PRESERVATIVE EFFICACY TESTING: GUIDELINES TO AN AUTOMATED SIMPLIFIED TESTING SYSTEM

Introduction

The ultimate purpose of the Preservative Efficacy Test (PET) is to determine the effectiveness of the preservative(s) present in a cosmetic, toiletry, or pharmaceutical product.  USP Chapter <51> ANTIMICROBIAL EFFECTIVENESS TESTING describes the type of products to be tested (categories), the specified microorganisms to be used for testing, and the inoculum and log reduction amounts required per category of product.  The product to be tested is inoculated with a high number of bacteria, yeast, and mold, and the reduction in the initial inoculum amount is calculated over a 28 day period.

Current Methodology

Until now, the only way to perform PET was by the plate count method.  Typically, a product is inoculated with a high number of organisms (usually 105 - 107), and after 7, 14, 21, and 28 days, samples of that inoculated product are tested to determine the log reduction that occurred to the organisms when subjected to the preservative system.  Because it is not known how effective the preservative system is, serial dilutions have to be plated to determine the number of organisms remaining in one gram of product.  As a result every organism require a number of plates, dilution bottles and tips as shown in figure 1.

Advantages of BioLumix Simplified Automated System

The main advantages the customer gains using BioLumix are savings on time, labor, and materialsPreservative Efficacy Test

The BioLumix assay (Figure 2) takes about 75% less hands-on labor as compared to the standard plate count method, and the simplicity of the BioLumix method is unparalleled.  Ease of use, less materials (as seen in comparing Fig1 to Fig2), and less dilution reduces the chance of error.  After inoculation of product with pure cultures, the BioLumix method allows the operator to perform testing in 4 easy steps:

  1. Weigh 1.0 gram of inoculated product to a sterile sample bag.
  2. Add 9.0 mL neutralizing broth and allow to sit for up to 45 minutes.
  3. Add 1.0 mL to the BioLumix vial.
  4. Enter the vial into the BioLumix Instrument and begin the automated testing.

Pre-programmed calibration curves as shown in Figure 3 are used to generate the colony forming unit count per gram of product based on the detection time of the curve.  This eliminates tedious counting of multiple plates and interpretation of results when working with thick or waxy products, or products containing materials that may resemble bacterial or yeast colonies.  The BioLumix method also saves time in obtaining results.  For each day of sampling, BioLumix reduces the time-to-results for bacteria from 48 hours to 24 hours and from 5-7 days for yeasts and mold to 48 hours.  The BioLumix system uses a single vial to replace a number of plates, dilution bottles and pipet tips, thereby reducing disposable costs.calibration curves

Example of Results Obtained

The example, show the results of PET obtained with eye drops with and without benzalkonium chloride.  The results obtained by both the BioLumix system (BL) and the plate count methodology are shown in the two table below.

results obtained by both the BioLumix system (BL) and the plate count methodology

results obtained by both the BioLumix system (BL) and the plate count methodologyThe data by both methods shows that in the product with the inhibitor benzalkonium chloride (Table1) after 7 days all bacteria are reduced by more than 5 log cycles, as are the counts of Candida. It took 14 days for Aspergillus to be reduced by 5 log cycles.

When the product did not contain benzalkonium chloride (Table 2) and as a result was not properly preserved, only P. aeruginosa was reduced by 5 logs after 7 days. E. coli took 14 days to reduce the numbers by 5 logs. The other organism had a very slow reduction over time.  The counts of Aspergillus were reduced by less that 2 log cycles.  The results show that the two methods yield very similar results.

Many other products were tested with the BioLumix PET methodology including scrubs, creams, lotions, Shampoos, hand and facial cleansers to name a few, and equivalency with the plate count methodology and reproducibility of results was demonstrated for all products.

The BioLumix system allows the operator to save time, materials, and money by drastically cutting hands-on labor and time-to-results.  If offers labor reduction by 75-80% of the labor required for the Petri dish method, with much less disposable used, faster time to results and good correlation with current methodology.  It allows for the creation of a paperless laboratory.  The system automation provides automated data achieving, and automated reporting including log reduction calculations.