The AOAC use dilution test, its pass criteria and EPA's ATP
The use dilution test checks liquid disinfectants on 60 steel carriers. Hospital claims pass at no more than 3/60 and 6/60 positive carriers.
The use dilution test and EPA's pass criteria
The AOAC use dilution test is the carrier-based disinfectant efficacy test for liquid products on hard surfaces, run at the label use dilution (EPA MB-05-17). For an EPA hospital claim, each of three batches must pass with no more than 3 positive carriers out of 60 for Staphylococcus aureus, and no more than 6 out of 60 for Pseudomonas aeruginosa (OCSPP 810.2200).
How the use dilution test is run
EPA's SOP MB-05-17, revised February 21, 2023, covers dilutable and ready-to-use liquids. It is still the version on EPA's methods page, last updated March 10, 2026 (EPA methods). EPA cites AOAC 955.14 for Salmonella enterica, 955.15 for S. aureus and 964.02 for P. aeruginosa. The strains are ATCC 10708, ATCC 6538 and ATCC 15442 (EPA MB-05-17).
Labs soak polished type 304 stainless steel penicylinders (8±1 mm outer diameter) in culture, then dry them. Each of 60 test carriers goes into its own tube of 10 mL disinfectant for the label contact time. The test temperature is 20±1°C unless another temperature is specified. Carriers then move to a neutralizing broth and incubate for 48±2 hours at 36±1°C. Growth in the primary tube, or in a required secondary subculture, makes that carrier positive (EPA MB-05-17).
Control counts check the inoculum. Mean log density must be 6.0 to 7.0 for S. aureus and P. aeruginosa, and 5.0 to 6.0 for S. enterica (EPA MB-05-17).
Disinfectant efficacy pass criteria
For hard-surface disinfectants with public-health claims, 40 CFR 158.2220 requires OCSPP 810.2200 data (eCFR). The February 2018 guideline (compliance from August 28, 2019) calls for three batches at the lower certified limit, 60 carriers per organism, each batch on a different day. Products should meet the standard at a contact time of 10 minutes or less. Each batch has these limits (OCSPP 810.2200):
| Claim | Organisms | Max positive carriers per 60 |
|---|---|---|
| Limited spectrum | S. aureus or S. enterica | 3 (S. aureus), 1 (S. enterica) |
| Broad spectrum | S. aureus plus S. enterica, or S. aureus plus P. aeruginosa | 3 for S. aureus, plus 1 for S. enterica or 6 for P. aeruginosa |
| Hospital/healthcare | S. aureus ATCC 6538 and P. aeruginosa ATCC 15442 | 3 and 6 |
Additional bacteria claims use two batches at nominal concentration, 10 carriers each, and zero positives allowed (OCSPP 810.2200).
The Salmonella limits differ between documents. MB-05-17 allows up to two positives for S. enterica. For registration, EPA's 2019 FAQ says the standard "remains at one positive carrier out of 60" (EPA FAQ).
Older references allow only one positive for S. aureus and P. aeruginosa. AOAC approved the 3/60 and 6/60 limits on August 23, 2013, and EPA adopted them for registration and post-registration testing (EPA UDM revision). Spray and towelette methods still require a kill on 59 of each set of 60 carriers (OCSPP 810.2200).
Phenol coefficient test
AOAC 955.11 is the phenol coefficient method against Salmonella typhi (PR Notice 2001-4). It doesn't support an EPA end-use efficacy claim. EPA's 1982 guidelines called phenol coefficient tests "unacceptable as documentation of efficacy for end-use claims". They were acceptable only for products intended only for formulation purposes (EPA Subdivision G). The 2018 guideline doesn't list the method (OCSPP 810.2200). That same 2001 notice ended phenol resistance checks of test cultures and called for at least 10^4 CFU per carrier in AOAC carrier tests (PR Notice 2001-4).
EPA Antimicrobial Testing Program
The Antimicrobial Testing Program was EPA's post-market check of hospital disinfectants and tuberculocides. It began in 1991 after a GAO report (EPA APEP). Its purpose was to make sure hospital disinfectants, sterilants and tuberculocides on the market kept meeting efficacy standards. A failing product could lose its hospital or TB claims from the label, be voluntarily cancelled by the registrant, or face EPA stop-sale orders (EPA ATP). Samples were collected by federal or state inspectors, or bought on the market. Testing ran at EPA's microbiology lab in Maryland and at state agriculture labs in Michigan, North Carolina and Ohio (OIG 09-P-0152).
In 2005 and 2006 testing, 29.5% of hospital disinfectants failed, and 40% of tuberculocides failed. As of February 2009, EPA had tested 325 of 671 registered products (OIG 09-P-0152).
OIG 09-P-0152 described how EPA handled results, using the pre-2013 bar of no more than one positive carrier out of 60, not today's 3/60 and 6/60 limits:
- 1 or fewer positives: the manufacturer was notified.
- 2 to 3: notice, plus a label-claim change, a retest under different conditions at an independent lab, or a retest of a reformulated product.
- 4 or more: referral to EPA's enforcement office (OECA) for an enforcement case review.
The manufacturer had 45 days to respond, or the product could face suspension or cancellation under FIFRA section 6 (OIG 09-P-0152). Enforcement tools included Stop Sale, Use or Removal Orders and civil administrative complaints (OIG 09-P-0152).
EPA suspended the ATP in November 2017 (EPA APEP). Its planned replacement, the Antimicrobial Performance Evaluation Program, is a risk-based draft released for comment in August 2019. The APEP page, updated June 25, 2026, still says EPA expected a final strategy in 2020 and implementation in 2022. Check that page before treating APEP as an active testing program (EPA APEP).
The public results list is still the March 2018 PDF. "Agency Taking Action" includes products still under testing or review, as well as products where EPA is acting. It doesn't by itself mean the product failed (EPA tested products).
You can also ask RegAffairs AI about the use dilution test and see its cited sources.