Sporicidal Registration Requirements

Sporicides are used on hard, non-porous inanimate surfaces and objects to kill spores. Bacterial spores are considered the most difficult form of microbial life to destroy.

The U.S EPA requires the generation of efficacy data under controlled testing conditions in order to make sporicidal claims on antimicrobial products. To generate sporicidal efficacy data, the AOAC Official Method 966.04 Sporicidal Activity of Disinfectants should be followed. Adding a hard, non-porous surface sporicidal claim to a liquid product involves exposing 60 carriers of stainless steel penicylinders inoculated with spores of Bacillus subtilis (ATCC 19659) and Clostridium sporogenes (ATCC 3584) to the sporicide. Three batches of the product at the LCL of the A.I.(s) listed on the CSF for the product should be tested. In addition to this requirement, a sporicide must also demonstrate an absence of growth (complete kill) on 60 carriers (30 carriers per organism for one batch) in a verification test. Verification tests must be performed in a separate lab or with a new set of staff.

In Europe, manufacturers perform suspension tests in accordance with method EN13704 to claim sporicidal activity. To make a sporicidal claim, a >3 log reduction of Bacillus subtilis (ATCC 6633) must be achieved with a contact time of less or equal to 60 minutes.

The most up to date version of EN13704 in 2018 was issued with a number of changes from the previous version issued in 2002, including changes to spore preparation and susceptibility checks of test organisms. 

EN13704 spore preparation requires 80% of the vegetative cells to sporulate and AOAC 966.04 requires 95% of the vegetative cells to sporulate.  Once the spore suspension is made, EN13704 requires a susceptibility test to check the spore suspension performance against two standard biocides, Glutaraldehyde and Peracetic Acid. The tests use two strengths per biocide, high and low concentrations. To meet EN13704 acceptance criteria, the spore suspension must demonstrate survival (<3 log reduction) at the low concentration and kill (>3 log reduction) at the high concentration. AOAC 966.04 requires a Hydrochloric Acid resistance test to check the spore suspension performance against a 2.5 M HCl standard. To meet AOAC 966.04 acceptance criteria, the spores must resist 2.5 M HCl for greater than or equal to 2 minutes.

The use of spore suspensions in the AOAC sporicidal test, and the careful preparation and use of spore suspensions in EN13704 ensures that products that claim to be sporicidal are suitably challenged during efficacy testing.

Quaternary Ammonium Compound Sporicidal Efficacy

Quaternary Ammonium Compounds (QACs) are cationic surface-active agents and have a broad range of activity against microorganisms, albeit that they are generally more effective against Gram-positive bacteria at lower concentrations than Gram-negative bacteria. They are considerably less effective against spore formers³. 

Many biocides are bactericidal or bacteristatic at low concentrations for non-sporulating bacteria, including the vegetative cells of Bacillus and Clostridium species. High concentrations of some biocides may achieve sporicidal efficacy (e.g. for glutaraldehyde and CRAs). 

By contrast, even high concentrations of alcohol, phenolics, QACs, and chlorhexidine lack a sporicidal effect (although sporicidal efficacy may be possible for chlorhexidine when used at elevated temperatures)⁴. 

QACs are sporostatic; they inhibit the outgrowth of spores (the development of a vegetative cell from a germinated spore) but not the actual germination processes (development from dormancy to a metabolically active state)².

QACs are likely to show some degree of efficacy against vegetative cells of Bacillus and Clostridium species, however QACs are not capable of destroying the spore itself.

Sporicidal claims require testing to be performed against spores, not vegetative cells. To maintain control within the cleanroom setting, biocidal agents with sporicidal activity against the spores themselves should be employed.

[1] McDonnell, G, Russell AD. Antiseptic and Disinfectant: Activity, Action and Resistance. Clin Micrbiol. 1999 Jan; 12(1):147-179.
[2] Russell AD. Bacterial spores and chemical sporicidal agents. Clin Microbiol Rev. 1990 Apr; 3(2):99-119.
[3] Sandle, T. The CDC Handbook: A Guide to Cleaning & Disinfecting Cleanrooms. 1–27.
[4] Shaker L A, Russell A D, Furr J R. Aspects of the action of chlorhexidine on bacterial spores. Int J Pharm. 1986;34:51–56.