4. Using the pour plate technique, plate out, in duplicate the last 7 dilutions using the appropriate media. Allow solidifying. Invert the Petri dishes and incubate the plates for the bacterial count at 30 -35°C for 2 days and the plates for the fungal count at 20°C – 25°C for 2 – 7days. Count the cfu per ml. Tabulate the counts obtained in the Test Data Sheet.
5. Inoculate each original product container or product tube (when original container is not suitable for inoculation with sterile syringe fitted with needle, transfer 10 -20 ml per capped bacteriological tube) with one of the standardized microbial suspensions using a ratio equivalent to 0.1 ml of inoculums suspension to 20 ml of product and mix.
6. The final concentration should be between 1×105 and 1×106 microorganisms per ml of product.
7. Determine the number of viable microorganisms by plate count method in each inoculums suspension. Prepare ten-fold serial dilutions using sterile normal saline (0.9 % w/v) with peptone (0.1 % w/v) and for Aspergillus niger uses 9 ml sterile saline with polysorbate 80 (0.05%) up to 10fold dilution and plating all the dilutions. Calculate the initial concentration of inoculums in the product (at zero hours).
8. Incubate the inoculated containers at 20 to 25°C.
9. Determine the viable count at 7,14 and 28 days respectively.

Frequency:

For each consignment.

DOCUMENTS / RECORDS (FORMS / ANNEXURE)

Preservative efficacy test report.