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 CulturesIncubator No: (30ºC – 35ºC) |
|||
Fungal CulturesIncubator No:(20ºC – 25ºC) |
||||
Incubation
Completed at / Date |
Bacterial CulturesIncubator No:(30ºC – 35ºC) |
|||
Fungal CulturesIncubator 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: |