Nowadays, product safety is critical from the consumer’s point of view. Testing for antimicrobial efficacy is thus required for products like pharmaceuticals, cosmetics, and other personal care products. Considering the current threat posed by microbial resistance and product contamination across the globe, there is a need to evaluate whether products can resist microbial growth over an extended period. This is evaluated through a regulatory requirement known as the Antimicrobial Effectiveness Test (AET), which helps manufacturers verify the safety and efficacy of their products.

What is the Antimicrobial Effectiveness Test (AET)?

The Antimicrobial Effectiveness Test, also called Preservative Efficacy Testing, is a standardized method of testing the ability of a product to inhibit or kill microbial growth over a certain period.  They are most often applied to products that tend to be spoiled by microorganisms, such as pharmaceuticals, cosmetics, and personal care items containing water.

The purpose of an AET test is to ensure that a product’s antimicrobial agents, such as preservatives, are enough to prevent contamination during the shelf life and usage of the product. It simulates microbial contamination in the product by exposing it to known bacterial, yeast, or mold strains. After exposing the product to these contaminants, it will be kept under observation to see the microbial levels over time. The test, therefore, finds whether the product could successfully reduce the population size of microorganisms, thus ensuring consumer safety.

How is the Antimicrobial Effectiveness Test Performed?

The steps involved in antimicrobial effectiveness testing  are:

1. Selection of Microorganisms

 According to regulatory guidelines, the more commonly used microorganisms are :

• Staphylococcus aureus (gram-positive bacteria)
• Escherichia coli (gram-negative bacteria )
• Pseudomonas aeruginosa (gram- negative bacteria)
• Candida albicans (yeast)
• Aspergillus brasiliensis (mold)

2. Inoculation of the Product

Once microorganisms are selected, the product is inoculated with a known amount of microbial suspension. The inoculum size typically ranges between 100,000 (10^5) CFU/milliliter or gram of the product.

This inoculation process is a form of simulating microbial contamination that the product might experience in its lifecycle, from production to consumer use. This step is essential in determining whether preservatives or antimicrobial agents in the product can inhibit or kill the microorganisms.

3. Incubation and Sampling

Once the product has been inoculated, it is incubated under conditions favorable to the growth of microorganisms. During the incubation phase, periodic samples are drawn off to monitor for microbial numbers. Samples are usually taken on day 7, day 14, and sometimes day 28; however, this sampling may differ according to the type of product being evaluated.

4. Analysis of Results

The efficacy of the preservatives or antimicrobial agents is determined by comparing counts of microorganisms over the shelf life period to inoculum levels before the treatment. Results are often expressed as a decrease in microbial count on a logarithmic scale. Regulatory guidelines state the level of log reduction that is required for the product to pass the test.

For bacteria- an average 3-log reduction (99.9%) in 7 days is usually required.

For yeast and mold- 1-log reduction is typically acceptable within the same time frame.

Log reduction measures how effectively the product reduces the microbial population.

Antimicrobial Effectiveness Test Requirements and Conditions

The Antimicrobial Effectiveness Test, AET, shall fully comply with all the requirements and conditions to ensure that the results are viable and meet regulatory standards.

1. Regulatory Compliance

The results of the AET test will be primarily based on compliance  issued by the regulatory authorities, which are:

• United States Pharmacopeia (USP <51>)
• Chapter 5.1.3 European Pharmacopoeia (Ph. Eur.)
• International Organization for Standardization (ISO)

These guidelines clearly define the nature of the microorganisms to be tested, the testing process, and the acceptance criteria for ensuring product safety.

2.Tested Organisms

The AET is designed to evaluate the ability of the product to inhibit the growth of common microbial contaminants. Regulatory guidelines usually indicate that testing against specific microorganisms may include the following:

Bacteria: Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus

Yeast: Candida albicans

Fungi: Aspergillus brasiliensis

3.Test Conditions

AET is performed under simulated environmental conditions to make the real conditions in which a product would be exposed during storage and usage. The following are some of the main test conditions:

Temperature: Testing is usually conducted at room temperature (20–25°C) to simulate standard storage conditions.

Duration: Testing is carried out over a specific period, usually between 7 and 28 days, in which the time taken and effectiveness of the preservative system at controlling microbial growth are observed.

Challenge Inoculation: A known culture of microorganism added at the start of the test to challenge

4. Product Formulation 

The nature of the product tested in AET testing microbiology would determine the testing conditions Some of the factors that affect the test are as follows:

• Levels of pH: Some preservatives perform well in specific levels of pH. This is particularly so with parabens, where the formulation is applied at a pH level between 3-8.
• Water Content: High water contents in formulations create an environment that is favorable for microbial growth. Therefore, stronger or more effective preservative systems are needed.

5. Shelf-Life Considerations

Antimicrobial effectiveness testing must ensure that the product maintains its antimicrobial activity up to the shelf life. The study must be conducted in such a manner that the study represents the shelf life of the product so that the preservatives or antimicrobial agents would be effective at the time of product expiration. This applies to those products that are stored for longer periods of time, and some products are sold in multi-dose containers where repeated exposure of the preservatives to the contaminants is expected.

Conclusion

Antimicrobial effectiveness tests play an important role in ensuring the safety, efficacy and shelf-life of products that are susceptible to microbial contamination like pharmaceuticals, cosmetics, and personal care items. Complying strictly with regulatory guidelines and conditions ensures that the preservative or antimicrobial agents within a product will act in an effective manner against microorganisms and protect it throughout its shelf life.

At Microbe Investigations Switzerland, we are dedicated to providing comprehensive antimicrobial testing services to ensure the safety and compliance of your products. To learn more about our testing services or to schedule a consultation, please contact our experts today.