Optimised Neutralisation Strategies for Validating the Virucidal Efficacy of Micro-Chem Plus™ Against High-Containment Negative-Sense RNA Viruses

Effective disinfectant validation is essential for ensuring biosafety in high-containment laboratories when lethal pathogens are being handled. Micro-Chem Plus™ (MCP) is widely used in high-containment facilities for pathogen disinfection and routine decontamination. However, it induces severe cytotoxicity in cell culture, which may lead to an overestimation of its virucidal efficacy during disinfectant validation assays. To resolve this problem, we systematically evaluated the effects of three neutralisation methods (dilution, chemical neutralisation, and chromatographic separation) on MCP. The results showed that a 400-fold dilution with assay medium completely neutralised MCP, but reliable detection required high viral titers (≥6 log₁₀ TCID₅₀/mL). Chemical neutralisation using Dey-Engley broth showed inherent cytotoxicity, while chromatographic separation (MicroSpin S-400 HR/DetergentOUT™ columns) was the most effective but necessitated an additional 8-fold dilution. Validation in a BSL-4 facility with the risk group 4 (RG-4) agent Ebola virus confirmed MCP's concentration- and time-dependent virucidal activity, achieving a ≥6 log₁₀ TCID₅₀ reduction within 1-5 min. This study establishes an optimised framework for disinfectant validation in high-containment laboratories, addressing critical gaps in current protocols.