PROCEDURE OF THE PRESERVATIVE EFFICACY TESTS. | MPK

PROCEDURE OF THE PRESERVATIVE EFFICACY TESTS.

PRESERVATIVE EFFICACY TEST

Department: Microbiology
Originated by:
		(Microbiologist)		Date
Reviewed by:
		(Incharge Microbiology Lab)		Date
Reviewed by:
		(Quality Assurance Manager)		Date
Approved by:
		(Head Quality Assurance)		Date
Next Review Date:

 

Change Record:

V/N	Date	Change Initiator	Description of Change
1.
2.			Addition of Limits, change of Annexure and Addition of references
3.			SOP upgradation, addition of log reduction calculations, change of Annexure

 

Distribution List:

Head of Quality Assurance

Incharge Microbiologist

1.0.      Purpose:

To describe the procedure of the Preservative Efficacy Testing.

 

2.0.      Scope:

This procedure is applicable to Microbiology lab.

 

                        Responsibilities:

 

Action	Responsibility
Follow and implement this SOP	Microbiologist
Compliance of SOP	Laboratory Incharge

 

4.0.      General Requirements:

 

4.1.   Equipments and Glasswares

         Incubator

         Cool Incubator

         Depyrogenator

         Autoclave    

         Water Bath

         Autoclaved Tips 1and 5 ml

         Depyrogenated Glass Petri plates 90mm

 

4.2.    Media

         Tryptone Soya Agar (TSA)

         Sabouraud Dextrose Agar (SDA)

         Peptone water 0.1 % (PW)

         Normal Saline (NS)

         Tryptone Soya Broth (TSB)

 

4.3.    Microorganisms

         Escherichia coli                   ATCC 8739

         Pseudomonas aeruginosa   ATCC 9027

         Staphylococcus aureus       ATCC 6538

         Candida albicans                 ATCC 10231

         Aspergillus niger                  ATCC 16404

   Environmental Isolates        Recovered from plates of Area Monitoring of Production Area

 5.0.      Procedure:

 

5.1. Preparation of Media and Inoculum

 

S. No	Action	Responsibility
5.1.1	Prepare and autoclave Media, (TSA, SDA, PW, NS), as per SOP #:	Microbiologist
5.1.2	The stock cultures of microorganisms will grow in the TSB at 32.5 ± 2.5 0C for 24 hrs and SDA at 22.5 ± 2.5 0C 5 days.
5.1.3	On next day cells will be harvested by centrifugation.
5.1.4	Discard the supernatant and resuspend the pellet in sterile saline for washing.
5.1.5	After washing again resuspend the pellet in sterile saline to obtain bacterial and C albicans cultures count of about 1 x 108 cfu / ml.
5.1.6	For the harvesting of the cells of A niger, use sterile saline containing 0.05% polysorbate 80 to obtain a count of about 1 x 108 cfu / ml.
5.1.7	The Inoculum concentration will be determined by turbidimetric or McFarland’s index measurements for the challenge microorganism. Referred Table 03.
5.1.8	Refrigerate the suspension, if it is not used with in 2 hours.
5.1.9	The bacterial and yeast suspension to be used with in 24 hours and the fungal suspension to be used with in 7 days.

 

5.2. Efficacy Test:

 

S. No	Action	Responsibility
5.2.1	Take 99 ml of product and inoculate 0.5 to 1.0 ml of standardized suspension of 1 x 108 cfu / ml, mix well. The whole solution with in the sterile container will be called as Inoculum container.	Microbiologist
5.2.2	The concentrations of test microorganisms that are added to the product are described in table 02.
5.2.3	The initial concentration of added Inoculum in the product will be determined by plate count method.
5.2.4	Incubate the Inoculum containers at 22.5 + 2.5 0C.
5.2.5	Pick the samples from each container at the appropriate intervals of 0, 14th and 28th day for bacterial and fungal count.
5.2.6	Record any changes in the cfu count at these intervals.
5.2.7	Use TSA for bacterial count and SDA for fungal count for plate count method.
5.2.8	Incubate TSA plates for 3 to 5 days at 32.5 + 2.5 0C and SDA plate for 5 to 7 days at 22.5 + 2.5 0C.

 

    5.3. Negative Controls:

 

S. No	Action	Responsibility
5.3.1	Media Control: Perform negative control tests of TSA, SDA, TSB and Peptone at each intervals of time when inoculated containers are checked for microbial count inoculated test microorganisms.	Microbiologist
5.3.2	Product Control: Perform negative control tests of Product at each intervals of time when inoculated containers are checked for microbial count inoculated test microorganisms.
5.3.2	Initial Inoculum Control: It will check only at the beginning of the test when Inoculum are added in the product, and also at the same time equal volume of microorganisms will added in the peptone having same volume as for the product. Then count will be determined by plate count.

 

 

5.4. Calculation for Log Reduction:

 

 

S. No	Action	Responsibility
5.4.1	Log reduction will be calculated for all days in which the tests were performed. Log Reduction can be calculated as: Log Reduction = Log (Initial Inoculum – Testing Day)	Microbiologist
5.4.2	After the Log Reduction calculation, Interpret the results as given in Table 01.

 

 

Table 01: Limits of tested Microorganisms in Compendial product Categories

 

S No.	Product Category	Product Description	Acceptable Limits
			Bacteria	Molds and Fungi
1	1	Injections, other parenteral including emulsions, otic products, sterile nasal products, and ophthalmic products made with aqueous bases or vehicles	Not less than 1.0 log reduction from the initial calculated count at 7 days, not less than 3.0 log reduction from the initial count at 14 days, and no increase from the 14 days count at 28 days	No increase from the initial calculated count at 7, 14, and 28 days
2	2	Topically used products made with aqueous bases or vehicles, non sterile nasal products, and emulsions, including those applied to mucous membrane	Not less than 2.0 log reduction from the initial count at 14 days and no increase from the 14 days count at 28 days	No increase from the initial calculated count at 14 and 28 days
3	3	Oral products other than antacids, made with aqueous bases or vehicles	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	No increase from the initial calculated count at 14 and 28 days
4	4	Antacids made with aqueous bases	No increase from the initial calculated count at 14 and 28 days	No increase from the initial calculated count at 14 and 28 days

 

 

Table 02: Inoculum size in each Product Category before and after addition in the   

                Product

 

 

S No.	Product Category	Inoculum size Before Addition in the Product	Inoculum size after Addition in the Product
1	1	1 x 108 cfu / ml	1 x 105 and 1 x 106 cfu / ml
2	2	1 x 108 cfu / ml	1 x 105 and 1 x 106 cfu / ml
3	3	1 x 108 cfu / ml	1 x 105 and 1 x 106 cfu / ml
4	4	1 x 108 cfu / ml	1 x 103 and 1 x 104 cfu / ml

 

Table 03: Preparation of McFarland Index:

 

McFarland Scale	CFU (x106 / ml)	1% BaCl2 + 1% H2SO4
0.5	<300	0.05 + 9.95
1	300	0.1 + 9.9
2	600	0.2 + 9.8
3	900	0.3 + 9.7
4	1200	0.4 + 9.6
5	1500	0.5 + 9.5
6	1800	0.6 + 9.4
7	2100	0.7 + 9.3
8	2400	0.8 + 9.2
9	2700	0.9 + 9.1
10	3000	1.0 + 9.0

 

 6.0.      Definitions:

 

Term/word/statement	Meaning/Description
Inoculum	Cells used in an inoculation, such as cells added to start a culture.
Antimicrobials	Antimicrobials are chemicals that kill microbes

 

7.0.      References Used:

 

S. No	Description
1.	USP30/NF25, 2007 <51>”antimicrobial effectiveness testing”.
2.	Pharmaceutical Microbiology Forum Newsletter – Vol. 12 (8), “McFarland Index”
3.	Second Edition, Microbial Limit and bioburden Tests by lucia clontz, 2009, “The Preservative of Pharmaceutical Products”, pg 40 – 47.
4.	Second Edition, Microbial Limit and bioburden Tests by lucia clontz, 2009, “Preparation of Working Cultures”, pg 165 – 166.

 8.0.      Frequency:

 

S. No	Description
1.	Every New Product / Validation Batches.
2.	As per requirement

 9.0.      List of Records Generated:

 

S. No	Description
1.	Report of Preservative Efficacy Testing.

 

10.0.    Referred Documents:

 

Document No.	Description
	N/A

 

11.0.    Annexure:

 

S. No	Description
1.	Report of Preservative Efficacy Testing.