3.7.2. Test for Efficacy of Antimicrobial Preservation (Challenge Test)

The effectiveness of antimicrobial protection was determined in accordance with the European Pharmacopoeia 10.0 (Ph. Eur. 10.0). The preparation demonstrates appropriate preservative properties if there is a significant decrease, or no increase, in the number of viable microbial cells in the inoculated preparation under the test conditions, after the specified time and at the specified temperature.

Test Microorganisms

With the reference to Ph. Eur. 10.0., the preservation efficacy test of the dermal product was performed with inoculation of the preparation with the standardized following microorganisms: Pseudomonas aeruginosa ATCC 9027, Staphylococcus aureus ATCC 6538, Escherichia coli ATCC 8739, Candida albicans ATCC 10231 and Aspergillus brasiliensis ATCC 16404. Each microbial strain was tested on the preparation separately.

Preparation of Inoculum

Each inoculum was prepared from a fresh culture of the aforementioned microorganisms. The individual cultures were incubated at the appropriate temperature for the appropriate time according to the indications of Ph. Eur. 10.0. The obtained microorganism suspensions had the following densities: for bacteria—10⁷–10⁸ CFU/mL, C. albicans—10⁶–10⁷ CFU/mL, A. brasiliensis—10⁶–10⁷ CFU/mL.

Method Description

The study began with an assessment of the effectiveness of the neutralizer, followed by an evaluation of the preservation efficiency of the preparation intended for cutaneous application.

The study was performed separately for each test sample and test microorganism. The test sample (20 g) was infected with the strain (0.2 mL) and stored at room temperature (20–25 °C). The detailed concentrations utilized for each strain, complying with the Ph. Eur. 10.0. criteria, are presented in the Supplementary Materials (Test reports S4–S6). At specified time intervals (Table 5), 1 g of sample was taken into 9 mL of neutralizer, thoroughly mixed and incubated (bacteria and C. albicans—32.5 ± 2.5 °C for 48–72 h, A. brasiliensis 22.5 ± 2.5 °C for 3–5 days). After the incubation period, the grown colonies were counted; the degree of log reduction was then determined and compared to the Ph. Eur. 10.0. criteria (Table 5—acceptance criteria).

In the antimicrobial preservation efficiency test, the number of surviving microorganisms was determined at specified time intervals over a 28-day period. For each microbial strain, the log reduction index was calculated as the number of viable microorganisms against the value obtained for the inoculum; this value was compared with the criteria defined by Ph. Eur. 10.0. Detailed information of the criteria A and B is described in Table 5. The detailed results of each strain reduction over time, as indicated by the Ph. Eur. 10.0. criteria, are presented in the Supplementary Materials (Test reports S4–S6).

Acceptance Criteria

The criteria for the evaluation of antimicrobial activity are presented in Table 5 (Section 2.6.2) and refer to the log reduction in each microorganism type.

The Ph. Eur. 10.0 expresses criteria A as recommended antimicrobial protection efficacy, implicating no additional preservatives are required. However, the antimicrobial protection efficacy level “B” might be accepted only in justified cases: for instance, if the A criterion cannot be achieved due to a significant risk of adverse reactions caused by the additional amounts of preservatives.