and document PET experiments.

QA Officer: To review PET reports and ensure adherence to pharmacopeial standards.

QC Manager: To evaluate data and approve the test results.

4. Accountability

Head – Quality Control shall ensure compliance with this SOP and address any deviations arising during preservative efficacy testing.

5. Procedure

5.1 Sample Selection

1. Select a representative batch of gel containing final preservatives in intended concentration.
2. Ensure the sample has passed routine microbiological limits before initiating PET.

5.2 Test Organisms

Use the following standardized microbial strains:

• Escherichia coli (ATCC 8739)
• Staphylococcus aureus (ATCC 6538)
• Pseudomonas aeruginosa (ATCC 9027)
• Candida albicans (ATCC 10231)
• Aspergillus brasiliensis (ATCC 16404)

5.3 Inoculum Preparation

1. Cultivate strains in appropriate media and harvest cells in sterile saline or buffer.
2. Adjust inoculum concentration to achieve ~10⁵ to 10⁶ CFU/mL.

5.4 Inoculation and Incubation

1. Inoculate test gels separately with each microbial suspension under aseptic conditions.
2. Store inoculated samples at 20°C–25°C protected from light.

5.5 Sampling Schedule

Evaluate microbial viability at Day 0 (immediately after inoculation), Day 7, Day 14, Day 21, and Day 28.

5.6 Enumeration

1. Withdraw ~1 g sample at each time point and dilute in neutralizing buffer.
2. Plate on appropriate agar and incubate under suitable conditions for recovery.
3. Count colonies and compare to initial inoculum.

5.7 Acceptance Criteria

Comply with acceptance criteria as per USP <51>, EP or product-specific regulatory guidelines. For example:

• Bacteria: Not less than 2 log reduction at Day 14 and no increase thereafter.
• Fungi: No increase from initial count at any time point.

5.8 Documentation

1. Record all data in PET Worksheet and Microbiology logbook.
2. Attach raw data, microbial plates photographs (if required), and recovery charts to the report.

6. Abbreviations

• PET: Preservative Efficacy Testing
• CFU: Colony Forming Units
• USP: United States Pharmacopeia
• EP: European Pharmacopeia

7. Documents

1. Preservative Efficacy Test Worksheet – Annexure-1
2. PET Summary Report Template – Annexure-2
3. Microbial Inoculum Preparation Record – Annexure-3

8. References

• USP <51> Antimicrobial Effectiveness Testing
• European Pharmacopeia – PET Guidelines
• WHO Guidelines on Pharmaceutical Microbiology

9. SOP Version

Version: 2.0

10. Approval Section

	Prepared By	Checked By	Approved By
Signature
Date
Name
Designation	Jr. Production Chemist	QA Executive	Head – Manufacturing
Department	Gel Manufacturing	Quality Assurance	Manufacturing

11. Annexures

Annexure-1: Preservative Efficacy Test Worksheet

Sample Name	Batch No.	Organism	Initial Count (Day 0)	Day 7	Day 14	Day 21	Day 28	Remarks

Annexure-2: PET Summary Report Template

Test Organism	Log Reduction Day 14	Log Reduction Day 28	Complies with USP/EP	Conclusion
E. coli
S. aureus
P. aeruginosa
C. albicans
A. brasiliensis

Annexure-3: Microbial Inoculum Preparation Record

Date	Organism	Culture Media	Harvest Method	CFU/mL Achieved	Prepared By	Checked By

Revision History

Revision Date	Revision No.	Details	Reason	Approved By
05/06/2022	1.0	Initial Release	New SOP	QA Head
02/06/2025	2.0	Updated structure and annexures	Compliance Update	QA Head