Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) testing define a test material's potency in terms of the concentration at which it will inhibit growth of (Minimum Inhibitory Concentration, or MIC) or completely kill (Minimum Bactericidal Concentration, or MBC) 1 x 106 (one million) challenge microorganisms during an 18-to-20-hour period of incubated (35 ± 2°C) exposure.
The MIC method measures the effect of decreasing concentrations of antiseptic over a defined period of time in terms of inhibition of microbial population growth. The concentration of drug required to produce the effect is defined as the Minimum Inhibitory Concentration, and is normally several hundred to thousands of times less than the concentration found in the finished dosage form.
The standard reference used as the basis for performing the Minimum Inhibitory Concentration (MIC) determination is the most current version of Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow Aerobically (currently, CLSI Doc. M7-A9, Clinical Laboratory Standards Institute [formerly, National Committee for Clinical Laboratory Standards, or NCCLS]) or anaerobically (CLSI Doc. M11-A8).
The product is serially diluted 1:2 in a growth medium suitable for supporting growth of each challenge microorganism, usually Mueller-Hinton Broth (MHB) or Mueller-Hinton Agar (MHA), to produce a dilution series of product in medium out to 1:32,768 (per BSLI SOP L-2045). However, depending on the type of product, the dilution series may not be carried out so far. To the tubes in the series each containing a 2.0 mL aliquot of the product serially diluted in growth medium now is added 1.0 mL of a 1 x 106 CFU/mL suspension of a challenge species. Following the 18 to 24 hours of incubated exposure, the tubes containing the challenge microorganism/product/medium suspension are examined to determine the highest dilution of product (and conversely, the lowest concentration of product) that completely inhibits growth of the microorganism, as determined by the naked eye. This dilution value (and/or product concentration value) is recorded as the Minimum Inhibitory Concentration value.
If an MBC determination is desired, measured quantities are transferred from the dilution suspension that represents the Minimum Inhibitory Concentration, and from the two or three dilution suspensions preceding the Minimum Inhibitory Concentration dilution, to plates of solid growth medium, which are then incubated for 24 hours at 37E± 2E. Following incubation, the plates are examined for growth of the challenge microorganism. That dilution of product (and/or the product concentration) that produces no growth is recorded as the MBC. Note that MIC/MBC testing procedures can be performed using 1:2 serial dilutions of product in nutrient agar medium (solid phase testing) or using 1:2 serial dilutions of product in nutrient broth medium (suspension phase testing). Unless the Sponsor specifically requires the agar-dilution method, BSLI will perform the MIC testing using the macrotube broth-dilution method, because it is much less materials- and time-intensive.
For products that must be tested under an IND, the FDA clearly specifies on page 31444 of the TFM what microorganisms must be used to challenge the products in vitro. For the Minimum Inhibitory Concentration study, the TFM states that the finished product, the product active principal, and the product vehicle must each be challenged with 25 ATCC strains and 25 fresh clinically isolated strains of each the 12 Gram-negative and 11 Gram-positive bacterial species and the two yeast groups of species (Candida albicans and Candida spp., not albicans). This, then, comprises a very large study that involves testing 1,250 strains of the total 25 bacteria and yeasts that are specified in the TFM versus each of the three product configurations.
In recent years, the FDA has backed off somewhat from this onerous requirement – although the finished product must be tested versus the 1,250 strains, as stated, the product active principal and the product vehicle need only be challenged with five ATCC strains and five fresh clinically isolated strains of each of the 25 bacteria/yeast species. Even so, this totals 250 strains that must be tested for both the active and the vehicle, and the overall TFM MIC study is quite massive and expensive – in total, 1,750 individual Minimum Inhibitory Concentration determinations. The FDA also allows some species substitutions (e.g., species of Bacteroides other than B. fragilis, and species of coagulase-negative Staphylococcus other than S. epidermidis, S. hominis, S. haemolyticus, and S. saprophyticus) to accommodate the scarcity of ATCC strains and/or clinical isolates of some of the species required by the TFM. And, acknowledging that, in general, alcohols are not antimicrobially active after about two 1:2 dilutions, the FDA does not require MIC data for products in which an alcohol is the only active component.
Although BSLI has performed several of the full-blown Minimum Inhibitory Concentration studies required by the TFM for IND-registered products, the majority of Minimum Inhibitory Concentration and occasional MBC studies are performed for proof-of-concept and marketing purposes. In these kinds of studies, BSLI generally recommends to clients that ATCC strains of all 25 of the species/strains listed in the TFM, plus a clinical isolate of each, be tested (i.e., a total of 50 challenge strains).