A preservative efficacy test (PET), also called a challenge test, evaluates whether your preservative system can control microbial growth when a product is contaminated during normal use. Shampoo bars may look like low-risk solid products, but they are wetted, handled, and stored in humid bathrooms, so a well-designed PET is essential if you are making a preserved or water-containing formulation.

1. Choose a recognized method and acceptance criteria

For cosmetics and shampoo bars, two common standards are used:

  • ISO 11930 - Cosmetic preservative efficacy standard, often preferred for personal-care products sold internationally.
  • USP <51> - Antimicrobial Effectiveness Testing, commonly used in the United States.

Both can be adapted for solid bars. ISO 11930 is generally more stringent for bacteria and fungi, so many cosmetic manufacturers use it as the default.

2. Prepare the test organisms

Use standard challenge organisms, typically:

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

Prepare fresh cultures and standardize the inoculum to approximately 108 CFU/mL per organism. You will add enough inoculum to achieve a final concentration of about 105-106 CFU per gram or per sample.

3. Prepare the shampoo bar sample

Solid bars require special handling because the preservative may not be evenly distributed and the matrix does not mix like a liquid. Two practical approaches are:

  • Shaved/ground method: Aseptically grate or shave a representative sample of the bar into a sterile container. Mix gently before testing. This gives a more homogeneous sample.
  • Surface inoculation method: For a more realistic use simulation, inoculate the intact bar surface. This can be done alongside the ground method.

If using the ground method, weigh about 10 g of shaved product per test organism and place it in a sterile container.

4. Inoculate the sample

Add the standardized microbial suspension directly to the sample. For example, add 0.1 mL of a 108 CFU/mL suspension to 10 g of product to achieve approximately 106 CFU/g. Mix thoroughly with a sterile spatula or glass rod to ensure contact between the inoculum and the product.

For surface testing, apply a known volume of inoculum over a marked area of the bar surface and allow it to air dry briefly in a sterile container.

5. Incubate and sample at defined intervals

Store the inoculated samples in the dark at 20-25 °C, which is typical for cosmetic PETs. At the following time points, remove a sample, neutralize the preservative, and plate for surviving organisms:

  • Day 0 - immediately after inoculation, within 30-60 minutes
  • Day 7
  • Day 14
  • Day 28

At each interval, extract the sample using a validated neutralizer such as Dey-Engley broth or a lecithin/polysorbate 80 solution. The neutralizer must be shown to stop preservative activity without killing the challenge organisms. Then perform serial dilutions and plate counts on appropriate microbiological media.

6. Include essential controls

Every PET must include:

  • Inoculum viability control: Shows the challenge organisms survive in the absence of preservative.
  • Neutralizer efficacy control: Confirms the neutralizer works.
  • Product sterility control: Confirms the uninoculated product does not introduce background contamination.

7. Interpret the results

Compare the surviving counts at each time point with the initial count. Typical acceptance criteria are:

ISO 11930 criteria for cosmetics

  • Bacteria: At least a 3-log reduction by day 7, and no increase from day 7 through day 28.
  • Yeast and mold: At least a 1-log reduction by day 7, and no increase from day 7 through day 28.

USP <51> category for topical/cosmetic products

  • Bacteria: At least a 2-log reduction by day 14, and no increase from day 14 through day 28.
  • Yeast and mold: No increase from the initial count at day 14 and day 28.

If the product fails, you may need to increase the preservative concentration, change the preservative system, adjust pH, reduce free water, or reformulate to improve preservative distribution in the solid matrix.

Practical tips for shampoo bars

  • Test both ground and intact-surface samples if the product will be used wet and stored solid.
  • Measure water activity; bars with very low water activity are less prone to microbial growth, but the wet surface can still be a risk.
  • Validate your neutralizer before running the full test, because solid product particles and preservatives can interfere with recovery.
  • Use multiple bars from different production batches if you want to assess batch-to-batch preservative performance.