SOP on Preservative Efficacy Test of Oral Preparations

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1.0 Objective:

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

2.0 Scope:

2.1 This SOP is applicable for testing of Efficacy of Preservatives added in products manufactured in pharmaceutical plant.

3.0 Responsibility:

3.1 Microbiologist

3.2 Head-Microbiology

4.0 Accountability:

4.1 Head – Quality Control

5.0 Procedure:

5.1 Ensure the all the media employed should be tested for their growth promoting properties.

6.0 INOCULUM PREPARATION:

6.1 Inoculate the surface of the Soybean casein Digest Agar and Sabouraud Dextrose Agar media plates / slants with 1 ml of the microorganisms from the recent culture suspension as given in Table-1.

6.2 Incubate the plates/ Slants at suitable conditions as given in the Table-!.

6.3 Harvest the cells except 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.

6.4 For harvesting of Aspergillus Niger, Use Normal saline with 0.05% Polysorbate 80.

6.5 From the Stock solution, do ten fold serial dilutions using sterile normal Saline as diluent to get a count of 1 x 108 CFU / ml.

6.6 Determine the number of CFU per ml of these harvested suspensions by plate count Method using suitable Agar media and incubate at appropriate temperatures.

6.7 Use the initial count of suspensions to calibrate the size of inoculum to be used in the Test.

6.8 Use the Bacterial and Candida albicans suspensions within 24 hours of harvest.

6.9 Fungal suspensions shall be refrigerated and used up to 7 Days.

6.10 For microorganisms and media to be used, incubation conditions, microbial recovery time, refer to Table-1 given below.

Table-1

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

 

7.0 Test Procedure:

7.1 Use five Product containers if the volume per container is sufficient.

7.2 If the volume is not sufficient, transfer the product into five sterile screw capped containers.

7.3 Reconstitute the product container if it is in dry powder form, as per the instruction on the label.

7.4 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 between1 × 105 and 1 × 106 cfu per mL of the product.

7.5 Add the inoculum into the product containers using Sterile Syringe or Pipette.

7.6 Determine the initial count of the inoculated containers by Plate count Method.

7.7 Incubate the inoculated containers at 22.5 ± 2.5

7.8 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.

7.9 Observe and record any changes in appearance of the sample during the sampling as per Annexure-I.

7.10 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.

7.11 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.

7.12 Express this in terms of Log Reduction as per Annexure-I and report as Complies if results are complying to the criteria as given in Table-2.

8.0 Acceptance Criteria:

Table –2

S.

No.

Micro organism 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 Molds 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.

 

9.0 List of Annexure / Format:

S.

No.

Format Title Format

Number

Annexure

Number

No. of Pages
1 Preservative Efficacy Test report of Oral Preparations

 

10.0           References (if any):

10.1             USP-32

11.0 Reason for Revision:

11.1     Not Applicable

12.0 Abbreviations:

SOP                      : Standard Operating Procedure

QC                        : Quality Control

Annexure-I

Preservative Efficacy Test Report of Oral Preparations

Name of the Product
Batch No.
A.R.No
Date of analysis
Date of report

MEDIA DETAILS:

Day of Analysis 1st Day 14th day 28th day
Media used      
Lot No.:  

 

 

 

 

   
Autoclave Lot No.:  

 

 

 

   

 

Incubation Details:

Incubation Period

 

1st  Day 14th  day 28th  day
Incubation

Started at

/ Date

Bacterial Cultures

Incubator No:

(30ºC – 35ºC)

     

Fungal Cultures

Incubator No:

(20ºC – 25ºC)

     
Incubation

Completed at / Date

Bacterial Cultures

Incubator No:

(30ºC – 35ºC)

     

Fungal Cultures

Incubator No:

(20ºC – 25ºC)

     

 

Name of the Microorganism Day of Analysis

 

Dilution Considered in the Calculation

 

Test Count  

% recovery

= Final Count*   x 100

   Initial Count*

Escherichia coli

ATCC 8739

1st  Day      
14th  Day      
28th  Day      
Pseudomonas aeruginosa ATCC 9027 1st  Day      
14th  Day      
28th  Day      
Staphylococcus aureus

ATCC 6538

1st  Day      
14th  Day      
28th  Day      
Candida albicans

ATCC 10231

1st  Day      
14th  Day      
28th  Day      
Aspergillus niger ATCC 10231 1st  Day      
14th  Day      
28th  Day      
Day of Sample withdrawal Appearnce of the Product:
1st  Day  
14th  Day  
28th  Day  

* Initial Count denotes the Count of 1st Day Analysis and Final Count indicates 14th  and 28th  Day Analysis.

                                                    Acceptance Criteria
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.
Yeast and Molds No increase from the initial calculated count at 14 and 28 days.

 

 

Conclusion: The preservative added is effective/ not effective and the product complies/does not comply.

Analyzed By :

Date:

Checked by:

Date:

Reviewed by:

 

Date:

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