PREVENTION Ag Silver Composite Materials: EH & S Considerations

© 2012 NanoHorizons Inc.CONFIDENTIAL Ancient Times Egyptians used in drinking water Roman soldiers used in wound healing Chinese used in eating utensils Crede's prophylaxis for infant eye drops 1920's - Over 3 million prescriptions written annually for nanoscale silver-containing (colloidal silver) medications. 1960's - Silver compounds begin to be used in burn wards 1980's - Silver ion based additive for plastics become widespread 1990’s – Silver impregnated medical devices introduced 2000's – Polymer compatible silver additives developed Silver has a long history as a safe and effective antimicrobial Aqua-Precision

© 2012 NanoHorizons Inc.CONFIDENTIAL Encapsulation of silver particles Prevention Ag additives are delivered as a traditional ready-to-use powder. As produced, Prevention Ag is a composite powder, ie. particles encapsulated in a micron size polymer stabilizer Standard PPE (as listed in MSDS) is adequate. micron sized Prevention Ag powders encapsulated Prevention Ag particles Aqua-Precision

© 2012 NanoHorizons Inc.CONFIDENTIAL Air Quality Monitoring at The Prevention Ag manufacturing plant (2009, 2010) during manufacture and hopper loading showed silver levels at ~10% of the OSHA Permissible Exposure Limit (PEL) for General Industry (0.01 mg/m 3 TWA) and ~1% of the American Conference of Governmental Industrial Hygienists (ACGIH) Threshold Limit Value (TLV) (0.1 mg/m 3 TWA) for Silver, Metal. In vivo studies 1 have confirmed that the longstanding 0.1 mg/m 3 NOEL for airborne silver compounds is adequate for silver. 1 Sung, et al. Subchronic Inhalation Toxicity of Silver and Silver Nanoparticles. TOXICOLOGICAL SCIENCES 108(2), 452–461 (2009). Exposure Monitoring Aqua-Precision

© 2012 NanoHorizons Inc.CONFIDENTIAL Cytotoxicity Method – MEM Elution ISO Part ed. Testing Org – Biochem S.A.S. Predosa, Italy Test Item – Silver Treated Rayon Absorbent (0.024% Ag/wt) Conclusions – No Measurable Cytotoxicity 24 Hour ResultsScore Positive Control4 Negative Control0 MEM Control0 MEM Extract0 48 Hour ResultsScore Positive Control4 Negative Control0 MEM Control0 MEM Extract0 Biocompatibility Testing Aqua-Precision

© 2012 NanoHorizons Inc.CONFIDENTIAL Dpm (mean+SD)Stimulation IndexStatistical Significance* Negative Control817 ± 309NA Test Article992 ± No Positive Control10758 ± Yes * p<0.05 as compared to negative control Biocompatibility Testing Sensitization Method – Test for Irritation and Delayed Type Hypersensitivity ISO Part ed. Testing Org – NAMSA, Northwood, OH Test Item – Polyform AS (50% Ag/wt), Prepared 20%/wt in DMSO Conclusion – No Significant Proliferation of Lymphocytes Aqua-Precision

© 2012 NanoHorizons Inc.CONFIDENTIAL Rabbit No.Obs.Dermal Reactions Days Intact ER ED Intact ER ED Intact ER ED Abraded ER ED Abraded ER ED ER = ErythemaED = Edema Biocompatibility Testing Dermal Toxicity Method – FHSA Dermal Toxicity Study 7, Day 16 CFR 1500 Testing Org – NAMSA, Northwood, OH Test Item – Polyform AS (50% Ag/wt), Administered at 2g/kg Conclusion - 2g/kg Dose Would not be Considered Toxic Aqua-Precision

© 2012 NanoHorizons Inc.CONFIDENTIAL Animal Number SexBody WeightClinical Obs. Days 1-7 Necroscopy Day 7 Day 0Day Male243314Appeared NormalMacroscopically Normal 6957Male256328Appeared NormalMacroscopically Normal 6958Male263335Appeared NormalMacroscopically Normal 6959Male264347Appeared NormalMacroscopically Normal 6960Male250321Appeared NormalMacroscopically Normal Mean: Biocompatibility Testing Oral Toxicity Method – FHSA Oral Toxicity Study 7, Day 16 CFR 1500 Testing Org – NAMSA, Northwood, OH Test Item – Polyform AS (50% Ag/wt) Dosed at 5g/Kg Body wt Conclusions – Would not be Considered Toxic at 5g/Kg Dose Aqua-Precision

© 2012 NanoHorizons Inc.CONFIDENTIAL Rabbit No. Gender Reaction24 Hours72 Hours IntactAbradedIntactAbraded Male Erythema0000 Edema Male Erythema0001 Edema Male Erythema0000 Edema0000 * Primary Irritation Index = 1/12 = 0.1 Biocompatibility Testing Skin Irritation Method – FHSA Skin Irritation Study 7, Day 16 CFR 1500 Testing Org – NAMSA, Northwood, OH Test Item – Polyform AS (50% Ag/wt), Prepared at 20%/wt in Water Aqua-Precision

© 2012 NanoHorizons Inc.CONFIDENTIAL Biocompatibility Testing Cytotoxicity & Microbial Toxicity Testing Org – Rice University & University of Duisburg-Essen Test Item – Silver Salts (Silver Acetate & Silver Nitrate) – Silver particles (<50 nm diameter) Conclusion – Biological activity not affected by silver particles – Antimicrobial properties are directly related to the Ag + release (like all other silver-based antimicrobials) Conc. of Silver ExaminedSilver AcetateSilver Particles 15 μg/mL98%96% 20 μg/mL43%85% 25 μg/mL0%70% Conc. of Viable Human Cells (relative to control) after exposure to different types of silver. Aqua-Precision

© 2012 NanoHorizons Inc.CONFIDENTIAL Raad, I., et al., Anti-adherence activity and antimicrobial durability of anti-infective- coated catheters against multidrug-resistant bacteria. J. Antimicrob. Chemother., (4): p Valappil, S.P., J.C. Knowles, and M. Wilson, Effect of silver-doped phosphate-based glasses on bacterial biofilm growth. Applied and Environmental Microbiology, (16): p Kollef, M.H., et al., Silver-Coated Endotracheal Tubes and Incidence of Ventilator- Associated Pneumonia: The NASCENT Randomized Trial. JAMA, (7): p Weir, E., et al., The use of particles in anti-microbial materials and their characterization. Analyst, (7): p Roe, D., et al., Antimicrobial surface functionalization of plastic catheters by silver nanoparticles. J. Antimicrob. Chemother., (4): p Steven L. Percival, P.B.E.J.W., Assessing the effect of an antimicrobial wound dressing on biofilms. Wound Repair and Regeneration, (1): p Biocompatibility Testing Aqua-Precision