Document ID: EPA-HQ-OPP-2016-0467-0005
Agency: epa
Document Type: Supporting & Related Material
Title: 
Posted Date: 2016-08-10T04:00Z

Protocol for the Evaluation of Bactericidal Activity of 
                Hard, Non-porous Copper/Copper-Alloy Surfaces 
                                  (11/04/15)

I.	Overview

This document describes testing protocol recommended by the Environmental Protection Agency (EPA) to support the registration of hard non-porous copper and copper alloy-based surface products with non-food contact surface sanitizer claims.  The following items summarize the approach employed in this protocol to support these product claims: 

     A detailed product characterization is recommended to provide information on the product's physical durability and chemical stability as it relates to the proposed use patterns. The durability assessment includes an eight week abrasion and chemical exposure process.   
    
     Efficacy testing involves the evaluation of two product production lots against Staphylococcus aureus and Pseudomonas aeruginosa, with the test carriers from one of these lots having undergone the abrasion and chemical exposure process. The stainless steel control carriers do not receive the abrasion and chemical exposure.  
    
     An effective product is expected to achieve a 3 log10 reduction (LR) in viable bacteria (compared to the control) for each microbe within a 1 hr contact period.  Additional details on the performance standard are described in the Product Performance Data section.

     This protocol is designed to address sanitizer claims for copper and copper alloy surfaces, including a claim for "continuous reduction" of bacteria.

     All testing should be conducted under Good Laboratory Practice Standards.  

     Contact times less than 1 hour may be deemed appropriate upon consultation with EPA.

     Note that this protocol is applicable to non-porous copper-containing surface products that are intended for indoor use only. Applicants interested in pursuing registrations with outdoor uses or for porous copper-containing surface products should consult with the EPA regarding protocol development and testing to support these uses.

  This protocol has been established for use with copper-containing materials; however, upon consultation with EPA, the protocol may also be appropriate for testing other solid, non-food contact surfaces for antimicrobial activity.

     Efficacy test results will be used to determine the sanitizing activity of the copper-containing surface product by comparing the reduction in viable bacteria on product carriers to the control carriers. 
   
     If alterations to this protocol are deemed necessary by an applicant, the modified test protocol should be submitted to the EPA for review in advance of data generation with all requested changes to the procedure clearly identified
   
     Product performance testing should be conducted on two production lots; one lot with sets of exposed carriers and the second lot with only unexposed carriers. The term "exposed" refers to carriers subjected to the physical and chemical assessment, while "unexposed" carriers refers to those not subjected to the physical and chemical assessment.  Table 1 provides an overview of the carrier testing requirements.
  
 Table 1. Carrier distribution for testing of copper-containing surfaces  
                                  Product Lot
                               Product Carriers
                               per Test Microbe*
                               Control Carriers
                               per Test Microbe*
   Lot 1
                              15 exposed carriers
                   (5 for each of three chemical solutions)
                         3 unexposed product carriers
                   and 3 unexposed stainless steel carriers
   Lot 2
                             5 unexposed carriers
                     3 unexposed stainless steel carriers
 *Two microbes are evaluated for determining product efficacy (see Section IV. B)
 ** Product carriers exposed to abrasion and chemical solutions
 ***Product carriers not exposed to abrasion or chemical solutions

II. 	Product Characterization

            Since both the physical durability and chemical stability of a hard non-porous antimicrobial product are critical to the level of efficacy performance over time, a profile of certain product characteristics should be submitted in support of registration.  This section describes information that should be addressed in the product characterization submission.  The information provided in this submission should pertain to the specific product and product components (as identified in the Confidential Statement of Formula), including all proposed formulation types and potential product variations.  Attributes should include:  
   
 Define all product manufacturing and application processes, product compositions/ formulations, and proposed product use patterns/use sites.  For products that involve the application of copper-containing material to a non-copper substrate for the purpose of forming a copper coating (either pre- or post-sale), describe each type of substrate material in detail that is proposed for use with the product.  If the product label proposes use with metal substrates, a discussion of metal substrate compatibility with the copper/copper-alloy component (coating) is recommended.

 Describe the potential for physical disruption of the product surface (e.g., cracking, peeling, and chipping) resulting from normal use in relation to the proposed use patterns.  The results of standardized surface hardness testing would be relevant information for most hard surface products.  Describe the expected duration of use after product application/installation, and the potential for atypical physical or chemical challenges that could result from any of the proposed uses.  This information may be documented either as descriptive (qualitative) observations and/or as quantitative measurements.  

 Describe the product surface characteristics including the thickness of the surface layer (if applicable), typical surface morphology, and any intentionally manufactured features (e.g., gloss, matte).  If nanostructures/nanomaterials are known to be present or are likely to be present on the product surface, a thorough description of these characteristics or components should be provided.

III. 	Abrasion and Chemical Exposure Process Overview 
 
           As noted above, this protocol includes a product assessment of the effects of mechanical abrasion and of exposure to certain chemical solutions.  The abrasion and chemical exposure process is intended to represent a degree of normal and relevant physical wear, as well as reproduce potential effects resulting from repeated exposure of copper-containing surfaces to three different biocidal cleaning materials (chemical solutions).  A single abrasion and chemical exposure treatment involves six passes of an abrasive material against the product surface, followed by a 10 minute exposure of the product surface to a specific solution.  These exposure treatments are to be performed on one production lot five times a day and five days a week for eight consecutive weeks, resulting in a total of 200 total exposures (abrasion/chemical).  
	Specifically, the assessment involves the use of 15 copper product test carriers, 3 copper product control carriers, and 3 stainless steel control carriers.  The copper coupons should be derived from the same production lot.  For the controls, the copper product control carriers and the stainless steel control carriers do not receive the abrasion and chemical solution exposures. The fifteen copper product test carriers are subjected to mechanical surface abrasion followed by exposure to one of three different chemical solutions (solutions A, B or C) in groups of 5 (see Table 2). Following the chemical exposure, the exposed carriers should be rinsed thoroughly with deionized or distilled water, air-dried and stored at room temperature until the next exposure cycle. Each group of 5 product test carriers should be uniquely identified and exposed to the same chemical solution for each exposure treatment during the 8 week process.  
  
  
  Table 2.  Carrier exposure to chemicals
                                  Product Lot
                                  Solution A
                                       
                         Sodium Hypochlorite (Bleach)
                                  Solution B
                                       
                               Hydrogen peroxide
                                  Solution C
                                       
                             EDTA/ phosphoric acid
                                   Controls 
               (do not receive abrasion and chemical exposures)
                                     Lot 1
                                5 test carriers
                                5 test carriers
                                5 test carriers
                           3 copper product carriers
                        and 3 stainless steel carriers
    
    Product performance testing should be initiated within 3 days of completion of the 8 week exposure regimen.  All carrier storage conditions (temperature and humidity range) should be included in the study report.  As indicated in Section I, two groups of 15 product test carriers (one 15-carrier group per microbe) that have undergone the abrasion and chemical exposure process and 6 control carriers (21 total carriers) are evaluated for efficacy according to the laboratory methodology identified in Section IV.  Each group (2) of 21 carriers is tested against one of the two test microbes identified in the method. 
    Using a second production lot of copper, testing should be conducted on 5 unexposed product test carriers and 3 unexposed stainless steel control carriers. 
    Identification of product test carriers by the type of chemical solution used during the exposure process should be maintained throughout product performance testing.  
 	Abrasion and Chemical Exposure Treatment Process 
	Carriers should be selected and prepared as described in Section IV (A) of this document for the abrasion and chemical exposure process.  Note that all copper product test and control carriers must be cut from the relevant hard, non-porous copper-containing materials anticipated for final production.  Individual carriers should be oriented with the copper treated surface side-up (i.e., the sanitizing copper-containing surface); this orientation should be maintained throughout the exposure treatment.  All test and control carriers should be maintained under comparable conditions during each abrasion and chemical solution exposure treatment.  The exposure treatment of the test carriers should be performed at room temperature.  

	The abrasion and chemical exposure treatment should be performed 5 times per day with each at least 30 min between each exposure treatment.  As indicated, these daily exposure treatments should be performed 5 days per week for 8 consecutive weeks, after which a visual inspection of all carrier surfaces should be performed.  Any visual changes to the product test carrier surfaces in comparison to the unexposed product controls (such as discoloration, pitting or the presence of scratches) should be recorded and included in the final study report submitted to EPA.
    
    	1. 	Chemical Solution Preparation
   The treatment solutions to be applied during the chemical exposure process are identified below.  All solutions should be clearly labeled, and new solutions should be prepared each day of treatment.
 Solution A:  Solution A is a 3000 +- 150 ppm sodium hypochlorite solution (e.g., Sigma-Aldrich reagent grade sodium hypochlorite) prepared in distilled or deionized water.    All details related to the source product and dilution process (if applicable) should be included in the study report. The final concentration of the solution should be verified and recorded. 

 Solution B:  Solution B should contain hydrogen peroxide (between 3.0% and 6.0%) and ethaneperoxoic acid as active ingredients.  This solution should be an EPA-registered antimicrobial pesticide product that allows spray application to hard surfaces.  The solution concentration for the ethaneperoxoic acid component is not limited to a defined range, but should be indicated in the study report.  All details related to the product selected and the dilution process (if applicable) should be included in the study report.

 Solution C:  Solution C should contain between 5.0% and 5.2% ethylenediamine-tetraacetic acid, tetrasodium salt (CAS# 64-02-8) and phosphoric acid between 8.0% and 8.3%.  Distilled or deionized water should be used as the diluent.  

	2. 	Conducting the Abrasion Treatment

 The abrasion exposure should be performed with a Gardco, Model D10V abrasion tester.  A 3M Scotch-brite, General Purpose Hand Pad 7447 should be used as the abrasive material.  Attach the pad to the abrasion tester as specified in the product use manual. Replace pad daily. 

 The weight of the fully assembled abrasion boat (Gardco WA-2225) should be between 1000 g and 1085 g.  

 One abrasion cycle should consist of six (6) passes of the abrasive pad against the carrier test surface (the pad should contact the carrier surface six times).  

 The abrasion tester speed should be set between 2.25 to 2.50 for a total surface contact time of approximately 6 seconds per treatment.  

 Following the abrasion cycle, the carriers should be wiped with a clean, dry cloth and subjected to the chemical solution. 

 Proceed to the chemical solution treatment.   

	3. 	Conducting the Chemical Solution Treatment
        
       After preparing the chemical solutions (solutions A, B and C), place the test carriers, treatment (copper surface) surface up, on a flat surface (e.g., inside a Petri dish). Apply each chemical solution to 5 test carriers by spraying two to three pumps at 6-8 inches of the appropriate solution to each carrier surface (i.e., enough liquid to cover the carrier surface). Utilize a trigger sprayer such as a Quorpak trigger spray bottle (VWR part number 16344-101) or similar device.

       Allow each test carrier to be in contact with the appropriate chemical solution for approximately 10 min at room temperature.  

       After the 10 min contact period, rinse thoroughly with distilled or deionized water, allow to air dry, and store at room temperature.  

       Following the 200 abrasion-chemical exposures, rinse all carriers thoroughly with distilled or deionized water, allow to air dry, and store at room temperature.  Proceed to product performance testing.  Product performance testing should be initiated within 3 days of the final abrasion-chemical exposure process.  

IV.	Test Methodology

       Carriers.  
         
	For testing of two microbes from one production lot, prepare 30  exposed product test carriers (from copper-containing product), six (6) unexposed product control carriers, and 6unexposed stainless steel control carriers; and from a second production lot, prepare ten (10) unexposed product test carriers and six (6) stainless steel control carriers. Extra carriers should be prepared for sterility assessment. The steel stock sheets used for the stainless steel control carriers should physically match the product carriers as closely as possible with respect to thickness, degree of polish and/or brushed surface machining, etc.  The composition of the copper-containing test carriers must be representative of the final product and meet the specifications for the target chemistry formulation. The chemical composition of the treated test product carriers must be documented. 

 Product test material and stainless steel stock material sheets should be die/machine cut into individual approximate 1in x1in square carriers in similar fashion to minimize variation in size and cut edge artifacts.  
 Each carrier should be physically screened to insure uniformity.  Carriers with visible surface or edge abnormalities (e.g., corrosion/rust, chipping, gouges or deep striations, etc.) should be discarded. Note: The screening should be conducted prior to the abrasion/chemical exposure.    
 Soak physically screened carriers in a suitable detergent solution (e.g., Liquinox) for 2-4 hr to degrease and then rinse thoroughly in distilled or deionized water.  Gently wipe with a clean lint free cloth (e.g., KIMTECH PURE W4 Dry Wipers) and allow to completely dry. The rinsing should result in a surface free of residual detergent without any residual antimicrobial properties. 
 To prepare carriers for testing, immerse in 95-98% ethanol for 5-10 min to decontaminate.  Using sterile forceps, remove individual carriers and place face up in matted, pre-sterilized Petri dishes (one carrier per dish). Allow carriers to dry in a Biological Safety Cabinet with lid open. Flaming, autoclaving, or exposure to UV radiation are not desirable techniques for sterilizing coupons and may alter the antimicrobial properties of the treated surfaces. Individual carriers should be oriented with the treated (i.e., copper surface) up; this orientation should be maintained. Handle carriers aseptically. 
 To monitor the occurrence of microbial contamination, randomly select a control and treated carrier from each batch and incubate in appropriate growth medium as a sterility control.  No growth is the desired outcome. 
 Provide details of physical screening and sterility check along with vendor or source in the final report; coupon thickness and degree of surface brush or polish should also be reported.  
 Use cleaned decontaminated carriers within one week of preparation.
 Copper and stainless steel coupons are considered single use.
 Production lot (batch) identity must be maintained throughout the testing process.
 Note: If a copper-containing material is applied (e.g., spray application) directly to the surface of a porous or non-porous substrate to form the "antimicrobial product", then the application process and characteristics of the final deposition (e.g., thickness of copper material and the substrate) must be fully described and documented in the submission. The test carriers must be representative of the anticipated final product.    
          
B.	Test Cultures.  The test microbes are Staphylococcus aureus (ATCC 6538) and Pseudomonas aeruginosa (ATCC 15442).

 1) Stock Cultures. Initiate new stock cultures from lyophilized cultures from ATCC at least every 18 months. Open ampule of freeze dried organism per manufacturer's instructions.
 Using a tube containing 5-6 mL of TSB, aseptically withdraw 0.5 to 1.0 mL and rehydrate the lyophilized culture.  Aseptically transfer the entire rehydrated pellet back into the original tube of broth.  Mix thoroughly.  Incubate broth culture at 36 +- 1ºC for 24 +- 2 hours.
 After incubation, streak a loopful of the suspension on TSA to obtain isolated colonies.  Incubate the plates for 18-24 h at 36 +- 1°C.
 Select 3-5 isolated colonies of the test organism and re-suspend in 1 mL of TSB.  For S. aureus, select only golden yellow colonies.  Multiple phenotypes are present for P. aeruginosa  -  the stock culture should be representative of all phenotypes present on the streak isolation plate.   Spread plate 0.1 mL of the suspension on each of 6-10 TSA plates.  Incubate the plates for 18-24 h at 36 +- 1°C. 
 Following the incubation of the agar plates, place approximately 5 mL sterile cryoprotectant solution on the surface of each plate.  Re-suspend the growth in the cryoprotectant solution using a sterile spreader without damaging the agar surface.  Aspirate the suspension from the plate with a pipette and place it in a sterile vessel large enough to hold about 30 mL.  Repeat the growth harvesting procedure with the remaining plates and continue adding the suspension to the vessel (more than 1 tube may be used if necessary).  Mix the contents of the vessel(s) thoroughly; if more than 1 vessel is used, pool the vessels prior to aliquoting culture. Immediately after mixing, dispense 0.5-1 mL aliquots of the harvested suspension into cryovials; these represent the frozen stock cultures.
 Store the cryovials at -70 +- 5ºC for a maximum 18 months then reinitiate with a new lyophilized culture. 
 Conduct Quality Control check of the pooled culture concurrently with freezing.  For example, streak a loopful on a blood agar plate, and selective media such as MSA and Cetrimide.  Incubate all plates at 36 +- 1ºC for 24 +- 2 hours. Record the colony morphology as observed on the blood agar plates and selective media plates (including the absence of growth). Perform a Gram stain from growth taken from the blood agar plates and observe the Gram reaction by using bright field microscopy at 1000x magnification (oil immersion).
   Note: Alternative preparation procedures for stock and test cultures may be used for test organisms not mentioned herein; however, the methodology must be clearly specified in the study protocol, and approved by EPA in advance. 
    
   2) Test Cultures 
 For S. aureus, defrost a single stock culture cryovial at room temperature and briefly vortex to mix.  Each cryovial should be single use only.  Add 10 uL of the thawed stock to a tube containing 10 mL tryptic soy broth (TSB) and then vortex to mix.  Incubate at 36+-1°C for 18-24 hrs.  Following incubation, use the broth culture to prepare a final test suspension. Briefly vortex the culture prior to use.
 For P. aeruginosa, defrost a single cryovial at room temperature and briefly vortex to mix.  Each cryovial should be single use only.  Add 10 uL of the thawed stock to a tube containing 10 mL TSB and then vortex to mix.  Incubate at 36+-1°C for 18-24 hr.  Inspect culture prior to use. Remove visible pellicle on surface of medium and around associated interior surfaces of the tube by pipetting or with vacuum suction.  Using a serological pipette, withdraw the remaining broth culture (approx. 7-8 mL), avoiding any sediment on the bottom of the tube, and transfer to a new tube. Following removal of pellicle, use the broth culture to prepare a final test suspension. Briefly vortex the culture prior to use.
            
 Dilute (in Phosphate Buffered Saline) or concentrate the culture appropriately to achieve the target carrier counts (4-5 logs/carrier). Centrifuge the 18-24 h broth cultures to achieve the desired level of viable cells on the dried carrier.  Centrifuge at ~5,000 gN for 20+-5 min and re-suspend the pellet in 10 mL Phosphate Buffered Saline (PBS).  Note: Remove the supernatant without disrupting the pellet.  For S. aureus, disrupt the pellet using vortexing or repetitive tapping/striking against a hard surface to disaggregate the pellet completely prior to re-suspending it in 10 mL. If necessary, add 1 mL of PBS to the pellet to aid in the disaggregation.
 Purity of the final test cultures (with soil load) should be determined by streak isolation on Tryptic Soy Agar (TSA) with 5% sheep's blood, or other appropriate plating medium, incubate (36+-1°C for 48+-4 hr), examine for purity. 
 It is recommended that the titer of the final test culture (with soil load) be determined for informational purposes. Plate dilutions on TSA plates (or TSA with 5% sheep's blood) or other appropriate medium and incubate (36+-1°C for 24-48 hr) and enumerate.  Count the number of colonies to determine the number of organisms per mL (i.e., CFU/mL) of inoculum present at the start of the test.     

C.	Soil Load (three-part). 

 The recommended standard soil load to be incorporated in the test suspension is a mixture of the following stock solutions in PBS:
 Bovine serum albumin (BSA): Add 0.5 g BSA to 10 mL of PBS, mix and pass through a 0.2 um pore diameter membrane filter, aliquot and store at -20 +- 5°C.
 Yeast Extract: Add 0.5 g yeast extract to 10 mL of PBS, mix, and pass through a 0.2 um pore diameter membrane filter, aliquot and store at -20 +- 5°C.
 Mucin: Add 0.04 g mucin (bovine or porcine) to 10 mL of PBS, mix thoroughly until dissolved, and autoclave (15 minutes at 121°C), aliquot and store at -20 +- 5°C. 
 The stock solutions of the soil load are single use only and should not be refrozen once thawed; store up to one year at -20 +- 5°C.
 Vortex the test suspension for 10-30 seconds or until re-suspended (no more than 60 seconds) to evenly distribute the cells.
 To obtain 500 μL of the final  test suspension with soil load, vortex each component and combine the following (or appropriate ratio):
 25 uL BSA stock
 35 uL yeast extract stock
 100 uL mucin stock
 340 uL microbial test suspension
 Following the addition of the soil load, vortex the final test suspension for 10 seconds and immediately prior to use.

D.	Efficacy Test Procedure

 Evaluate fifteen (15) exposed product test carriers (from one production lot) with three (3) stainless steel control carriers and three (3) unexposed product control carriers against each test organism; and from the second production lot  test five (5) unexposed product test carriers and three (3) unexposed stainless steel control carriers against each test organism.  
 Control carriers should be evaluated concurrently with the test carriers. 
 The exposure (contact time) of the inoculum to the carrier surface begins immediately upon inoculation; therefore, the contact time begins when final test suspension (with soil load) is deposited onto a test carrier. 
 Record the initiation of the contact time and inoculate each carrier at staggered intervals with 20 uL of final test culture using a calibrated pipette (a positive displacement pipette is desirable).
 Spread the inoculum to within approximately 1/8 inch of the edge of each carrier, using a sterile transfer loop or the pipette tip, for example. Use an appropriate interval (e.g., 30 sec) to allow sufficient time for careful spreading of the inoculum.
 The contact time begins immediately following carrier inoculation. Record the lab temperature and relative humidity during the one hour exposure period. 
 Allow carriers to remain in a horizontal position under ambient conditions with the lid on the Petri plate for 60+-5 min.   
 Following the exposure period, sequentially and aseptically transfer carriers to 20 mL of the appropriate neutralizer solution  -  this represents the 10[0] dilution.
 Record the exposure period end time when the treated and control carriers (or a set of carriers) are placed into the neutralizer solution.
 After all the carriers have been transferred into the neutralizer, sonicate for 5 min +-30 secs to suspend any survivors from the carriers, swirl to mix.
 Within 30 min of sonication, prepare serial dilutions of the neutralized solution (10[0] dilution) out to 10[-4] for the treated carriers.  Transfer the control carriers to neutralizing subculture media and sonicate as for test carriers. Prepare serial dilutions of the neutralizing subculture medium and plate the appropriate dilutions in duplicate to yield countable numbers (up to 300 colonies per plate). Incubate and enumerate with the treated carrier plates.
 Plate 0.1 mL in duplicate using spread plating or pour plating technique on TSA plates (or TSA with 5% sheep blood).
 Incubate the plates at 36+-1°C for 48+-4 hr. 
 Following incubation, count colonies and record the results.

E.	Study Controls

 Purity Control.  Perform a "streak plate for isolation" on TSA plates (or TSA with 5% sheep blood) for each final test culture, and following incubation examine in order to confirm the presence of a pure culture. The acceptance criterion for this study control is a pure culture demonstrating colony morphology typical of the test organism.
 Soil Load Sterility Control. Streak plate or add a sample of the three part soil load to a growth medium, incubate, and visually examine for growth. The acceptance criterion for this study control is lack of growth.
 Carrier Sterility Control. Add a representative un-inoculated test and stainless steel control carrier to the neutralizing subculture medium. Incubate and examine for growth in the subculture medium containing each carrier. The desired outcome for this study control is lack of growth.
 Neutralizer Sterility Control. Incubate an unused tube of neutralizing solution and visually examine for growth.  If the neutralizing solution does not support growth, then plate 1.0 mL or 0.1 mL using spread plating or pour plating technique on TSA plates (or TSA with 5% sheep blood). The acceptance criterion for this study control is lack of growth.

F.	Neutralization Confirmation 

 Perform a neutralization confirmation control to demonstrate the neutralizer's ability to inactivate the test carrier. This should be conducted with both test microbes prior to the efficacy evaluation and reported separately. The neutralization of the test carriers is confirmed by using unexposed test and unexposed stainless steel control carriers and the neutralizer as in the test procedure. 
 Add a test carrier (one per production lot) to a tube of neutralizer solution (20 mL).
 Hold the carrier in the neutralizing solution for approximately10 min. 
 Add a 1.0 ml aliquot of a diluted suspension of the test organism yielding 10-100 CFU/0.1 ml of neutralizing subculture medium to the neutralizer, mix well.  Hold for approximately 10 min. Duplicate plate   0.1 mL aliquots of this mixed solution using spread plating or pour plating technique on TSA plates (or TSA with 5% sheep blood). 
 A numbers control (provides baseline level of CFU for comparative purposes) is performed utilizing stainless steel control carriers; process as indicated for the test carriers. 
 The resulting plates are incubated as in the test and enumerated. The acceptance criterion for this study is the difference between the treated and control counts should be <=50%.

G. 	Product Performance Data 

Impact of Abrasion and Chemical Exposure  -  Production Lot 1. 

 Comparative visual observations should be used to identify any deleterious effects caused by the abrasion and chemical exposure for production lot 1; report findings in the study report.

 The effects of the abrasion and chemical exposure on mean log reduction should be presented for production lot 1  -  this is based on the mean log reduction values for the exposed product carriers compared to the mean log reduction values for unexposed product carriers. The mean control counts associated with the stainless steel control carriers are used for the log reduction calculations.  

 The mean log reduction values (i.e., per abrasion/chemical treatment per microbe) for the exposed product carriers compared to the unexposed product carriers should be within 0.5 log; in addition, the mean log reduction for the exposed product carriers should not be less than the performance standard of 3 logs for any abrasion/chemical treatment group for either of the test microbes.
   
Production Lot 2. Mean log reduction data for production lot 2 should be calculated and presented by comparing viable counts for the 5 product carriers and 3 stainless steel control carriers. 

Stainless Steel Control Counts Acceptance Criteria.  The acceptance criterion for the control carriers is 4-5 logs CFU/carrier. All study controls must perform according to the criteria detailed in the study controls description section.

Product Efficacy.  For the test substance to be considered a sanitizer, a >=99.9% reduction (>=3 log reduction) in the numbers of each test microbe (the difference between product test carriers and the stainless steel control carriers) must be demonstrated following the exposure time (60 min) for each production lot. 

H.	Calculations/Data Analysis

 Calculate the mean log reduction in viable cells for each microbe for the following treatments: 1) exposed product carriers (per chemical) for production lot 1, 2) unexposed product control carriers (one 3-carrier set per microbe) for production lot 1, and 3) product carriers for production lot 2.  Log reduction values are calculated based on the difference in log densities associated with the product test carriers compared to the stainless steel control carriers.  

 For determining the number of viable bacteria per carrier: 

CFU/carrier = (average number colonies/plate @ dilution) x (dilution factor) x (volume of neutralizer)
                                    (Volume plated)

 For determining the geometric mean number of organisms surviving on three control carriers (unexposed copper or stainless steel) where X equals CFU/control carrier:
   
      Geometric Mean = Antilog of Log10X1 + Log10X2 + Log10X3
							3

 Example: For determining the geometric mean of number of organisms surviving on five product test carriers where Y equals CFU/test carrier:

Geometric Mean = Antilog of Log10Y1 + Log10Y2 + Log10Y3 + Log10Y4 + Log10Y5
						               5
      (Adjust equation 3 above accordingly for three carriers for the unexposed product test carriers for lot 1)
 
 % reduction = [(a-b)/a] x 100 

Where: 

            a = geometric mean of the number of organisms surviving on the inoculated control carriers 
      
      b = geometric mean of the number of organisms surviving on the test carriers.

 Log10 Difference = (Log10 Stainless Steel Numbers Control)  -  (Log10 Product Test carriers) 

V.	Product Labeling

    	Label Claims 

 The following label claim is supported by the protocol: 

"This surface kills at least 99.9% of bacteria after a 1 hour contact time when maintained in accordance with the product care and use directions."

 Claims are limited to indoor use of hard, non-porous copper/copper-alloy containing surfaces. 

    	Required Label Language

 Care and Use of Antimicrobial Copper Surfaces in Health Care Facilities: 

	"Product Care and Use:  Antimicrobial copper alloy surfaces must be cleaned and disinfected according to standard practice. Health care facilities must maintain the product in accordance with infection control guidelines; users must continue to follow all current infection control practices, including those practices related to disinfection of environmental surfaces.  This copper surface material has been shown to reduce microbial contamination, but does not necessarily prevent cross contamination. This product must not be waxed, painted, lacquered, varnished, or otherwise coated by any material."

 Care and Use of Antimicrobial Copper Surfaces for Non-Health Care Facilities:  

	"Product Care and Use:  Routine cleaning to remove dirt and filth is necessary for standard hygiene and to assure the effective antibacterial performance of the antimicrobial copper surface. Cleaning agents typically used for environmental surfaces are permissible. The use of an antimicrobial copper surface does not replace standard good hygienic practices and/or infection control procedures. This product must not be waxed, painted, lacquered, varnished, or otherwise coated by any material."