Nitrogen Dioxide Sterilization Follows Log-Linear Microbial Inactivation Kinetics Using <i>Geobacillus stearothermophilus</i> Biological Indicators

The primary purpose of this study was to determine the inactivation kinetics of <i>Geobacillus stearothermophilus</i> biological indicators (BIs) exposed to nitrogen dioxide (NO₂) gas in the presence of humidity. BIs inoculated with 6 log₁₀ <i>G. stearothermophilus</i> spores were used as a test substrate. Three BI lots manufactured from each of three different BI spore crops were evaluated. Test cycles were run at room temperature with approximately 80% relative humidity. Direct enumeration methods were used to quantify the resistance of spores with surviving populations greater than approximately 50 colony forming units (CFU). Fraction negative methods were used to estimate the surviving spore populations in the quantal region_. The methods were combined in order to show spore inactivation from 6 log₁₀ to approximately -2 log₁₀ The <i>D</i> value and coefficient of determination (r²) were calculated. Over 100 direct enumeration and fraction negative cycles were completed at a fixed NO₂ concentration varying only exposure time. Process parameters were maintained over all cycles. Empirical data confirmed a log-linear relationship over an 8 log₁₀ population range with r² values greater than 0.8, allowing for extrapolation of the curve to achieve a sterility assurance level (SAL) of 10^-6 Study outcomes were comparable for all manufactured BI lots. NO₂ sterilization follows first-order log-linear microbial inactivation kinetics, which is consistent with a mechanism of action based on a single active species. This is the first study to report on the microbial inactivation kinetics of NO₂ sterilization.