PRESERVATIVE EFFICACY TEST

 

Preservative Efficacy Testing (PET)

Preservative Efficacy Testing (PET) evaluates the effectiveness of a cosmetic or over-the-counter (OTC) pharmaceutical product’s preservative system, which is designed to inhibit the growth of microorganisms that could be introduced during manufacturing or by users during regular use.

By simulating these conditions, Preservative Efficacy Testing helps determine if products are capable of remaining free from harmful microbes, safeguarding consumer health and maintaining product integrity.

This testing is a critical safety and quality assurance measure for manufacturers in the health and beauty industries.

PRINCIPLE OF TEST:

Antimicrobial preservatives are added to products to prevent or limit microbial contamination, which can occur during normal conditions of storage and use.  The efficacy of an antimicrobial preservative may be enhanced or diminished by the active constituent of the preparation, or by the formulation in which it is incorporated, or by the container and/or closure being used as the final packaging material.

The test method must be qualified for the product under evaluation to ensure the correct diluent is used in assays for surviving microorganisms.

The product is inoculated with specified number of each challenge organism.  The inoculated product is held at room temperature for 28 days.  It is examined by the duplicate plate count method to determine the number of viable microorganisms which survive at each specified time interval.

A preservative efficacy test (PET), also known as an antimicrobial effectiveness test (AET) or challenge test, is a crucial procedure used to evaluate the ability of a product's preservative system to prevent the growth of microorganisms that may be introduced during manufacturing or consumer use.

The goal of a PET is to ensure that a product remains safe for its intended shelf life by effectively controlling microbial contamination. This is particularly important for multi-dose products (e.g., lotions, creams, eye drops) where repeated opening and use can introduce microorganisms.

Here is a general overview of a typical preservative efficacy test protocol:

1. Preparation of Test Materials

·         Test Sample: The product to be tested is divided into separate containers, one for each test microorganism. It is often recommended to use the final container closure system to simulate real-world conditions.

·         Test Microorganisms: A panel of standard microorganisms is used to represent common contaminants. The most common strains include:

o    Pseudomonas aeruginosa (ATCC 9027)

o    Staphylococcus aureus (ATCC 6538)

o    Escherichia coli (ATCC 8739)

o    Candida albicans (ATCC 10231)

o    Aspergillus brasiliensis (ATCC 16404)

o    Note: Other relevant environmental isolates from the manufacturing facility may also be included.

·         Preparation of Inoculum: The microorganisms are grown under specific conditions to a desired concentration, typically to produce a suspension of about 1×108 colony-forming units (CFU)/mL.

2. Inoculation and Incubation

·         Inoculation: A small volume (e.g., 0.5% to 1.0% of the product volume) of the prepared microbial suspension is added to each container of the test product. The final concentration of microorganisms in the product should be between 1×105 and 1×106 CFU/mL.

·         Mixing: The inoculated product is thoroughly mixed to ensure a homogeneous distribution of the microorganisms.

·         Incubation: The inoculated samples are incubated at a prescribed temperature, typically in the range of 20−25∘C or 22.5±2.5∘C, for a period of at least 28 days.

3. Sampling and Microbial Enumeration

·         Sampling Intervals: Samples are withdrawn from each container at specific time intervals to determine the number of surviving microorganisms. Standard time points often include:

o    Initial count (time 0)

o    Day 7

o    Day 14

o    Day 28

o    Some protocols may include additional time points, such as Day 2, Day 21, or re-challenge on a later date.

·         Neutralization: A crucial step is to neutralize the antimicrobial activity of the product's preservative system before plating the samples. This ensures that the microorganisms are not killed on the agar medium, allowing for accurate enumeration of the surviving population. Neutralizers such as polysorbate 80 and lecithin are commonly used.

·         Plate Counting: The neutralized samples are serially diluted and plated onto appropriate agar media. The plates are then incubated, and the colonies are counted. This process allows for the calculation of the number of viable microorganisms per gram or milliliter of the product at each time interval.

4. Calculation and Acceptance Criteria

·         Log Reduction Calculation: The log reduction is calculated by comparing the initial microbial count to the count at each sampling interval. The formula is as follows:

o    Log Reduction=log10​(Initial Count)−log10​(Count at Time t)

·         Acceptance Criteria: The product is deemed adequately preserved if the microbial counts meet the specific criteria outlined in the relevant pharmacopoeia or standard. The criteria vary depending on the product category (e.g., injections, topical products, oral preparations).

o    For example (simplified criteria):

§  Bacteria: A certain log reduction (e.g., 1 or 2 log) is required at Day 7, followed by a further reduction or no increase at Day 14 and Day 28.

§  Yeast and Mold: The count should not increase from the initial count, or a specified log reduction may be required.

Important Considerations

·         Suitability Testing: Before the official test, a "suitability of recovery" or "neutralizer validation" test is performed. This confirms that the chosen neutralization method effectively inactivates the preservative without harming the test microorganisms, ensuring accurate enumeration.

·         Environmental Isolates: Incorporating microorganisms isolated from the manufacturing environment can provide a more robust test, as these strains may have a higher resistance to the preservatives used.

·         Product-Specific Factors: The effectiveness of a preservative can be influenced by the product's pH, the presence of other ingredients, and the packaging. A thorough PET should account for these factors.

·         Shelf-Life and Stability: PET is often performed as part of a product's stability study to ensure the preservative system remains effective over its entire shelf life.