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.
