Sop for Efficacy of Antimicrobial Preservative

This document describes (Standard Operating procedure) Sop for Efficacy of Antimicrobial Preservative

 Sop for Efficacy of Antimicrobial Preservative

I. Purpose & Scope:

• The purpose of this SOP is to lay down a procedure for Efficacy of Antimicrobial Preservative for Pharmaceutical products
• This Standard operating procedure shall be applicable for Efficacy of Antimicrobial Preservative for Pharmaceutical products.

II. Responsibilities:

• All Quality Control and microbiology department personnel shall be responsible to follow and implement this SOP i.e. Efficacy of Antimicrobial Preservative.

III: Introduction and Procedure :

• Antimicrobial preservative are substances added to non sterile dosage forms to protect them from microbiological growth or from microorganism that are introduced inadvertently during or subsequent to the manufacturing process.
• In case of sterile articles packaged in multiple dose containers, antimicrobial preservatives added to inhibit the growth of microorganism.
• Antimicrobial effectiveness must be demonstrated for multiple dose topical and oral dosage forms and for other dosage forms such as ophthalmic, otc, nasal, irrigation, and dialysis fluids.

Requirements for Efficacy of Antimicrobial Preservative:

• Sample for analysis for preservative efficacy test.
• Cultures of the specified micro-organisms.(Candida albicans , Aspergillus brasiliensis, Escherichia coli , Pseudomonas aeruginosa, Staphyllococcus aureus.)
• Tubes containing 9ml sterile saline solution for dilutions.
• Pipettes, Nichrome loop, Petri plates, Soybean casein digest agar (SCDA) , Sabouraud dextrose agar (SDA) .

Product Categories for Efficacy of Antimicrobial Preservative:

For the purpose of testing, compendial articles have been divided into four categories (see following table 1).

Table – 1: Compendial Product Categories

Product Category	Product Description
1	Injections, other parenterals including emultions, OTC products, sterile nasal products and ophthalmic products made with aqueous bases or vehicles.
2	Topically used products made with aqueous bases or vehicles, non sterile nasal products and emulsions.
3	Oral products other than antacids, made with aqueous bases or vehicles.
4	Antacids made with an aqueous base.

Preparation of the cultures for Challenge test : 

• For performing the challenge test the following cultures should be used, Candida albicans ATCC 10231, Aspergillus brasiliensis ATCC 16404, Escherichia coli ATCC 8739, Pseudomonas aeruginosa ATCC 9027, Staphyllococcus aureus ATCC 29737.
• Prepare the initial cultivation of the test organism on the slants of Soyabean Casein Digest Agar or any other medium, not less nutritive than the said medium.
• Incubate the slants at 30-35°C for bacteria, and at 20-25°C for yeast/ moulds.
• Harvest the bacterial and yeast growth, and dilute it suitably with sterile saline to bring the count to about 1´10⁸ cells per ml.
• Similarly, harvest brasiliensis culture with sterile saline solution containing 0.05% w/v of Polysorbate 80, and adjust the spore count to about 1´10⁸ per ml.
• Determine the number of colony-forming units (cfu) per ml of each suspension. This value serves to determine the size of inoculum to be used in the test.

Performing the preservative challenge Test :

• The test can be conducted either in five original containers if sufficient volume of product is available in each container or in five sterile, capped bacteriological containers of suitable size into which a sufficient volume of product has been transferred.
• Inoculate each container of the product with standardized microbial suspension of one organism, using 0.1ml of inoculum for every 20ml of the product or inoculums used is between 0.5% to 1.0% of the volume of the product. The final concentration should be in the range of 1´10⁵ to 1´10⁶ organisms per ml of the product.
• Mix the product and suspension properly to give a homogenous mixture.
• Determine the number of viable organisms by the plate-count method in each of the mixture by serially diluting the mixture to about 10^-6
• Also take the count cfu/ml of the product only. The initial count of the organism also detect.
• Incubate the inoculated containers at 20 -25°C.
• Determine the viable count of each mixture by pour plate method on 7^th, 14^th, and 28^th day, after inoculation.
• Positive control run simultaneously with test.

Interpretation of Results for Efficacy of Antimicrobial Preservative :

• • The requirement for antimicrobial effectiveness is met if the criteria specified under table (Table 2) are met.

 

 Table 2: Criteria for Tested Microorganism

Products Category	For Bacteria	For Yeast and Moulds
1	i) Not less than 1 log reduction from initial calculated count at 7th days, ii)Not less than 3 log reduction from the initial count at 14th days. iii)No increase from the 14th days count at 28th days.	i)No increase from the   initial calculated count at 7th,14th and 28th day.
2	i) Not less than 2 log reduction from the initial count at 14th days and no increase from the 14 days count at 28th days.	i) No increase from   the initial calculated count at 14th and 28th days.
3	i) Not less than 1 log reduction from the initial count at 14th days and no increase from the 14 days count at 28th days.	i) No increase from the initial calculated count at 14th and 28th days.
4	i) No increase from the initial calculated count at 14th and 28th days.	i) No increase from the initial calculated count at 14th and 28th days.

 

                                                                    Annexure I

                               Report of Preservative Efficacy Test

Name of Product       :		Date of Testing            :
Batch Number           :		A.R.Number                :
Incubation Temp.  :		Incubator ID.               :
Analysed By             :		Date of Completion    :

 

1) Organism Used: Staphylococcus aureus

 

Date	Day	Media lot No.		Dilution					Log Reduction
			101	102	103	104	105	106

 

 

2) Organism Used: Pseudomonas aeruginosa

 

Date	Day	Media lot No.		Dilution					Log Reduction
			101	102	103	104	105	106

 

3) Organism Used: E.coli

 

Date	Day	Media lot No.		Dilution					Log Reduction
			101	102	103	104	105	106

 

4) Organism Used: Candida albicans

 

Date	Day	Media lot No.		Dilution					Log Reduction
			101	102	103	104	105	106

 

5) Organism Used: A.brasiliensis

 

Date	Day	Media lot No.		Dilution					Log Reduction
			101	102	103	104	105	106

 

 

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Analysed By (QC):                                                Checked By (Head QC) :                                      Verified By (QA) :

Date:                                                                        Date:                                                                      Date :