ASTM E1054

Neutralization Evaluation Test

Summary

The effectiveness of antimicrobial agents incorporated into disinfectants, sanitizers, and antiseptics is measured by their ability to kill microorganisms within a specified exposure (contact) time. Therefore, determining the antimicrobial effectiveness requires complete and immediate inactivation (neutralization) of the antimicrobial agent. Ineffective or incomplete neutralization allows the antimicrobial agent to continue killing the microorganism beyond the exposure time resulting in an overestimate of antimicrobial activity. 

Neutralization may be achieved through physical methods (filtration, dilution, secondary subcultures) and/or chemical means (addition of reagents to the recovery broth/diluent with known neutralizing properties). For each efficacy test, neutralization procedures should be conducted at the completion of the exposure time to demonstrate that the neutralizer employed inactivates the active ingredients and does not possess any antimicrobial activity itself. 

ASTM E1054 provides guidelines for validating the effectiveness of neutralization with recovery on semi-solid medium, liquid medium, or by filtration. Each recovery procedure is divided into four separate tests based on the following:

Test Overview:

• Test A – Neutralizer Effectiveness: the neutralizer is inoculated with a low concentration of the test microorganism. An aliquot of the test product is added to the inoculated neutralizer, the solution is mixed and sampled at two intervals (immediately and at 30 minutes).
• Test B – Neutralizer Toxicity: the neutralizer is inoculated with a low concentration of the test microorganism. An aliquot of sterile buffer solution is added to the inoculated neutralizer (resulting in the same dilution ratio as used in Test A), the solution is mixed and sampled at two intervals (immediately and at 30 minutes).
• Test C – Test Organism Viability: sterile buffer solution is inoculated with a low concentration of the test microorganism. The solution is mixed and sampled at two intervals (immediately and at 30 minutes).
• Test D – Test Material Control: the prepared product is inoculated with a low concentration of the test microorganism. The solution is mixed, held for an exposure time necessary to allow detection of antimicrobial effect and sampled.

Neutralization is considered adequate if the results from Test A are not significantly different from the results of Test C, and if the results from Test D are significantly smaller than the results from Test C. 

The neutralizer is considered non-toxic if the results from Test B are not significantly different from the results of Test C.