Summary Virucidal Efficacy of CuVerro V Antimicrobial Copper Film Page 1 of 4 • Surface-based efficacy tests were conducted to determine the efficacy of new, unused CuVerro antimicrobial copper film against Phi6 and MS2. • Efficacy of new CuVerro V film was >4.4 Log Reduction against Phi6 and MS2 for contact times ranging from 5 minutes to 2 hours. • Efficacy was determined for new CuVerro V film and film that had been deployed for 90 days in LA Metro vehicles (rail car and bus). • Efficacy of post-deployed CuVerro V film was >5.4 Log Reduction against MS2, for contact times ranging from 5 minutes to 30 minutes. • No viable virus was recovered from CuVerro V test surfaces in any of the tests conducted with new or post-deployment film. Introduction: Los Angeles Metro (LA Metro) sent samples of CuVerro V antimicrobial self-adhesive film (EPA Reg# 85353-5) to the U.S. Environmental Protection Agency, Office of Research and Development, Center for Environmental Solutions and Emergency Response, Homeland Security and Materials Management Division, for efficacy testing against the enveloped bacteriophage Phi6 and the non-enveloped bacteriophage MS2. Testing of the CuVerro V film was performed in two phases. The first phase included testing new, unused CuVerro V film using both Phi6 and MS2. The second phase included testing new, unused CuVerro V alongside CuVerro V film which had been deployed by LA Metro for 90 days. Results from previous tests have shown MS2 to be more resistant to several decontamination techniques compared to Phi6. For this reason, only MS2 was used the second phase of testing. Methods and Materials: New, unused CuVerro V film was adhered to stainless steel coupons (2 cm x 4 cm) for testing. Post- deployment (90-days) CuVerro V film was collected from railcar and bus locations, adhered to aluminum foil sheets, and then cut into 2 cm x 4 cm sections prior to testing. New, unused CuVerro V film was also adhered to aluminum foil and cut into 2 cm x 4 cm sections and evaluated for efficacy alongside post- deployment CuVerro V film. Stainless steel coupons or aluminum foil sections (also 2 cm x 4 cm) were used as positive control surfaces during efficacy tests for stainless-steel-backed and foil-backed tests, respectively. For all test and control samples, virus particles were analyzed in an extraction buffer consisting of 10 mL of 10 mM ethylenediaminetetraacetate (EDTA) in 10% Dey Engley (DE) broth prepared in IX phosphate-buffered saline (PBS), unless otherwise indicated. Three replicate coupons were included for all efficacy test conditions. Triplicate coupons were included in neutralization tests with Phi6. A single test coupon was included in subsequent neutralization verification tests with MS2, due to MS2 being less sensitive to chemical inactivation. It was anticipated that effective neutralization conditions for Phi6 would be more than sufficient for MS2 (this assumption was supported by the data). MS2 and Phi6 Efficacy Tests CuVerro V test coupons and positive control coupons were inoculated with 1 x 10s virus (either MS2 or Phi6) particles (stabilized in 5% Fetal Bovine Serum (FBS)) using a 10 piL inoculum droplet spread over the coupon surface. After the prescribed contact time, both test and control coupons were placed in 10 mL of extraction buffer and subjected to continuous agitation on a vortex mixer for 2 minutes. A tenfold dilution series was prepared as appropriate in IX PBS for each sample. Aliquots of either 1 mL (undiluted), ------- Summary Virucidal Efficacy of CuVerro V Antimicrobial Copper Film Page 2 of 4 100 pil (undiluted), or 100 pil from each dilution were plated in triplicate using a conventional soft agar overlay method. Escherichia coli (ATCC 15597) and Pseudomonas syringae were used as the bacterial hosts for MS2 and Phi6, respectively. Plates were incubated overnight at 35 °C (MS2) or ~21 °C (Phi6) and manually enumerated the following day. Inoculation controls (i.e., direct inoculation of extraction buffer) were prepared at the same time of coupon inoculation, then plated as described for test coupons. The inoculum titer was determined each test day by performing a tenfold dilution series in IX PBS directly from the inoculum and plating 100 piL aliquots in triplicate. Neutralizer Verification Neutralizer tests were conducted to verify the ability of neutralization chemicals to completely quench antimicrobial activity of the tested products. These tests were first performed with Phi6. The neutralization verification test was performed by continuously vortex mixing non-inoculated CuVerro V test samples (cut into ~2 cm x 4 cm pieces and adhered to SS coupons) for 2 minutes in extraction and neutralization buffer (10 ml of 10 mM EDTA in 10% Dey Engley Broth), then letting the samples sit undisturbed at ambient temperature for at least 5 minutes. Samples were then inoculated with 1 x 10s Phi6. Inoculation controls were prepared by directly inoculating extraction buffer. Following inoculation, samples were plated after a >10-minute hold time at ambient temperature. Neutralized test sample recoveries should approximate (i.e., ± 0.5 log) control recoveries to demonstrate complete neutralization (quenching) of antimicrobial activities in the test samples following the contact time. Neutralization was further verified in the second phase tests by removing 5 ml aliquots from non- inoculated samples (i.e., negative controls) and inoculating with MS2 using 10 pil aliquots of 1 x 10s virus particles. Inoculation controls were prepared by directly inoculating extraction buffer. Following inoculation, samples were plated after a >10-minute hold time at ambient temperature. These neutralized test sample recoveries should also approximate (i.e., ± 0.5 log) control recoveries to demonstrate complete neutralization (quenching) of antimicrobial activities in the test samples following the contact time. Results Summary: Table 1 - Efficacy of New CuVerro V Film Against MS2 and Phi6 Efficacy of New CuVerro V Antimicrobial Film against MS2 and Phi6 Bacteriophages Mean Log PFU/Sample Std. Dev. Mean Log Reduction Pooled Error Phi6 - 30- and 120-minute contact times Inoculum 6.6 NR Inoculation Control 6.7 0.0 Stainless Steel Control - 30-minute contact 5.7 0.2 Stainless Steel Control - 120-minute contact 5.4 0.1 CuVerro V on Stainless Steel - 30-minute contact 1.0* 0.0 >4.7 0.1 CuVerro V on Stainless Steel - 120-minute contact 1.0* 0.0 >4.4 0.1 ------- Summary Virucidal Efficacy of CuVerro V Antimicrobial Copper Film Page 3 of 4 Phi6 - 5-, 10-, and 15-minute contact times Inoculum 6.4 NR Inoculation Control 6.3 0.0 Stainless Steel Control - 5-minute contact 6.3 0.0 Stainless Steel Control - 10-minute contact 6.2 0.0 Stainless Steel Control - 15-minute contact 6.3 0.0 CuVerro V on Stainless Steel - 5-minute contact 1.0* 0.0 >5.3 0.0 CuVerro V on Stainless Steel - 10-minute contact 1.0* 0.0 >5.2 0.0 CuVerro V on Stainless Steel - 15-minute contact 1.0* 0.0 >5.3 0.0 MS2 - 30- and 120-minute contact times Inoculum 6.5 NR Inoculation Control 6.6 0.1 Stainless Steel Control - 30-minute contact 6.1 0.1 Stainless Steel Control - 120-minute contact 5.7 0.2 CuVerro V on Stainless Steel - 30-minute contact 1.0* 0.0 >5.1 0.0 CuVerro V on Stainless Steel - 120-minute contact 1.0* 0.0 >4.7 0.1 *Detection Limit Value, NR - Non-Replicated test sample, standard deviation not applicable Table 2 - Efficacy of New and 90-Days Deployed CuVerro V Film Against MS2 Efficacy of New and Post-Deployment CuVerro V Antimicrobial Film against MS2 Bacteriophage Mean Log PFU/Sample Std. Dev. Mean Log Reduction Pooled Error Rail Location Film - 30-minute contact time Inoculum 6.7 NR Inoculation Control 6.8 0.1 Aluminum Foil Control - 30-minute contact 6.6 0.1 New CuVerro V on Aluminum Foil - 30-minute contact 1.0* 0.0 >5.6 0.1 Post-Deployment (Rail Location) CuVerro V on Aluminum Foil - 30-minute contact 1.0* 0.0 >5.6 0.1 Rail Location - 5- and 10-minute contact times Inoculum 6.5 NR Inoculation Control 6.6 0.1 Aluminum Foil Control - 5-minute contact 6.6 0.0 Aluminum Foil Control - 10-minute contact 6.4 0.2 New CuVerro V on Aluminum Foil - 5-minute contact 1.0* 0.0 >5.6 0.0 New CuVerro V on Aluminum Foil - 10-minute contact 1.0* 0.0 >5.4 0.1 Post-Deployment (Rail Location) CuVerro V on Aluminum Foil - 5-minute contact 1.0* 0.0 >5.6 0.0 ------- Summary Virucidal Efficacy of CuVerro V Antimicrobial Copper Film Page 4 of 4 Post-Deployment (Rail Location) CuVerro V on Aluminum Foil - 10-minute contact 1.0* 0.0 >5.4 0.1 Bus Location - 5- and 10-minute contact times Inoculum 6.9 NR Inoculation Control 6.9 0.0 Aluminum Foil Control - 5-minute contact 6.8 0.0 Aluminum Foil Control - 10-minute contact 6.8 0.1 New CuVerro V on Aluminum Foil - 5-minute contact 1.0* 0.0 >5.8 0.0 New CuVerro V on Aluminum Foil - 10-minute contact 1.0* 0.0 >5.8 0.0 Post-Deployment (Bus Location) CuVerro V on Aluminum Foil - 5-minute contact 1.0* 0.0 >5.8 0.0 Post-Deployment (Bus Location) CuVerro V on Aluminum Foil - 10-minute contact 1.0* 0.0 >5.8 0.0 *Detection Limit Value, NR - Non-Replicated test sample, standard deviation not applicable Table 3 - Neutralizer Effectiveness Neutralizer Effectiveness Results (10 mL of 10 mM EDTA in 10% Dey Engley Broth) Phi6 MS2 Mean Log PFU/Sample Std. Dev. Mean Log PFU/Sample Std. Dev. Inoculum 6.4 NR Inoculation Control 6.4 0.1 CuVerro V on Stainless Steel Coupons 6.4 0.0 Inoculum 6.3 NR Inoculation Control 6.4 0.0 CuVerro V (new) on Aluminum Foil 6.3 NR CuVerro V (post-deployment) on Aluminum Foil 6.4 NR MR - Non-Replicated test sample, standard deviation not applicab e ------- |