(M1030-03-16) Method Development for Antimicrobial Efficacy Test in Anhydrous Based Products

Purpose: Traditional methods for Antimicrobial Efficacy Tests listed in USP < 51 >, ISO-11930, EP 5.1.3, and JP < G4-3-170 > are designed to assess the antimicrobial properties of nonsterile aqueous formulations and utilize aqueous-based vehicle inoculums. Traditional method limitations to non-aqueous-based formulations include volatility, hydrophobicity, and water -oil compatibility and can be challenging to overcome. If an anhydrous-based formulation is tested for AET using an aqueous-based inoculum, the results are inconsistent. Modifications and development of standard methods need to be adopted to overcome these incompatibilities. Utilizing an anhydrous-based vehicle for inoculums, such as mineral oil and Tween 20, the efficacy of anhydrous-based formulations’ antimicrobial properties can be adequately challenged.

Methods: A comparative study was conducted for Antimicrobial Efficacy for an anhydrous product utilized and aqueous-based inoculum, saline, versus two anhydrous-based inoculums, mineral oil and Tween 20. Inoculums were prepared by adding organisms to aliquots of the inoculums using sterile loops ad mixing to ensure homogeneity. For anhydrous-based inoculums, homogeneity was achieved using sterile glass beads. Traditional methods of standardizations were used for the aqueous based inoculum, which include use of a spectrophotometer and McFarland standards. For mineral oil inoculum, standardization and adjustments were made using McFarland standards to ensure inoculum was approximately 10⁷ to 10⁸ CFU/mL. For Tween 20 inoculum, standardization was achieved by conducting a pre-read to ensure inoculum as approximately 10⁷ to 10⁸ CFU/mL. Products were inoculated with inoculums to not exceed 1% of the total volume of the product. At each timepoint, product was mixed well before plating and plates were incubated per their respective organisms. Sterile glass beads were used to facilitate the product’s homogeneity in neutralizing broths prior to plating.

Results: Control plates confirmed theoretical initial inoculum levels in formulations were between 10⁵ to 10⁶ CFU/mL as required in USP < 51 > for both aqueous and anhydrous-based inoculums. Results for anhydrous-based formulation inoculated with aqueous-based inoculum were inconsistent, with growth varying across time points. During plating time points, the separation of aqueous-based inoculum and anhydrous formulation was noted. No confirmation could be done on whether the aqueous-based inoculum was properly homogenized with the formulation during the entire study. As the log reductions were more consistent for the anhydrous-based inoculum and a steady decrease in growth was observed, it can be confirmed the inoculum was properly homogenized with the formulation during the entire study.

Conclusion: For Antimicrobial Efficacy Testing of anhydrous products, it is important to consider the formulation’s inherited anhydrous properties. Usage of aqueous-based inoculums for anhydrous-based formulations could lead to inconsistent results. Anhydrous-based inoculums, such as mineral oil and/or Tween 20, are better suited to inoculate anhydrous-based products, providing consistent results and confirmation on homogeneity during the study.

References: PCPC M6- A Method for Preservation Testing of Atypical Personal Care Products
ISO 11930 Cosmetics — Microbiology — Evaluation of the antimicrobial protection of a cosmetic product
EP 5.1.3 Efficacy of Antimicrobial Preservation
JP Preservatives-Effectiveness Tests

Tabel 1: Results of the AET test