BioScience Laboratories, Inc. works in the customer service environment dealing with in-vitro and in-vivo (clinical efficacy) evaluations of many simple and complex antimicrobial products. One of the most critical aspects of a study’s performance is providing useful and sufficient neutralization/inactivation of the products being tested. In brief, proof of neutralization is necessary to ensure the validity of the test method.
When an antimicrobial product is to be evaluated, the neutralization process must be validated. If no neutralizer is used, a product continues to kill microorganisms, even after the samples have been plated and incubated. For example, an investigator could not state that a product kills X number of microorganisms in 30 seconds, because without a neutralizer, there is nothing to prevent the product from continuing to work beyond the 30-second time frame. This results in over-stated efficacy, because the product appears to kill more microorganisms than it actually does.
Depending upon the active ingredient(s) in a test product, neutralization is accomplished in one of four ways: 1) chemically, through the addition of various neutralizing agents (i.e., lecithin, Tween 80, catalase, etc.), 2) by dilution of the active ingredient to a sub-inhibitory level, 3) by combining the first two conditions, or 4) by filtration. One of the first questions BioScience Laboratories, Inc. asks a potential customer is “what is the active ingredient(s) in your product?”
BioScience Laboratories has over 25 years' experience working with many single antimicrobial ingredients, as well as combinations of products, and utilizes an effective neutralization formula or dilution level that works. However, there are cases of new ingredients and ingredient combinations for which a study design provides a “best guess” neutralization formula for immediate inactivation of a given product. It may require additional research to develop, confirm, and validate a neutralization method when confronted with new or innovative products.
Turning our focus to published documents, the ASTM E1054-08 (2013), method for Standard Test Methods for Evaluation of Inactivators of Antimicrobial Agents, provides excellent guidance on methodologies for demonstration of adequate neutralization. All neutralization verification procedures should, at minimum, include the following:
1) An arm/phase that provides data demonstrating that the neutralizing fluid immediately and completely neutralizes a set concentration of a product. If neutralization is not complete or immediate, residual “killing” activity may occur – a bacterial reduction reported for a 15-second exposure time may actually be a reduction for 30 seconds, 1 minute, or longer. If this happens, it is possible that a product may be reported as being more effective than it actually is.
2) An arm/phase demonstrating that the neutralizing fluid itself is not toxic to the organism(s) tested. If the neutralizer exhibits some antibacterial activity, it may actually “boost” the apparent antimicrobial activity of a product. Again, the possibility exists that a product may be reported as being more effective than it actually is.
3) An arm/phase providing data showing the starting bacterial challenge population. The data from this arm/phase are the basis of comparison for the previous phases – in general, neutralization procedures should employ a LOW population of bacteria, in terms of CFU/mL. If a challenge population is too high, a neutralizing system may appear to be effective when, in fact, it is not.
4) REPLICATION – this allows for a statistical comparison of the data – PROOF that the neutralizing system is effective.
In summary, verification of neutralization must be performed for all in-vitro and clinical efficacy evaluations of antimicrobial products to ensure the validity of the data. Depending upon the type of evaluation and the familiarity with the active ingredient(s), this verification may be conducted in advance of an efficacy evaluation for an unfamiliar active ingredient, or concurrent with the efficacy evaluation for a familiar active ingredient – as long as it is conducted using the same test formulation.