microbial growth over a defined period as per pharmacopeial standards.

2. Scope

This SOP applies to Analytical Method Development and Quality Control personnel performing preservative efficacy testing for eye drop formulations during formulation development, stability studies, and product release phases.

3. Responsibilities

• Microbiologist: Performs inoculation, sampling, enumeration, and interpretation of results.
• QC Analyst: Assists with dilutions, incubation, and data logging.
• QA Executive: Reviews preservative efficacy test results and approves final report.

4. Accountability

The Head of Analytical Method Development is accountable for ensuring that PET is executed using validated methodology in accordance with USP <51> and EP 5.1.3.

5. Procedure

5.1 Microorganisms Used

Challenge the formulation with the following ATCC organisms:

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

5.2 Inoculum Preparation

1. Grow each microorganism separately on suitable media (e.g., TSB, SDA) for 18–24 hours.
2. Harvest cells in sterile saline and adjust turbidity to match 0.5 McFarland standard.
3. Determine CFU count using plate count method to achieve a final challenge of 1×10⁵ to 1×10⁶ CFU/mL in the product.

5.3 Inoculation of Test Product

1. Using aseptic technique, inoculate the eye drop formulation with each organism to achieve the desired CFU concentration.
2. Mix well without compromising sterility.

5.4 Sampling Schedule

Collect 1 mL aliquots from each inoculated test unit at the following time intervals:

• T = 0 (initial count)
• Day 7
• Day 14
• Day 28

5.5 Enumeration of Microorganisms

1. Perform serial dilution of each sample.
2. Plate suitable dilutions on selective agar (e.g., MHA, SDA) and incubate:
   • Bacteria: 30–35°C for 3–5 days
   • Fungi: 20–25°C for 5–7 days
3. Count colonies and express results in CFU/mL.

5.6 Acceptance Criteria (USP <51>)

• Bacteria: ≥ 3 log₁₀ reduction by Day 14 and no increase thereafter
• Fungi: No increase from initial count at Day 14 and Day 28

5.7 Data Analysis and Interpretation

1. Calculate log reduction from initial count (T=0) at each time point.
2. Evaluate trends across all organisms and verify against pharmacopeial standards.
3. If criteria not met, investigate formulation preservative system and repeat validation.

6. Abbreviations

• PET: Preservative Efficacy Testing
• CFU: Colony Forming Units
• SDA: Sabouraud Dextrose Agar
• USP: United States Pharmacopeia
• EP: European Pharmacopoeia

7. Documents

1. Preservative Efficacy Data Log – Annexure-1
2. Inoculum Preparation Log – Annexure-2
3. PET Validation Report – Annexure-3

8. References

• USP <51>: Antimicrobial Effectiveness Testing
• EP 5.1.3: Efficacy of Antimicrobial Preservation
• ICH Q6A: Specifications – Test Procedures and Acceptance Criteria

9. SOP Version

Version: 2.0

10. Approval Section

	Prepared By	Checked By	Approved By
Signature
Date
Name	Dr. Nisha Verma	Ajay Kulkarni	Sunita Reddy
Designation	Microbiologist	QA Reviewer	Head – AMD
Department	Analytical Method Development	QA	Analytical Method Development

11. Annexures

Annexure-1: Preservative Efficacy Data Log

Organism	Day 0 (CFU/mL)	Day 7	Day 14	Day 28	Log Reduction	Status
S. aureus	1.0×105	1.2×102	<10	<10	≥ 3.0	Pass

Annexure-2: Inoculum Preparation Log

Organism	Medium	Growth Time	CFU/mL Adjusted	Prepared By
C. albicans	SDA	24 hrs	1.1×106	Microbiology Analyst

Annexure-3: PET Validation Report

Preservative efficacy testing performed on batch OPH-249 met the requirements of USP <51> with ≥ 3 log reduction in bacteria and no fungal growth by Day 28. Preservative system validated for product shelf life up to 24 months.

Revision History:

Revision Date	Revision No.	Details	Reason	Approved By
21/05/2025	2.0	Updated log reduction criteria and added EP reference	Annual Review	Sunita Reddy
10/04/2022	1.0	Initial SOP Release	New SOP	QA Head