Saili Dixit Gadkari

 NEUTRALIZATION VALIDATION IN MICROBIOLOGY A LOOK AT THE PURPOSE, VALUE, AND PITFALLS OF THE METHOD Validation is an important aspect of scientific experimentation. It is employed in almost all industries and is a crucial part of GMP that ensures your product to be of a particular quality. All methods used in any kind of industry are validated to be rest assured that the facility, systems, equipment, processes, procedures used are in control, which thereby imply a quality product. Microbiology is an integral part of various testing capabilities to detect, identify and quantify microorganisms from numerous sources and help in the proper prevention of contamination and disease occurrence. To aid in this process, neutralization validation is a critical procedure that is applied to the products of interest. A product possessing antimicrobial properties, due to a specific preservative in it, or by its inherent characteristics must be neutralized to determine if it harbours any viable organisms. WHAT IS NEUTRALIZATION VALIDATION? Neutralization validation is the method that validates a neutralizer, for its ability to inactivate or disable an antimicrobial agent. This is important in studies where presence and volume of the microbial load, if any, is to be estimated. Many factors affect the quantification of a solution’s antimicrobial activity and all these should be considered while designing the validation study. Some of these factors include; Nature of the challenge microorganisms Way in which the inocula of these organisms has been prepared Conditions of preparation and storage of inocula, conditions of conducting the neutralization validation test and of the microbial recovery The conditions of the validation must be the same as that of the test assay and all conditions should be standardized The neutralizer in combination with the product under study should not give rise to a toxic substance Neutralization of antimicrobials can be achieved by employing three most common methods of, 1) Chemical inhibition 2) Dilution 3) Filtration and washing USP chapter 1227 mentions some of the common neutralizers for chemical biocides. The compendial methods of neutralization validation (NV) are more or less similar. These include USP 1227 and ASTM E 1054. They state the method to be performed as follows; A validated method for neutralizing the antimicrobial characteristics of a product must meet two criteria, neutralizer efficacy and neutralizer toxicity. NEUTRALIZER EFFICACY - The neutralizer employed effectively inhibits the antimicrobial properties of the product. NEUTRALIZER TOXICITY - In doing the above, the neutralizer itself should not impair the recovery of the viable organisms. To establish the above criteria, three separate evaluations are carried out as follows, NEUTRALIZER EFFICACY: Suspension of test microorganism is prepared such that the suspension contains a low number of test microorganisms. A low number is used so that the microorganisms themselves do not help quench the active ingredient. An aliquot of the active ingredient suspension is added to the neutralizer under study, followed by addition of test substance containing active antimicrobial agent. Neutralizer effectiveness is assessed by qualitative and quantitative techniques measuring test microorganism growth NEUTRALIZER TOXICITY: A suspension containing a low number of test microorganisms is prepared. An aliquot of the active ingredient suspension is added to the neutralizer under study, followed by addition of Phosphate Buffered Saline. Neutralizer toxicity is assessed by qualitative and quantitative techniques measuring test microorganism growth. TEST ORGANISM VIABILITY: A suspension containing a low number of test microorganisms is prepared. An aliquot of this suspension is added to Phosphate Buffered Saline. Test organism viability is assessed by qualitative and quantitative techniques measuring test microorganism growth. The microbial recoveries are carried out on agar media for tests like Antimicrobial effectiveness test, Microbial limit tests and done using membrane filtration for Sterility tests. Advantages of NV: This method accounts for various antimicrobials, within numerous substrates and provides effective neutralization options The method accounts for effectiveness, toxicity, test organism viability and test material control within the appropriate neutralizer Sets the pattern for complete active antimicrobial agent inactivation The method is conducted in triple replicate to account for variability within antimicrobial test substance Disadvantages of NV: The method studies cells which have not been previously exposed to antimicrobials, and thus cells with a sub-lethal damage may not be accounted for thereby affecting the overall viability or recovery. Applications of NV: This method is commonly used for validation of neutralizers in Antimicrobial effectiveness tests Used to validate Microbial limit tests Used in Sterility tests Used to validate disinfectant efficacies Used to validate antimicrobial effects of medical devices All in all, NV is a beneficial and commonly applied method in Microbiology.