European Pharmacopoei 11.0, Chapter 5.1.11.Bactericidal,fungicidaloryeasticidalactivityof medicinal antiseptics products.

Test accredited by ENAC (Spanish National Accreditation Entity).

Test with the Certificate of Good Laboratory Practices (GLPs).

This section of the Pharmacopoeia describes a suspension test that can be used for the determination of antimicrobial activity in antiseptic drugs miscible with water and intended for administration by direct contact with the skin or mucous membranes. The extent of testing depends on the claimed antimicrobial activity of the product. The test determines if a product exhibits bactericidal, yeasticidal and/or fungicidal activity.

To evaluate the bactericidal activity, the strains of bacteria Staphylococcus aureus, Enterococcus hirae, Escherichia coli and Pseudomonas aeruginosa are used, for the yeasticidal activity, the test is performed with Candida albicans, and for the fungicidal activity, with Candida albicans and Aspergillus brasiliensis. The test strains are mixed with the product at the concentration requested by the client and with the indicated interfering substance (3g/L bovine albumin, for a final assay concentration of 0.3g/L bovine albumin) and kept at 33ºC, for 5 minutes for bactericidal activity and 15 minutes for yeasticidal or fungicidal activity. According to the Pharmacopoeia, additional contact times may be chosen, according to the stated use of the antiseptic medicinal product.

To consider the product bactericidal, a logarithmic reduction of 5 or higher in the number of colony forming units (CFU) must be demonstrated. To consider the product levuricidal or fungicidal, a logarithmic reduction of at least 4 in the number of viable cells in the number of colony forming units (CFU) must be demonstrated.

The procedure can be carried out by the dilution-neutralization method or by the filtration method. The concentration of the antiseptic product test solution shall be, if possible, 1.25 times the in-use test concentration because it is diluted to 80 per cent during the test and the method validation. Therefore, the maximum concentration of the test, when used pure (100%), is 80% due to the dilution that it undergoes in the test mixture.