USP <51> – Preservative Challenge Test

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The USP Chapter 51 Preservative Challenge Test is the most common method used to gauge preservative effectiveness. Much like a Preservative Challenge Screen, it is used to evaluate the effect of preservatives in cosmetics, personal care products, and drug products. Preservatives are antimicrobial ingredients that are added to aqueous product formulations to help maintain the safety of the product by inhibiting the growth and reducing the amount of microbial contaminants.

The USP <51> challenge test utilizes 5 microorganisms (3 bacteria and 2 fungi) for challenge testing. Each of the microorganisms are known strains of pathogenic microorganisms and they represent a wide range of microbial physiologies.

The first time a product is challenge tested for antimicrobial effectiveness under the USP <51> method, a neutralization and recovery validation is required to ensure that the microorganisms are able to be recovered from the product if they are present.

Summary of the USP <51> Challenge Test Procedure

Prior to initiation of USP <51>, the product should be evaluated using the USP <61> – Microbial Enumerations Test for the presence of pathogens after manufacturing.

  • The product is separated out into 5 containers, each being challenged with one of the 5 method-specified microorganisms (S. aureus ATCC 6538, E. coli ATCC 8739, P. aeruginosa ATCC 9027, C. albicans ATCC 10231, and A. brasiliensis ATCC 16404) at a concentration of >1×105CFU/g or ml.
  • The initial concentration of each microorganism is determined by inoculating a control substance and using standard dilution and plating techniques.
  • At the time of test initiation, a separate volume, typically 1 ml or 1 g, of the product is diluted in a volume of chemical neutralizer broth, to be used in the neutralization and recovery validation.
  • The inoculated product is held at room temperature for a period of no less than 28 days.
  • The product is evaluated at specific intervals within the 28 day period. Evaluation intervals depend on the category of the product specified by the method. Click here for USP <51> product categories.
  • At each contact time, the inoculated product is chemically neutralized and plated using standard dilution and plating techniques.
  • After 48 hours of incubation, surviving microorganisms are counted, and the log reduction of each microorganism at each interval is reported.
  • The effectiveness of the preservative system is determined based on the USP <51> passing criteria.
  • OPTIONAL Customization: The product is re-inoculated with more microorganisms and allowed a longer incubation period (specified by study sponsor).
When to Conduct a USP <51> Preservative Effectiveness Test

It is important to determine if the preservative chosen for a product is compatible with the formulation soon after manufacture.

It may also be helpful to perform a second challenge test within 2-3 months of manufacture or as appropriate to the intended shelf-life as part of your stability testing regimen. This will aid in ensuring that, as the product ages, the preservative system is not breaking down and becoming less effective.

It is also necessary to re-evaluate preservative effectiveness in your product whenever the formulation, an associated manufacturing process, or product packaging changes occur.