Comparative evaluation of hydrogen peroxide sporicidal efficacy by different standard test methods

There are different sporicidal standard tests with various specifications to deal with products that are claimed for sporicidal activity. The aim of this study was to compare the 7% H₂O₂ sporicidal efficacy against Bacillus subtilis spores using different standard test methods.

The 7% H₂O₂ sporicidal efficacy against Bacillus subtilis spores was determined according to the AOAC MB-15-04 standard of carrier test and two standard suspension tests (BS EN 13704, AFNOR NF 72-230) in both clean and dirty conditions and by using different interfering substances including bovine serum albumin, yeast extract and skimmed milk.

The results of suspension tests with 3 × 10⁵ and 2 × 10⁷ CFU/ml of B. subtilis spore concentration demonstrated that the higher spore counts lead to lower efficacy of 7% H₂O₂. Also, the sporicidal activity of 7% H₂O₂ was reduced in the presence of interfering substances. Bovine serum albumin, yeast, and skimmed milk showed similar interfering effects in suspension test with 3 × 10⁵ CFU/ml. While, in suspension tests with higher initial spore count (2 × 10⁷ CFU/ml) severity of interfering effects were intensified and distinct. Our results indicated that the carrier sporicidal test in comparison with suspension tests required more contact time to kill B. subtilis spores.

The results of this study showed that it is reasonable to use interfering substances and inoculated carriers in accordance with actual conditions of product usage in a sporicidal test. Interfering substances may reduce the contact surface between H₂O₂ and test spores; therefore, the sporicidal efficacy of H₂O₂ was diminished. So applying suspension test in clean condition to verify the claim of sporicidal activity is strongly discouraged.