EPA Registration 1677-226 

Vol.2 
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I BACKGROUND 

 The product, Virasept (EPA Reg. No. 1677-226), is a registered one-step 

disinfectant (bactericide, virucide, and fungicide), tuberculocide, and deodorizer for use 

in hospitals, nursing homes, institutions, schools, beverage/food processing plants, and 

daycare centers. The submitted efficacy data is to support addition of Clostridium 

difficile sporicide claims. Efficacy data was generated at Ecolab Research Center 

located at 655 Lone Oak Drive in Eagan, MN 55121. 

 The submitted data package contained a letter from the registrant (dated July 29, 

2009), Statement of No Data Confidentiality Claims, one efficacy study (MRID No. 

478186-01), and the proposed label. 

II USE DIRECTIONS 

 The product is designed to disinfect floors, walls, tables, chairs, countertops, bed 

rails, bathroom fixtures, sinks, shelves, racks, carts, and the exterior of refrigerators 

composed of glazed tiles, linoleum, vinyl, glazed porcelain, plastics, metal, stainless 

steel, chrome, glass, and painted surfaces. Directions on the proposed label provided 

the following instructions for the preparation and use of the product as a disinfectant 

against Clostridium difficile spores: 

Apply Virasept with a coarse trigger sprayer, cloth, mop, brush, scrubber, or by soaking 

to thoroughly wet surface. Virasept is effective against Clostridium difficile endospores 

after a 10 minute exposure time.

III AGENCY STANDARD FOR PROPOSED CLAIM 

Sporicidal Disinfectant against Clostridium difficile

The Agency has established interim guidance for the efficacy evaluation of 

antimicrobial products (e.g., dilutable products, ready-to-use products, spray products, 

towelettes) that are labeled for use to treat hard, non-porous surfaces in healthcare 

settings contaminated with spores of Clostridium difficile. The effectiveness of such a 

product must be substantiated by data derived from one of the following four test 

methods: Most recent version (2006) of AOAC Method 966.04: AOAC Sporicidal 

Activity of Disinfectants Test, Method I for Clostridium sporogenes; AOAC Method 

2008.05: Quantitative Three Step Method (Efficacy of Liquid Sporicides Against Spores 

of Bacillus subtilis on a Hard Nonporous Surface); ASTM E 2414-05: Standard Test 

Method for Quantitative Sporicidal Three Step Method (TSM) to Determine Efficacy of 

Liquids, Liquid Sprays, and Vapor or Gases on Contaminated Carrier Surfaces; or 

ASTM E 2197-02: Standard Quantitative Carrier Test Method to Evaluate the 

Bactericidal, Fungicidal, Mycobactericidal, and Sporicidal Potencies of Liquid Chemical 

Germicides. Modifications to each test method will be necessary to specifically 

accommodate spores of Clostridium difficile. Because Clostridium difficile is an obligate 

anaerobe, testing should ensure adequate incubation conditions for the recovery of 

viable spores. The following toxigenic strains of Clostridium difficile may be used for 

testing: ATCC 700792, ATCC 43598, or ATCC 43599. All products must carry a precleaning step, thus no organic soil should be added to the spore inoculum. Results must 

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ice-cold LB broth by pipetting up and down repeatedly. The entire 100 μl was plated on 

BHI agar with 5% sheep blood by spread plating. Fraction B tubes were sonicated for 5 

minutes ±30 seconds using a floating microcentrifuge tube holder placed inside an 

ultrasonic cleaner. After sonication, 600 μl ice-cold LB broth was added to each Fraction 

B tube and tubes were vortexed for approximately 1 minute. Each carrier was then 

transferred using forceps from Fraction B tubes to corresponding microcentrifuge tubes 

labeled as Fraction C. Fraction C tubes contain 400 μl ice-cold LB broth. After removing 

the carriers, the Fraction B tubes were centrifuged for 6 minutes ± 30 seconds at 16,000 

x g. The supernatant was removed and the pellet resuspended in 100 μl ice-cold LB 

broth. The entire 100 μl was spread plated on BHI blood agar plates. Fraction C tubes 

were placed on an orbital shaker at approximately 140 rpm inside an incubator for 30 ± 2 

minutes at 35 ± 2°C. After incubation, Fraction C were vortexed for approximately 1 

minute. The entire 400 μl was spread plated on BHI blood agar plates. All plates were 

incubated at 35±2°C for 72±4 hours under anaerobic conditions. The identity of a 

minimum of one representative colony taken from at least one plate per test substance 

was confirmed by Gram stain. The total number of spores per carrier was calculated by 

adding the total number of viable spores per fraction for A, B, and C. The log density 

was then calculated by taking the log10 of the total number of spores per carrier. The log 

reduction of test carriers was determined by subtracting the log density of test carriers 

from log density of control carriers. The LR was calculated for each test substance 

batch. 

Note: Protocol deviations/amendments were documented. 

V RESULTS 

Carrier Numbers Control 

Carrier Enumeration (CFU/Plate) 

Carrier Fraction A Fraction B Fraction C 

10-4 10-5 10-6 10-4 10-5 10-6 10-4 10-5 10-6

1 235 6 2 TNTC 110 24 108 14 3 

2 138 3 2 TNTC 112 11 137 16 3 

3 88 15 2 TNTC 101 17 128 23 6 

Total Carrier Count Carrier 1 = 1.3 x 107Log Density Carrier 1 = 7.11 

Carrier 2 = 1.3 x 107 Carrier 2 = 7.11 

Carrier 3 = 1.1 x 107 Carrier 3 = 7.04 

Average Total Carrier Count 1.2 x 107

Average Log density 7.09

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*Commercial/financial information may be entitled to confidential treatment*

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