# EPA Registration 1677-226 Vol.2

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### Pages 6 through 11 contain the product confidential statement of formula and are not included in this copy.Page 2 of 5 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 difficilesporicide 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 subtilison 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 difficilemay be used fortesting: 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 mustPage 4 of 5 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 log 10 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 10 7 Log Density Carrier 1 = 7.11 Carrier 2 = 1.3 x 10 7 Carrier 2 = 7.11 Carrier 3 = 1.1 x 10 7 Carrier 3 = 7.04 Average Total Carrier Count 1.2 x 10 7 Average Log density 7.09

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