### Registration Decision for NSPW-L30SS (previously referred to as "N anosilva")

A Materials Preservative for Use in Textiles and Plastics

/a k o senger, Dire tor Of file of P sticide Programsii TABLE OF CONTENTS

Executive Summary ............................................................................................................. ...................... iv I. Regulatory Decision Summary ................................................................................................ ............... 1 II. Background ................................................................................................................ ............................ 1 2.1 Regulatory History ........................................................................................................ ..................... 1 2.2 FIFRA Scientific Advisory Panel (SAP) Meeting ............................................................................. . 2 2.3 Proposed Decision Document ................................................................................................ ............ 3 III. Product Description, Testing, and Use .................................................................................... ........... 3 3.1 Product Description ....................................................................................................... ..................... 3 3.2 Product Use ............................................................................................................... ......................... 3 3.3 Product Testing and Waivers ............................................................................................... ............... 3 IV. Hazard Characterization of Nanosilver ..................................................................................... ........ 5 4.1 Acute Human Toxicology of NSPW-L30SS ...................................................................................... 6 4.2 Subchronic and Chronic Toxicologi cal Studies Available for Nanosilver ......................................... 6 4.2.1 Oral .................................................................................................................... .......................................... 6 4.2.2 Inhalation............................................................................................................... ...................................... 8 4.2.3 Dermal .................................................................................................................. ....................................... 9 4.2.4 Reproductive and Developmen tal Toxicity of Nanosilver ................................................................... ..... 10 4.2.5 Mutagenicity of Nanosilver .............................................................................................. ......................... 11 4.2.6 Silver Ions ............................................................................................................. .................................... 13 4.3 Absorption, Distribution, Metabolism, and Excretion (ADME) of Nanosilver ............................... 14 4.3.1 Injection of Nanosilver ................................................................................................. ............................. 14 4.3.2 Oral Administra tion of Nanosilver ....................................................................................... ..................... 15 4.4 Safety Factor for Infants and Children (FQPA Safety Factor) ......................................................... 16 4.5 Antimicrobial Resistance .................................................................................................. ................ 16 4.6 Point of Departure Selections and Target Margin of Exposure ........................................................ 16 4.6.1 Point of Departures for NSPW-L30SS ...................................................................................... ................ 17 4.6.2 Target Margin of Exposure ............................................................................................... ........................ 20 4.6.3 Margin of Exposure ...................................................................................................... ............................. 20 V. Occupational and Consumer Risk Assessment ................................................................................. 21 5.1 Occupational Risk Assessment for Mixing an d Loading the NSPW-L30SS Liquid Suspension .... 21 5.1.1 Occupational Exposure Preparing the Master Batch Contai ning NSPW-L30SS ...................................... 22 5.1.2 Occupational Margins of Exposure ........................................................................................ ................... 22 5.1.3 Conclusions for Occupationa l Risk ....................................................................................... .................... 24 5.1.4 Occupational Exposure and Health Data Re quirements ...................................................................... ...... 25 5.2 Consumer Risk Assessment for Pr oducts Incorporating NSPW-L30SS .......................................... 25 5.2.1 Consumer Exposures to Plastics an d Textiles Incorpora ting NSPW-L30SS ............................................ 26 5.2.2 Consumer Margins of Exposure to Plastics Incorporating NSPW-L30SS ................................................ 30 5.2.3 Consumer Margins of Exposure to Textiles Incorporat ing NSPW-L 30SS ............................................... 34 5.2.4 Aggregate MOES for Plastics and Textiles Incorporat ing NSPW-L 30SS ................................................ 37 5.2.5 Conclusions for Consumer Risk ........................................................................................... ..................... 39 5.2.6 Consumer Exposure and Health Data Requirements .......................................................................... ...... 40 VI. Environmental Risk Assessment ............................................................................................. .......... 40 6.1 Environmental Fate ........................................................................................................ .................. 40 6.1.1 Nanosilver .............................................................................................................. ................................... 41 6.1.2 Silver Ions ............................................................................................................. .................................... 41 6.1.3 Impacts to Wastewater Treatment/Septic Systems .......................................................................... .......... 41 6.2 Environmental Toxicity .................................................................................................... ................ 42 6.2.1 Silver Ions ............................................................................................................. .................................... 42 6.2.2 Aquatic Toxic ity of Nanosilver .......................................................................................... ....................... 42 6.3 Aquatic Risk Assessment ................................................................................................... .............. 46 6.3.1 Aquatic Risk Quotient ................................................................................................... ............................ 46 6.3.2 Conclusions for Aquatic Risk............................................................................................. ....................... 48iii 6.4 Ecological Effects and Environm ental Fate Data Requirements. ..................................................... 49 VII. Regulatory Action ........................................................................................................ ..................... 50 7.1 Legal Framework ........................................................................................................... ................... 50 7.2 Findings and Registration Decision ........................................................................................ .......... 51 7.3 Basis for Conditional Registration ........................................................................................ ........... 52 7.3.1 Data Genera tion ......................................................................................................... ............................... 52 7.3.2 Public Interest ......................................................................................................... ................................... 53 7.3.3 No Unreasonable Adverse Effects ......................................................................................... ................... 54 References:.................................................................................................................... ............................. 56 Appendix A – Evaluation of Nanosilva LLC’s Data Waiver Requests ................................................ 61 Appendix B – Data Requirements, Enforceable Sc hedule, and Conditions of the Conditional Registration for NSPW-L30SS ................................................................................................... ............. 64

LIST OF TABLES Table 1 – Product Testing for the NSPW-L30SS Liquid Suspension .......................................................... 4 Table 2 – Acute Toxicity Profile for the NSPW-L30SS Liquid Suspension ................................................ 6 Table 3 – Toxicity Endpoints for Use in the Risk Assessment of NSPW-L30SS Nanosilver .................... 18 Table 4 – Unit Exposures for Mixing and Loading the NSPW-L30SS Liquid Suspension ....................... 22 Table 5 – Occupational Handlers Inhalation MO Es for the Nanosilver in NSPW-L30SS ......................... 23 Table 6 – Occupational Handlers Dermal MO Es for the Nanosilver in NSPW-L30SS ............................. 24 Table 7 –Silver Concentrations in Fluid C ontacting NSPW-L30SS Treated Plastic Coupons................... 27 Table 8 – Concentration of Silver Released from Shirts Incorporating NSPW-L30SS .............................. 29 Table 9 – Amount of Silver Released from Shirts Incorporating NSPW-L30SS ....................................... 30 Table 10 – Incidental Oral MOEs for Pl astic Toys Incorporating NSPW-L30SS ...................................... 31 Table 11 – Dermal MOEs for Flooring Incorporating NSPW-L30SS ........................................................ 32 Table 12 – Incidental Oral MOEs for Flooring Incorporating NSPW-L30SS ............................................ 34 Table 13 – Inhalation MOE for Drying Te xtiles Incorporating NSPW-L30SS .......................................... 35 Table 14 – Dermal MOEs for Textiles Incorporating NSPW-L30SS ......................................................... 36 Table 15 – Incidental Oral MOES for Textiles Incorporating NSPW-L30SS ............................................ 37 Table 16 – Aggregate MOEs for Toys, Flooring and Textiles Incorporating NSPW-L30SS ..................... 38 Table 17 – Nanosilver Toxicity to Aquatic Species ............................................................................. ....... 43 Table 18 – Reported Characteristics of Nanosilver Mate rials in the Aquatic Species Toxicity Studies .... 44 Table 19 – Risk Quotients for Nanosilver in Surface Water ..................................................................... . 48iv 

### Executive Summary
EPA is granting a conditional registrati on for the Nanosilva LLC product NSPW-L30SS (previously referred to as “Na nosilva”) under section 3(c)(7)(C) of the Federal Insecticide, Fungicide and Rodenticide Act (F IFRA). EPA’s basis for the condi tional registration is that:

 NSPW-L30SS contains nanosilver as an activ e ingredient and the nanosilver in NSPWL30SS is not in any currently registered pesticide;

 Use of NSPW-L30SS will not cause unreasonable adverse effects on the environment during the four year period when newly required data are being developed;

 Insufficient time has elapsed for the newly required data to be generated and submitted; and

 Use of NSPW-L30SS is in the public interest. NSPW-L30SS, which contains 1% nanosilver by weight, is th e subject of an application submitted by Nanosilva LLC in August 2009 (M RID 47828900). Nanosilva LLC requested that the nanosilver in NSPW-L30SS be registered as a new active ingredient because it was not an active ingredient in any curre ntly registered pesticide pr oduct. NSPW-L30SS is a liquid suspension containing silica-sulfur-nanosilver part iculates where the nanosilver is attached to amorphous silica via a thiolate bond. EPA is making a registration de cision for non-food contact uses of NSPW-L30SS incorporated into plastics and textiles to suppress the growth of bacteria, algae, fungus, mold and mildew, which cause odors, discoloration, stains, and dete rioration. The plastics an d textiles will contain less than 0.003% silver by weight. EPA determined that workers, consumers, and the environment could be exposed to:

1. silver ions released from NSPW-L30SS; 2. NSPW-L30SS; and/or 3. nanosilver that might break away from NSPW-L30SS. In evaluating the risk from exposure to silver ions, EPA relied on th e existing reregistration decision for silver which concluded that the human health or ecological risk from exposure to silver ions derived from plastics and textile s incorporating NSPW-L30S S are not of concern. For purposes of evaluating the risk from shor t-term exposure to NSPW-L30SS, Nanosilva LLCsubmitted results from acute animal-toxicity tests completed using high-level doses of NSPWL30SS showing that there were no mortalit ies or abnormalities in test animals after administration of NSPW-L30SS by oral, dermal , and inhalation exposure routes. NSPW-L30SS caused moderate irritation to the eyes of test animals, and was not a sk in sensitizer. Based on these results, shipping containers filled with NSPW-L30SS are required to carry a label stating “CAUTION” where contact with eyes or clothi ng should be avoided. Nanosilva LLC submitted a request to waive the required in termediate-term toxicity studies because silver leaches from plastic coupons incorporating NSPW-L30SS below the limit of detection (LOD) and because of the lack of toxicity noted in the acute animal-t oxicity tests completed using high-level doses of NSPW-L30SS. To evaluate occupational exposure to the nanosilv er that might break away from NSPW-L30SS, EPA calculated the daily-dose to workers assuming that all the silver in NSPW-L30SS was freely available as nanosilver. EPA believes that this assumption overestimates the daily-dose ofv nanosilver that a person could pot entially receive when working with NSPW-L30SS. To evaluate consumer exposure to the nanosilver that might break away from plastics and textiles incorporating NSPW-L30SS, Nanosilva LLC submitted studies showing that silver leaches from plastic coupons and shirts incorporating NSPW-L 30SS below the LOD. Thus, there is little or no exposure to nanosilver from plas tics and textiles incorporating NSPW-L30SS. EPA assumed that silver leaching from plastic c oupons and shirts inco rporating NSPW-L30SS was at one-half the analytical LOD and was in the form of nanosilver as found in NSPW-L30SS.

There are no intermediate- or l ong-term human or environmental toxicity studies available for NSPW-L30SS or for the nanosilver released from products incorporating NSPW-L30SS. In the absence of intermediate-term toxicity studies, EPA evaluated the risk from occupational and consumer exposure using intermediate-term hazard da ta available in the sc ientific literature for other nanosilvers. Because of the potential diffe rence between the nanosilver in the available toxicity data and the nanosilver in NSPW-L 30SS, along with the lack of an acceptable developmental and reproductive toxicity study for nanosilver, EPA used a maximum 10-fold database uncertainty factor when evaluating the risk from exposure to NSPW-L30SS and the nanosilver released from produc ts incorporating NSPW-L30SS. Impact to the environment was assessed based on ecotoxicity studies available in the scientific literature for other nanosilvers and environmen tal exposure from indoor use (assuming that 300 million people (U.S. population) each purchased one t-shirt containing NSPW-L30SS) and from the plastic lumber use (assuming that 1% of the projected yearly production of plastic lumber in the U.S. would contain NSPW-L30SS). All t-shirts in the textile scenario were assumed to be washed weekly for 52 weeks, releasing 1.6% of the initial silver load as nanosilver per wash to waste water and in the plastic scenario all of the silver in the plastic lumber was assumed to be released during the year. Using toxicity data on nanosilver, conservati ve occupational exposur e assumptions, leaching data showing minimal consumer exposure to nanosilver, and maximum values for risk uncertainty factors, EPA is able to determine that for the period of conditional registration, the use of NSPW-L30SS will not cause unreasonable adverse effects on the environment during the period when newly required da ta are being developed. As a condition of the registration, EPA is re quiring that Nanosilva LLC conduct a number of studies during the period of conditional regi stration. The required information includes characterization studies to better characteri ze NSPW-L30SS and the nanosilver in NSPWL30SS; stability and dissolution studies to de termine if nanosilver breaks away from NSPWL30SS and the rate nanosilver dissolves; and a new plastic leaching study to address specific uncertainties and confirm EPA’ s risk findings. In addition, EP A is requiring the following inhalation route-specific studies:

 90-Day Inhalation Toxicity (Rat) (OC SPP GLN 870.3465) modified to include a functional observational battery, moto r activity and detailed neuropathology

 Reproduction/Developmental Toxicity Screen ing Test via inhala tion route (Modified OCSPP GLN 870.3550/ OECD TG 421) Although there is no risk concern for workers w ho use close-system loading when mixing and loading the NSPW-L30SS liquid suspension, EPA is requiring these studies to account for potential physiochemical differences of the nanosilver and because there are currently no acceptable studies on the reproductive and developm ental toxicity for nanosilver. The EPA isvi also requiring a freshwater acu te invertebrate study using Daphnia magna to confirm Agency assumptions and ecological risk findings. Additio nal particle characte rization, human health effects, ecological effects and e nvironmental fate studies may be triggered based on the results of these studies. The testing requireme nts are based on a tiered approach. These studies must be completed within four ye ars after issuing the condi tional registration. This time period was chosen to allow time for protocol reviews prior to initiation of the studies, completion of the studies, and Agency review of the study results. EPA will evaluate these data as they are submitted to confirm EPA’s determ ination that the use of NSPW-L30SS will not cause unreasonable adverse effects to human health and the environment during the conditional registration period while these data are bei ng developed. If Nanosilva LLC fails to take appropriate steps to initiate the required studies, or if Nanosilva LLC fails to submit the protocols or data, EPA will issue a notice of intent to cancel NSPW-L30SS’s registration under FIFRA section 6(e). In addition, the conditional regist ration for NSPW-L30SS will automatically expire four years after being issued. Nanosilva LLC should request an amendment to remove the expiration date once they have subm itted the required data if they wish to continue to sell and distribute NSPW-L30SS in the United States.

EPA believes that the use of NSPW-L30SS is in the public in terest. Use of NSPW-L30SS may lead to less environmental loading of silver as co mpared to currently registered products with the same use patterns.1 I. Regulatory Decision Summary EPA is granting a conditional registration fo r the Nanosilva LLC product named “NSPW-L30SS” under section 3(c)(7)(C ) of the Federal Insect icide, Fungicide and R odenticide Act (FIFRA). NSPW-L30SS, which contains 1% nanosilver by wei ght, is the subject of an application submitted by Nanosilva LLC in August 2009 (MRID 47828900) . Nanosilva LLC requested that the nanosilver in NSPW-L30SS be registered as a ne w active ingredient because it was not an active ingredient in any cu rrently registered pesticide product.

As described in the risk assessment sections of this document, EPA has determined that use ofNSPW-L30SS will not cause unreasonable adverse effects on the environment during the fouryear period when newly require d data are being developed. In August, 2009 Nanosilva LLC submitted acute t oxicity studies required under 40 CFR part 161 1

, a study that determined the amount of silver l eaching from plastic coupons incorporating NSPWL30SS, and a request to waive some required stud ies. The waiver request was based on the low exposure to NSPW-L30SS given that the product is embedded in plastic and the submitted leaching study for a plastic incorporating NSPW-L30 SS indicated that silver was not released at concentrations above the analytical limit of detection (LOD). In May 2013, Nanosilva LLC also submitted a study showing that silver was not releas ed at concentrations above the analytical LOD from textiles incorporating NSPW-L30SS. EPA is granting the registration request with requirements for new data as terms and condi tions on the NSPW-L30SS registration. Nanosilva LLC, however, has not had sufficient time to gene rate and submit these newly required data.

EPA has determined that use of NSPW-L30SS is in the public interest. EPA believes that Nanosilva LLC’s product may lead to less environm ental loading of silver as compared to currently registered products containing silver salts with the same use patterns. EPA is requiring Nanosilva LLC to conduct a number of studies as a condition of registration. These studies must be completed within a time du ration of four years which was chosen to allow time for protocol reviews prior to initiation of the studies, comple tion of the studies, and Agency review of the study results. EPA will evaluate these data as they are submitted during the period of the conditional registration to c onfirm EPA’s risk assessment. If Nanosilva LLC fails to take appropriate steps to initiate the required studies, or if Nanosilva LLC fails to submit the protocols or data, EPA has the authority to use the proced ures of FIFRA section 6(e) to cancel Nanosilva LLC’s registration. In addition, Nanosilva LLC’s conditional registration for NSPW-L30SS will automatically expire four years after being issued. Nanosilva LLC will need to submit an application to obtain an unconditional registration if they wish to continue to sell and distribute NSPW-L30SS in the United States. II. Background 2.1 Regulatory History In August 2009, Nanosilva LLC submitted an application for the registration of a new antimicrobial pesticide product named NSPW-L 30SS, a nanosilver-based product that was proposed for use as a materials preservative addi tive with no food contact uses. Nanosilva LLC’s application claimed that NSPW-L 30SS contains a new nanosilver act ive ingredient not contained

1 At the time Nanosilva LLC applied to register NSPW-L30SS , test guidelines were listed in 40 CFR part 161. In 2013, updated test guidelines were finalized in 40 CFR part 158W.2 in any currently registered silv er-based pesticide pr oducts and should be gi ven a registration under FIFRA section 3(c)(7)(C) (MRID 47828900). 2.2 FIFRA Scientific Advisory Panel (SAP) Meeting In November, 2009 EPA convened a meeting of the FIFRA Scientific Advisory Panel (SAP) to address a number of questions associated with assessing the hazard of and exposure to nanosilver and other nanoscale metal-based pesticides (FIFRA SAP, 2009). In general, the SAP advised that the toxicity of nanosilver could differ from and might be higher th an other forms of silver (e.g., silver ions). The SAP was unsupportive of bridging among silver-b ased materials with different properties. However, the SAP indicated that bridging would be appropriate for materials of similar size and essentially identical physical pr operties and that bridging between silver ions released from nanosilver and the existing database for silver ions is feasible (FIFRA SAP, 2009). The SAP cautioned about extrapolating from one nanosilver formulation to another when assessing hazards because differences in particle formulation (e.g., coating and inert ingredients) could affect biological activity, among other things.

The SAP commented that not e nough literature was available to draw any firm conclusions regarding human (occupational or consumer) and environmental exposures to nanosilver under realistic use scenarios. Potentially, three major ro utes exist for human expo sure to nanoparticles: oral, inhalation, and dermal. Only a few studies in rodents were known that investigated the toxicity of nanosilver from exposure by these rout es. Nor was there much information on the level of human exposure to nanosilver by these routes, for either workers or consumers using products containing nanosilver. The same situation existed for the environmental fate and transport of nanosilver. The ability to measure concentrations of nanosilver in the environment along with the environmental exposure pathways, bioavailability, toxic ity, and potential impact of nanosilver on ecological systems were not well quantified. Furthe rmore, little or no information on the fate of nanosilver in soils and sediments was found. As a result, the SAP recomm ended a case-by-case approach to hazard and exposure assessment (i.e., product-by-product). The SAP also advised that existing requirements may need to be adjusted to obtain data appropria te to assess the fate, degradation, metabolism, mobility, dissipa tion, and accumulation of nanomaterials. The SAP report further suggested th at existing information on convent ional silver could be useful but would not necessarily be sufficient in assessing potential nanosilver risks. The SAP recommended that EPA treat nanos ilver differently from its conve ntional silver counterpart in evaluating proposed nanosilver product applicati ons (in terms of both data requirements and the conduct of risk assessments). Moreover, the Pane l recommended that EPA require additional data on the physical chemistry, exposure potential, and th e potential hazard to human health and the environment. Historically, EPA has considered applications for pesticide products that claim to be identical or substantially similar in composition to a regist ered product as so-called “me-too registrations” under FIFRA registration authorities. Until recentl y, EPA generally has not focused on the size or surface coating of an ingredient as attributes relevant to determini ng if the product in an application is identical or substantially similar in composition to a registered pesticide product. However, a nanoscale ingredient may have pr operties that are di fferent from those of conventionally-scaled ingredients and properties that differ from the atoms or molecules from which the nanoscale ingredient is constructed. Th erefore, a nanoscale ingredient may also have different environmental health and safety properties. Accordingly, for a product containing an3 ingredient that is a na noscale version of a convent ionally-sized active or in ert ingredient contained in an already-registered product or a different nanoscale version of a nanoscale material that is an active or inert ingredient in an already registered pesticide pr oduct, EPA may require additional data to assess the nanoscale material and to make the requisite statutory findings. 2.3 Proposed Decision Document
On August 27, 2013, EPA posted a draft version of this Decision Document to the Pesticides Public Regulatory Docket for public comment . EPA received comments on the draft Decision Document along with supporting information fo r a period of 30 days until September 26, 2013. EPA is posting responses to the ten comments recei ved regarding this decision in the Pesticides Docket. III. Product Description, Testing, and Use 3.1 Product Description NSPW-L30SS is a liquid suspension containing s ilica-sulfur-nanosilver pa rticulates where the nanosilver is attached to amor phous silica via a thiola te bond. The particles are formed by reacting silver nitrate and polyvinylpyrro lidone (PVP) with spherical silica particles that have been modified with thiol groups and are suspended in ethanol (Lee et al. 2007). The diameter of the spherical silica core particles are 320 nm on av erage (Lee et al., 2006). The nanosilver particles form with time and have an average diameter of 6.9 nm (minimum diameter of 3 nm and maximum diameter of 12 nm) after 10 minutes and 10.6 nm (minimum diameter of 5 nm and maximum diameter of 18 nm) after 2 hours (Lee et al., 2007). The liquid su spension contains 1% nanosilver by weight with average diameters between 6.9 and 10.6 nm (minimum diameter of 3 nm and maximum diameter of 18 nm). The label states that NSPW-L30SS, which contains 1% nanosilver by weight, is to be formulated into a polymeric intermediate known as a master batch at rates up to 2% of NSPW-L30SS. The application rate for treatment of the finished co nsumer product is 5.0 - 15.0% of the master batch that contains NSPW-L30SS. Thus, the final produ ct to which a consumer may be exposed will contain less than 0.003% nanosilver by weight.

3.2 Product Use NSPW-L30SS is registered to be incorporated into non-food contact plas tics and textiles to
suppress the growth of bacteria, algae, fungus, mold and mildew, which cause odors, discoloration, stains , and deterioration. 3.3 Product Testing and Waivers As an applicant for registration of a new active ingredient in 2009, Nanosilva LLC was required to submit all the applicable information a nd studies under 40 CFR part 161. Nanosilva LLC submitted results from testing of NSPW-L30SS to determine the product identity and composition, physical and chemical properties, an d acute toxicity as summarized in Table 1. Some of these tests were completed using sta ndard EPA test guidelines 2 prior to consulting with EPA. Although these test results provided useful information for NS PW-L30SS, the guidelines on which they are based have not been adapted generally for use with na noscale particles. EPA anticipates that these guidelines will require revision going forward in term s of their application to nanoscale materials.

2 Available at http://www.epa .gov/ocspp/pubs/frs/home/guidelin.htm4 As a result, until revisions are completed, it is recommended that future applicants for products containing nanoscale materials consult with the EPA prior to performing any tests. Table 1 – Product Testing for the NSPW-L30SS Liquid Suspension

Guideline Number Guideline Name Required under 40 CFR Nanosilva LLC Submission (MRID) Product Identity and Composition 830.1550 Product identity and composition 161.155 47828901 830.1600 Description of materials to produce product 161.160 47828902 830.1620 Description of produc tion process 161.162 47828903 830.1650 Description of formul ation process 161.165 47828904 830.1670 Discussion of formulation of impurities 161.167 47828905 830.1700 Preliminary analysis 161.170 47828906 830.1750 Certified limits 161.175 47828907 830.1800 Enforcement analytical method 161.180 47828908 Physical and Chemical Properties 830.6302 Color 161.190 47828909 830.6303 Physical state 161.190 47828910 830.6304 Odor 161.190 47828911 830.6317 Storage stability 161.190 47828912 830.6320 Corrosion characteristics 161.190 47828912 830.7000 pH 161.190 47828913 830.7100 Viscosity 161.190 47828914 830.7220 Boiling Point/Boiling Range 161.190 47828915 830.7300 Density 161.190 47828916

830.7520 Particle size, fiber length, and diameter distribution Information in Lee et al., 2006 and 2007 used to address data requirement 830.7840 Water solubility 161.190 47828917 Health Effects (Toxicology) 870.1100 Acute Oral Toxicity 161.340 47828918 870.1200 Acute Dermal Toxicity 161.340 47828919 870.1300 Acute Inhalation Toxicity 161.340 47828920 870.2400 Acute Eye Irritation 161.340 47828921 870.2500 Acute Dermal Irritation 161.340 47828922 870.2600 Skin Sensitization 161.340 47828923

In addition to the test results listed in Table 1, Nanosilva LLC submitted the following study to support the registration of NSPW-L30SS: Plastic Leaching Study

 Leaching protocol for NSPW-L30SS-treated LLDPE polymer in food and food simulated matrices as functions of time, temperature a nd chemistry of the matrix with determined migration values (MRID 47828925) Nanosilva LLC also submitted a request to waive the following data requirements:

1. Hydrolysis (OCSPP Guideline Number (GLN) 835.2120) 2. Aquatic Invertebrate Acute Toxicity Test , Freshwater Daphnids (OCSPP GLN 850.1010) 3. Fish Acute Toxicity Test, Freshw ater and Marine (OCSPP GLN 850.1075) 4. Avian Acute Oral Toxicity Test (OCSPP GLN 850.2100) 5. 90-Day Dermal Toxicity (OCSPP GLN 870.3250)5 6. Prenatal Developmental Toxi city Study ( OCSPP GLN 870.3700) 7. Bacterial Reverse Mutati on Test (OCSPP GLN 870.5100) 8. Detection of Gene Mutations in Soma tic Cells in Culture (OCSPP GLN 870.5300) 9. In Vitro Mammalian Cytogeneti cs (OCSPP GLN 870.5375) 10. Mammalian Bone Marrow Chromosome Aberration Test (OCSPP GLN 870.5385) 11. Immunotoxicity (OCSPP GLN 870.7800)

Waivers for the above studies were requested because of the lack of acute toxicity noted in the six acute toxicity studies (M RID 47828918 through 47828923), because NSPW-L30SS has a specific gravity greater than water, because NSPW-L30SS ha s low solubility in water as determined by the water column generator method (MRID 47828917), due to the non-leachi ng characteristic of NSPW-L30SS, and because silver was below the level of detection (LOD) in leaching studies conducted with a plastic incorporating NS PW-L30SS (MRID 47828925). Evaluation of Nanosilva LLC’s Data Waiver Requests is detailed in Appendix A. In response to EPA’s review of the leachi ng and product chemistry studies, Nanosilva LLC subsequently submitted the following documents:

Nanosilver Content of Plastic Coupons Containing NSPW-L30SS

 Determination of Silver C ontent and Silver Recovery Rate for NSPW-L30SS (MRID 48652901) Description of Production and Formulation Process

 Lee, J.M., Kim, D.W., Jun, Y.D., Oh, S.G. 2006. Preparation of silica-silver heterogeneous nanocomposite particles by one-pot prepara tion strategy using pol yol process: Sizecontrolled immobilization of silver nanoparticles. Materials Research Bulletin 41:1407- 1416.(MRID 48379901 and 48379903)

 Lee, J.M., Kim, D.W., Kim, T.H., Oh, S.G. 2007. Facile rout e for preparation of silicasilver heterogeneous nanocomposite pa rticles using alcohol reduction method. Materials Letters 61:558-1562. (MRID 48379902 and 48379904) Textile Leaching Study

 Nanosilva LLC prepared and submitted on A ugust 20, 2012 a draft protocol titled “The Quantification and Characterizati on of Silver Released from Textiles Treated with NSPWL30SS (NSPW-L30) as a Results of Washing”

 The Quantification and Characterization of S ilver Released from Textiles Treated with NSPW-L30SS As a Result of Simulate d Laundering Conditions. Study Number: 110112.0001 Revision 2 (MRID 49045301)

IV. Hazard Characterization of Nanosilver Nanosilver is a broad-spectrum antimicrobial agent that works by releasing ionic silver, but also exhibits particle-specific effects (Wang et al . 2013). In November 2009 EPA convened a meeting of the FIFRA Scientific Advisory Panel (SAP) to address a number of ques tions associated with assessing the hazard of and expos ure to nanosilver and other nanoscale metal-based pesticides (FIFRA SAP, 2009). In general, the SAP advised th at the toxicity of nanos ilver could differ from and might be higher than other forms of silver (e .g., silver ions). The Panel agreed with EPA that particle size has a substantial im pact on particle properties, incl uding rate and concentration of silver ion release, where the effects of size ar e generally most observable for particles with dimensions below 20 nm and largely below 10 nm (FIFRA SAP 2009, p. 6). In addition to size,6 other properties such as shape, charge, and su rface coating have the potential to impact the biological response to nanosilver. 4.1 Acute Human Toxicology of NSPW-L30SSNanosilva LLC submitted results from guideline acute animal-toxicity tests completed using highlevel doses of a liquid suspension containing NSPW-L30SS with 1% nanosilver by weight. As outlined in Table 2, there were no mortalities or abnormalities noted in test animals after administration of NSPW-L30SS by oral, dermal, or i nhalation routes at do se levels of up to 5,000 mg/kg and 2.05 mg/L, respectively; NSPW-L30SS caused moderate to no irritation to skin or eyes at dose levels of up to 0.1 mL and 0.5 mL, respectiv ely; and was not a skin sensitizer. According to EPA’s Toxicity Category system, which is used for product labeling purposes, shipping containers filled with NSPW-L30SS are require d to carry a label stating “CAUTION” where contact with eyes or clothing should be avoided. Table 2 – Acute Toxicity Profile for the NSPW-L30SS Liquid Suspension
Study Result Toxicity Category Acute Oral Toxicity No mortality or abnormalities after dose of 5,000 mg/kg IV Acute Dermal Toxicity No mortality or abnormalities after dose of 5,000 mg/kg IV Acute Inhalation Toxicity No mortality or abnormalities after dose of 2.05 mg/L IV Acute Eye Irritation Moderate to not irritating after dose of 0.1 mL III Acute Dermal Irritation Mild or slight irritation after dose of 0.5 mL IV Skin Sensitization Not a sensitizer N/A

4.2 Subchronic and Chronic Toxicologica l Studies Available for Nanosilver There are no repeated-dose subchronic or chroni c toxicity studies available for NSPW-L30SS or the nanosilver in NSPW-L30SS. However, there are repeated-dose toxicity studies available in the scientific literature for nanos ilver. Since nanosilver might be released from NSPW-L30SS and articles incorporating NSPW-L30SS, EPA considers the scientific literature studies on nanosilver toxicity relevant. EPA believes th e studies described in the followi ng sections are sufficient for assessing the risks from the use of NSPW-L30S S during generation of the requisite data.

4.2.1 Oral There are currently three studies in the scientific literature that investigate the oral toxicity of nanosilver in rats and two studies completed with mice. The first study reported findings after 28 days of repeated gavage admini stration of carboxymethyl cellulose -coated nanosilver with average diameter of 60 nm (minimum diameter of 53 nm and maximum diameter of 71 nm) to four-week old male and female Sprague-Dawley rats (n = 10 per dose) (Kim, et al, 2008). There were liver effects (dilation of the central vein, bile-duct hype rplasia and increased foci), a coagulative effect on peripheral blood, and an increas e in serum alkaline phosphatase (ALP) and cholesterol. There was a dose-dependent increase in silver distri bution in many tissues (liver, kidneys, stomach, brain, lungs, testes, and blood) a nd a two-fold higher accumulation of silver in the kidneys of female rats when compared with male rats was also reported for all dose groups. A no observed adverse effect level (NOAEL) of 30 mg/kg/day (lowest dose level) was reported based on the observed liver effects and increase in ALP and cholesterol at 300 mg/kg/ day (mid dose level). The second study was performed by the same group using four-week old Fisher rats (n= 10 per dose) for 90 days (Kim et al., 2010). This st udy involved repeated gavage administration of7 carboxymethyl cellulose-coated nanosilver with aver age diameter of 56 nm (minimum diameter of 25 nm and maximum diameter of 125 nm) to rats and reported similar findi ngs as the 28-day study including gender-related distri bution of silver in the kidne ys and a reported NOAEL of 30 mg/kg/day. However, intestinal pigmentation from exposure to nanosilver was reported, which was not observed in the 28-da y study (Kim et al. 2008). The third study involved feeding nanosilver (14 nm average diameter) stabilized with PVP or silver acetate to four-week old male (n= 6 per dose) and female (n=10 per dose) Wistar Hannover Galas rats for 28 days (Hadrup et al. 2012a). They also reported no observed effects on the microbiological status of the ra t’s gastrointestinal tract following ingestion of nanosilver. However, Hadrup et al., (2012a) di d report effects for silver acet ate at a dose of 14 mg/kg/day including an increase in ALP, d ecrease in plasma urea, and lowe r thymus weights. Hadrup et al. (2012b) compared the neurotoxic e ffects of PVP-stabilized nanosilver (average diameter of 14 nm) and silver acetate, both, in vivo and in vitro . Following 28 days of oral administration, nanosilver (4.5 and 9 mg/kg/day) and silver acetate (9 mg/kg/ day) significantly increase d the concentration of dopamine in the brains of Wistar female rats, while the brain concentration of 5- hydroxytryptamine (5-HT) was increased only by nanosilver at a dose of 9 mg/kg/day. The NOAEL for this study was 9 mg/kg/day, but as this was the top dose tested, no LOAEL could be established. However, in the 14-day range-finding component of the studies, the brain dopamine concentration decreased in rats treated with nanosilver at doses of 2.25 and 4.5 mg/kg/day. In the in vitro experiment, three solutions consisting of 1) na nosilver, 2) an ionic silver solution obtained by filtering a nanosilver suspension, and 3) silver acetate were test ed in neuronal-like PC12 cells. Nanosilver did not induce necrosis ; however, cell viability was decreased and apoptosis (involving both the mitochondrial and the death receptor path ways) was found with all three solutions with the silver acetate being most potent. There is one study of repeated administration of nanosilver with average diameter of 42 nm (minimum diameter of 25 nm and maximum diamet er of 55 nm) to mice for 28 days (Park, et al., 2010). The study reported that, after oral administ ration of nanosilver at the dose levels of 0.25 mg/kg/day, 0.5 mg/kg/day, or 1.0 mg /kg/day, the serum enzyme le vels of ALP and aspartate transaminase (AST) were significantly elevated in both male and female mice in the high dose group. The level of alanine transaminase (ALT) was also elevated in the high dose females. Histopathological analysis was performed in the high-dose groups and reveal ed that tissue change (i.e., slight cell infiltration) was observed in the cortex of the kidneys in both male and female mice, but no other histopathological changes were found in the portions of liver or small intestines that were examined. A NOAEL of 0.5 mg/kg/day was initially re ported, based on the observed findings of elevated ALP, AST and ALT and the hi stopathological changes in the kidneys at the 1.0 mg/kg/day dose level. However, EPA reviewed th is study again as part of this assessment and now concludes that although the results of the Park et al. (2010) study show ed evidence of changes in clinical chemistry, it lacks histological suppor t for the effects used as the basis for the study NOAEL/LOAELs. In the absence of histopathological findings, the clinical chemistry changes observed alone are insufficient evidence of an adverse effect. The updated NOAEL is 1.0 mg/kg/day, the highest dose tested. Liu et al. (2013) reported that nanosilver di d not affect spatial cognition or hippocampal neurogenesis in adult male ICR mice (n=15 pe r dose, n=10 for control). Adult mice were administered nanosilver with av erage diameter of 51.4 nm by D ynamic Light Scattering (DLS) and 26.3 nm (size range from 10 to 70 nm) by Transmission Electron Microscopy (TEM) via intraperitoneal injecti on, at doses of 0, 10, 25, or 50 mg/kg, once a day in the morning for 7 consecutive days. Another group of mice received scopolamine (3mg/kg) as a positive control for8 the behavioral studies. Investigators used the Mo rris water maze (MWM) test to evaluate spatial cognition and bromodeoxyuridine for detecting pro liferating cells to measure neurogenesis. The test results showed that both reference memory and working memory were not impaired in nanosilver exposed groups, compared with the control group, and no differences were revealed in hippocampal progenitor proliferation, new born ce ll survival, or different iation in nanosilver treatment groups, indicating that neurogenesis was also unaffected.

4.2.2 Inhalation The subchronic inhalation toxicity of nanosilver was evaluated in six-week old male and female Sprague-Dawley rats (n=10 per dos e) exposed to nanosilver with average diameters between 18 to 19 nm (minimum diameter of 2 nm and maximu m diameter of 65 nm) in a whole body inhalation chamber for 13 weeks (90-day) using OECD te st guideline 413 (Sung et al., 2009). Male and female rats exposed to th e high-dose level of 515 μg/m 3 (3.03×10 6 particles/cm 3 ) showed toxic effects in the liver (bile-duc t hyperplasia) and lungs (chron ic alveolar inflammation and macrophage accumulation in both males and female s, and erythrocyte aggregation). A follow-on study was performed to determine lung-function r ecovery of five-week old male and female Sprague-Dawley rats (n= 17 male and 12 female per dose) after exposure to nanosilver with average diameters between 14 to 15 nm (minimum diameter of 4 nm and maximum diameter of 47 nm) in a whole body inhalation chamber for 12 weeks, followed by a 12-week recovery period (Song et al., 2012). The pathological findings in the lungs were very similar to those observed in Sung et al. (2009) even though there were slight differences in pa rticle size and concentration. During the 12-week exposure period, there was a decrease in lung func tion of male animals exposed to all doses of nanosilver, including a decrease in tidal and minute volume, and peak expiratory flow. After the 12-week recovery period, the male rats in the mid- (117 μg/m 3 or 1.41×10 6 particles/cm 3 ) and high-dose (381 μg/m 3 or 3.24×10 6 particles/cm 3 ) groups did not achieve complete recovery of lung function. Remarkably, no advers e lung function effects were observed for female rats. The decrease in l ung function was accompanied by histopathological observations of perivascular mi xed cell infiltrate and chronic alveolar inflammation in male animals, but these effects were also observed in females even though they had no measurable decrease in lung function. Although female rats showed gradual clearance of silver from lung tissues and decreased inflammation after 12 weeks of recovery, persistent inflammation in lung tissues was still observed in the high-dose male rats throughout the 12-wee k recovery period. This lack of recovery indicates permanent or persis tent organ damage. Signi ficant increases in the amount of silver in tissues, such as lungs, liver , olfactory bulb, brain, kidneys, and blood, were also reported. Females had two to three times more silver accum ulation in their kidneys than males. Liu et al. (2012) investigated the effects of na nosilver on hippocampal synaptic plasticity and spatial cognition in adult male Wistar rats (n =8 per dose). Uncoated nanosilver with average diameter of 244.5 nm (size range from 33 to 380 nm) by DLS and with sizes ranging from 50 to 100 nm by TEM was administered to rats by nasa l drops at doses of 3 mg/kg and 30 mg/kg once every two days for 14 days. Spatial cognition as determined by the Morris water maze (MWM) test showed that animals rece iving nanosilver exhibited lowe r learning ability and memory retention, which was more prominent in the high -dose group (30 mg/kg). This finding was also supported by impaired synaptic plasticity as determined by long-term potentiation (LTP) recordings for these same animals. In addition, the quantity of reactive oxygen species (ROS) in hippocampal homogenate was significantly increase d in nanosilver- trea ted groups, and edema, nuclear shrinking phenomenon, and necr obiosis were shown in hemat oxylin and eosin (HE) stains of pyramidal neurons in the PP (perforant path ) and DG (dentate gyrus) regions of hippocampus.9 4.2.3 Dermal There are only two dermal toxicity studies ava ilable for nanosilver. One study was performed to determine liver, skin and spleen pathologies of fi ve to six week old male guinea pigs (n=6) after exposure to nanosilver (concentrat ions 100 μg/ml, 1000 μg/ml, and 10,000 μg/ml) with a particle size of less than 100 nm based on expos ure to aqueous solution using AgNO 3 as a positive control (Korani et al, 2011). The nanosilver suspension was a pplied by daily rubbing to an area of 5 cm by 5 cm (10% surface area) on the back of the anim al with no wipe off or removal of chemical mentioned. The applied dose in mg/kg was not determined in the study. According to email contact with authors, animals were restrained for 5 hours afte r application of the solution; however, the method of restrain t was not discussed. During the 13 week study, dose dependent and nanosilver specific effects were seen in the liver, the spleen and on the skin. Skin effects included decreased thickness of the epidermis and dermis, inflammation, increased levels of round cells, acidophilic cytoplasm in muscle fibers and increa sed levels of macrophages in the endomysium. Liver effects included overproductio n of Kupffer cells and degenera tion of hepatocytes in a dose dependent fashion and necrosis at the 10,000 μg/m l concentration. Other dose-dependent effects observed in the liver included the following: th inner red capsules, inflammation, accumulation of red blood cells, and white pulp atrophy along with hepa tic cord destruction. It does not appear that gauze or another dressing was used to cover the dosed area. No hematology, clinical chemistry or urinalysis was performed. Only the liver, spleen and skin were examined histopathologically. Another study was performed to determine the eff ect of skin exposure to nanosilver (size = 20 and 50 nm, concentration = 0.34, 3.4 and 34 μg/ml aqueous so lution) in 2 female pigs (Samberg et al, 2010). A 500 μL nanosilver suspension was placed on one of 14 spots on the back of a hairclipped pig, allowed to air dry, th en covered with a H illtop chamber (occlusion pad) and secured with non-irritating tape. This was followed by a body stocking covering the dorsum of each pig. Actual exposure was 0.6, 6, and 60 ng/mm 2 and total exposure was 0.17, 1.7 and 17 μg per dosing period. No gross pathological e ffects were noticed on the porcine skin. A concentration, but not particle size or washing state, dependent effect was seen on the dermal layers under microscopic investigation. Low dose effects were slight intra and intercellular epidermal edema. Intermediate effects were a more focal intr a and intercellular epidermal edema alongside focal dermal and epidermal inflammation. The highest dose caused severe edema, with severe focal dermal inflammation, epidermal hyperplasia and parakeratosi s. Precautions were taken to prevent oral dosing, including restraint/anesthesia and multiple cove ring layers. Only effects relating to the skin were examined.

None of the above studies are an acceptable substitute for a derm al subchronic study or a dermal irritation study. In no case was a full gross or microscopic histopa thology panel performed, even in the cases where dose dependent effects of nano-silv er particles were seen. None of these studies used the same size particles, coating type or washing state, making it pot entially difficult to generalize information from them onto other pro ducts. In Korani et al. (2011), the study lacked any clinical chemistry, hematology or urinalysis of the guinea pigs . This study also could not draw NOAEL/LOAEL conclusions given that effects were seen at all levels of dosing. In Samberg et al. (2010), no systemic histopathological evaluations were possible as multiple dose concentrations were tested on the same animal. However, there is one published report on nanosilver that is av ailable for use by EPA concerning nanosilver used in wound dressings for patients su ffering from burns. In this report, a burn patient using a nanosilver-coated wound dr essing developed clinical signs of argyria and elevated serum liver enzymes indicative of liver toxicity along w ith elevated silver conc entrations in blood and10 urine (Trop, 2006). This study indicate s to EPA that nanosilver can be systemically absorbed when a large area of the skin barri er is severely compromised. In the absence of any dermal toxicity studies, EP A normally uses extrapolation from another route of exposure (usually oral). However, use of th e oral endpoint for evaluating dermal toxicity requires knowledge regarding the amount absorbed through the skin. This information is typically provided by a dermal penetration study using w hole animals (usually rats), which allows determination of the fraction of topically applie d dose that is available for systemic absorption (i.e., the dermal absorption factor or DAF). There are in vitro techniques available that enable determination of dermal penetr ation of chemicals through isol ated animal or human skin. However, EPA does not rely on in vitro dermal absorption study data as the sole basis for deriving a DAF because standardized in vitro test methodology is not currently available. Without detailed, standardized methodology for in vitro absorption studies, EPA has observed variation in test results among laboratories. As a result, EP A assumes a default DAF of 100% when no in vivo data are available. Therefore, if in vivo dermal penetration studies are available, either those studies alone or in combination with in vitro studies are used for derivi ng a DAF of less than 100%. Currently, there is no guidelin e or scientific literature st udy conducted in animals for the in vivo dermal absorption of the nanosilver in NSPW-L30SS or of nanosilver available to EPA. However, there is a human clinical study, which is observationa l, that examined silver levels in serum and urine after application of burn wound cream containi ng silver sulfadiazine nanosilver (Wan et al., 1991). EPA used this information to derive a conservative DAF of 6.7% for nanosilver. A study completed by Brandt et al. (2012) demonstrated that nanosilver and silver sulfadiazine have similar skin absorption characteristics in mice af ter normalizing for silver dose. In addition an in vitro study with nanosilver in human skin is availa ble in the scientific literature. This study demonstrated that nanosilver penetration is ve ry low for both intact and abraded skin at 0.00066% and 0.0033%, respectively (Larese et al., 2009). 4.2.4 Reproductive and Developmental Toxicity of Nanosilver There are studies showing dose-dependent increases in the concentration of silver in the testes of rats after oral ingestion, inha lation, and injection of nanosilver (see Section 4.3); however, there are few studies available on the reproductive and developmental toxicity of nanosilver.

There is an in vitro study investigating the toxicity of 15 nm nanosilver on spermatogonia isolated from 6-day old mouse testes and immortalized with SV40 large T antigen (Braydich-Stolle et al., 2005). In this transformed (i.e ., immortalized) cell line, both nanosilver and silver ions individually caused altered cellu lar morphology, decreased mitochondr ial activity (as indicated by MTS cell viability assay), and increased apoptosis at doses up to 10 μg/ml; however, the effects from nanosilver were greater than observed for silv er ions. The conservative interpretation of these data is that nanosilver that reaches the testes may be able to cause decreased fertility due to toxicity to spermatogonia and this effect would be more severe for nanosilver than for silver ion. In another study, Austin et al. (2012) investigated the distribution of citrate-coated nanosilver with diameters between 30 and 60 nm and silver nitrat e in pregnant mice (n= 6 to 12 per dose) and developing embryos. Nanosilver suspensions and silv er nitrate were administered by intravenous injection (i.v.) on gestation da ys (GD) 7, 8, and 9 at nanosilver concentrations of 0, 0.4, and 0.73 mg/kg/day. Austin et al. (2012) re ported a significant increase in nanosilver content as compared to silver nitrate treated animals in nearly all tissues; nanosilver accumulation was significantly higher in liver, spleen, lung, tail (i njection site), visceral yolk sac, placenta and endometrium. This study did not find significantly higher nanosilver in the ovary. Nanosilver was identified in11 vesicles in endodermal cells of the visceral yolk sac. This study demonstrated that nanosilver distributed to major maternal organs and extra-em bryonic tissues, but the authors stated that very little silver reached developing embryos and no adverse morphological effects on the developing embryos were observed. In a 14-day repeated maternal dose study (Yu et al. 2013), nanosilver (7.5 nm) was administered via gavage to 11 pregnant Sprague -Dawley rats per dose from days 6 to 19 of gestation. No gross pathological effects or other developmental effe cts on the fetus were observed up to the maximum dose of 1000 mg/kg/day. There were no effects on maternal food consumption or body weight but there were some slight changes in liver catalase and glutathione reductase levels (all dose levels) and a decrease in glutathione at the maximum dose . No effects were seen in any organs, including the ovaries. The teratogenicity potential of nanosilver in preg nant rats was investigated by Mahabady et al. (2012). Nanosilv er of unknown size and surface co ating was administered to pregnant rats via intraperiton eal injection (i.p.) on GD 8 and 9. Fetuses collected on GD 20 from animals that received nanosilver were reported to have reduced weight an d length but there were no effects on the skeletal system as compared to animals treated with a saline control. Because Mahabady et al. (2012) di d not report key information about the nanosilver particles size and surface coatings, it is not possible to compare these results to the nanosilver in any other study or in any nanosilver-con taining product. 4.2.5 Mutagenicity of Nanosilver Currently, there are no studies in the scientific lite rature that investigate th e potential of nanosilver to cause cancer (i.e., carcinogenic ity). The exposures from this ch emical are not expected to be long term. However, the potential of nanosilver to induce change s in genetic material (i.e., mutagenicity, genotoxicity) has been i nvestigated with equivocal results in vitro , using traditional mutagenicity tests including the bacterial reve rse mutation assay (i.e., Ames test), the mouse lymphoma forward mutation test, the mammalian cell chromosome aberration test in Chinese hamster ovary cells, and the mouse lymphoma Comet assay for oxidative damage. These in vitro studies are equivocal as to the cytotoxicity a nd genotoxicity of nanosilver. Additionally, nanosilver was investigated in a series of in vivo studies. Results from these assays are summarized below.

In vitro reverse gene mutation assay in bacterial ce lls: The mutagenicity of nanosilver (average diameter of 10 nm) suspended in a 1% citric acid solution was determined at concentrations of up to 500 μg/plate in bacterial cells using the Ames test (Kim et al., 2012). Although cytotoxicity was observed at 31.25 μg/plate, nanosilver did not induce a mutagenic eff ect in the histidine-requiring strains of Salmonella typhimurium TA98, TA100, TA1538 and TA1537 or in tryptophan-requiring Escherichia coli WP2uvrA with or without the metabol ic activation system (±S9). A similar result was obtained by Li et al.(2012) for nanosilver with an average diameter of 5 nm (size range from 4 to 12 nm) prepared in TEM for the primary partic les and for particles with a diameter of 1608.7 ± 175.4 nm, prepared in culture media. In this te st, there was no increase in revertant mutant colonies of the standard S. typhimurium tester strains, which include d the strain used to detect oxidative damage (TA102) up to cytotoxic co ncentration (31.25-62.5 μg /plate -S9; 125-250 μg/plate +S9). However, Li et al. (2012) cautioned that because of cytotoxicity and the physical properties of nanosilver, this test system may lack the sensitivity required to detect the mutagenic action of the test material.

In vitro forward gene mutation assay in mouse lymphoma L5178Y/TK +/- cells with a Comet Assay: The mutagenicity of nanosilver, which had an average diameter of 5 nm (size range from 4 to 12 nm) prepared in TEM for the primary pa rticles and a diameter of 1608.7 ± 175.4 nm in the culture media, was evaluated in mouse lymphoma L5178Y at the TK +/- locus and the modes of12 action was assessed using standa rd alkaline and enzyme-modified Comet assays with a gene expression analysis (Mei et al., 2012). Nanosilv er induced dose-dependent cytotoxicity and mutagenicity with a marked increase in the mutation frequency at 4 and 5 μg/mL (<50% cell survival at ≥ 5 μg/mL) where nanosilver had a clastogeni c mode of action. Subsequent testing revealed no evidence of DNA damage (Comet test), but oxidative damage (modified Comet test), confirmed by gene expression analysis, which sh owed an expression pattern consistent with production of reactive oxygen species (ROS).

In Vitro Chromosome Aberration Test: The clastogenic effect of nanosilver (a verage diameter of 10 nm) suspended in a 1% citric acid at concen trations of less than 31.25 μg/mL was determined in Chinese hamster ovary cells (CHO-k1) after 6- and 24-hour exposures ±S9 using OECD Test Guideline 473 (Kim et al. 2012). Nanosilver did not induce any statistically significant increase in the number of cells with chromosome aberra tions, polyploidy, or endoreduplication when compared with the control group at concentra tions causing approximately 50% cytotoxicity.

In Vitro Mammalian Cell Assays: An in vitro mammalian cell micronucleus test (OECD Test Guideline 487) was used to determine the geno toxicity of nanosilver, which had an average diameter of 5 nm (size range from 4 to 12 nm) by TEM for the primary particles and a diameter of 1608.7±175.4 nm prepared in culture media. Nanosilv er at concentrations of up to 30 μg/mL induced a significant and dose-rela ted increase in micronuclei in human lymphoblastoid TK6 cells, indicating that nanosilver has weakly pos itive genotoxic potential (Li et al, 2012). The genotoxic effects of silver nanoparticles (43-260 nm) were further studied by Kim et al. (2011) on normal human bronchial ep ithelial (BEAS-2B) cells using the in vitro micronucleus and comet assays. As expected, ≈ 100% of the particles with diameter s in the range of 40-55 nm were taken up by the cells; ≥ 50% of the particles in the range of 60 to 100 nm were found within in the cells and lower percents of the la rger sixe particles were also de posited in the ce lls. Significant and concentration-related increases in DNA damage and micronuclei induction were found in the bronchial cells exposed to concentrations of 0.01 to 10 μg/mL silver nanopa rticles. Significant and dose-related Intracellular genera tion of reactive oxygen species (ROS) was also demonstrated at 0.01 to 10 μg/mL silver nanoparticles. Finally, the i nvestigators present data indicating that the DNA damage induced by silver nanoparticles in BEAS-2B cells was blocked by several wellknown ROS scavengers, primarily superoxide dismutase (SOD).

In Vivo Studies: In contrast to the above in vitro findings, the oral gava ge administration of carboxymethyl cellulose-coated nanosilver with aver age diameter of 60 nm (minimum diameter of 53 nm and maximum diameter of 71 nm) caused overt toxicity but failed to induce an increase in micronucleated polychromatic eryt hrocytes (MN PCEs) in the bone marrow of male and female rats after 28 days of treatment at doses 0, 30, 300, and 1,000 mg/kg/day (Kim et al, 2008). This study indicates that nanosilver is neither clastogenic nor aneugenic in vivo , although a limitation of this study is that no measurements were perfor med to determine if na nosilver reached the bone marrow. Nevertheless, subsequent studies disc ussed below provide confirmation that silver nanoparticles are readily absorbed and distributed throughout the rodent body. Negative results were also obtaine d by other researchers (Kim J S et al., 2011) using the inhalation route for a rat micronucleus assay. In this study, male and female rats were exposed by inhalation to concentrations of silver nanoparticles (18 nm) as high as 2.9 x 10 6 particles/cm 3 (equivalent to

≈ 1000 mg/kg/day), 6 hours/day for 90 days. Although silver nanoparticles were not toxic to the animals or cytotoxic to the target tissue ( bone marrow cells), a dose- dependent deposition of AgNPs was found in the blood, stomach, brain, liver, kidney, lungs, and testes of the treated rats.13 In a series of in vivo studies, Li et al. (2013) found that various types of silver nanoparticles ranging in size from 5 to 15 - 100 nm (coated w ith either PVP or sili cone), were neither clastogenic, aneugenic nor mutagenic in the mouse bone marrow micronucleus or Pig-a gene mutation assays. Tissue distribut ion was confirmed at the high est dose tested (25 mg/kg), following intravenous injection once or daily for 3 days. At 25 mg/kg, PVP-coated silver nanoparticles caused significant, ≈ 30% reductions in the percentage of reticulocytes. By contrast, no cytotoxicity occurred after treatment with silic on-coated Ag particles. Thus, there is compelling evidence that PVP-coated silver nanoparticles reached the target tissue but did not induce a mutagenic response in mouse bone marrow cells.

Similar results indicate that PVP- or silicone -coated 25 mg/kg silver nanoparticles reached the target organ but were not genot oxic. However, both PVP- and s ilicone-coated 25 mg/kg silver nanoparticles induced significant oxidative damage in the mouse liver, manifested as binding to 8- oxoguanine adducts in the m odified Comet assay.

In another in vivo test, Cho et al., (2013) exposed male rats to concentrations of silver nanoparticles (14-15 nm) ranging from 0.66 x 10 6 particles/cm 3 ( ≈ 49 μg/m 3 ) to 3.24 x 10 6 particles/cm 3 ( ≈ 381 μg/m 3 ) 6 hours/ay for 12 weeks. Lung cells were harvested and DNA damage was assessed using the Comet assa y. No significant changes in body weight or lung weight were observed. Histopathological examination of th e lungs revealed an increased incidence of perivascular and chronic inflammation, accompanie d by alveolaritis, granulomatous lesions and alveolar wall thickening and macrophage accumula tion. This inflammatory response paralleled a significant and dose-dependent increase in silv er nanoparticles and DNA damage in lung tissue. The genotoxic effect at the high dose was significant and ≈ 2X higher than effects seen in the control. In summary, the data from the in vitro studies (i.e., Ames bacteria l gene mutation and mammalian cell mutation and chromosome aberration assays) i ndicate that nanosilver is not expected to be mutagenic in bacteria. In mammalian cells, however, the in vitro micronucleus test and the mouse lymphoma comet assay suggest that nanosilver may have mutagenic potential. This is consistent with a recent review article which stated that in vitro data point to possible mutagenic properties of nanosilver (Bart ł omiejczyk et al., 2013). However, the l ack of mutagenicityin several wellconducted in vivo studies, showing cytotoxicity and evid ence that the nanopa rticles reached the target tissue, suggests that the in vitro mutagenic potential obse rved in some of the in vitro studies may be intrinsic to nanoparticles, but is not expressed in whole animals. There is evidence that silver nanoparticles are genotoxi c, expressing DNA damage in cu ltured human lung cells and in the lungs and liver of intact animals (comet a ssay) via intracellular ge neration of ROS. These findings are supported by the inflammatory proce sses described above in lungs. However, since initiation of ROS-induced oxidative DNA damage can be blocked by ROS scavengers, there is not a concern for mutagenici ty or genotoxicity.

4.2.6 Silver Ions Humans may also be exposed to silver i ons that would be released by NSPW-L30SS. Conventional silver, and th e silver ions it releases, can quali fy as pesticides under FIFRA when used for pesticidal purposes. The SAP concluded that the hazards of silver ions would be the same, whether they came from conventional silver or from silver nanoparticles. W ith respect to silver ions, EPA evaluated the toxicity and exposure to silver ions a nd determined that unreasonable adverse effects from use of silver-containi ng products are unlikel y (U.S. EPA, 1993). EPA reviewed a recent unpublished FDA study (DP# 427190) and determined that it did not change the conclusions of the Silv er risk assessment.14 4.3 Absorption, Distribution, Metabolism, and Excretion (ADME) of Nanosilver Studies were completed using either injection or oral administration of nanosilver to laboratory animals to determine the absorption, distribut ion, metabolism, and excretion (ADME) of nanosilver in whole animals. Based on the stud ies described in the following sections EPA believes biliary or fecal excretion of nanosilver is the primary elimination pathway. Silver is found primarily in the liver, spleen, and kidneys, but also in the thymus, brain, hear t, lungs and testes of animals dosed with nanosilver, silver nitrate, and silver acetate, where the organs of animals dosed with silver nitrate and silver acetate contained greater amount s of silver than did animals dosed with nanosilver. Animals can clear silver from bl ood and most organs given enough time but retain silver in the testes and brain. Animals treated with silver nitrate, silver acetate, and nanosilver all contain silver granules with dimensions on the nanoscale. Howe ver, it is unclear if intact nanosilver is absorbed into tissues or if nanosilver dissolves into i onic silver before being absorbed into tissues and forming nanoscale granules. 4.3.1 Injection of Nanosilver The translocation, distribution and accumulation of silver after a si ngle subcutaneous injection at 62.8 mg/kg of body weight of nanosilver with di ameters between 50 and 100 nm and microsilver with diameters between 2,000 and 20,000 nm was determined in Wistar female rats (n = 30 per group) (Tang et al, 2009). The silver content of feces between 2 and 24 w eeks after injection was significantly higher than in urine for both the nanosilver and microsilver-injected animals, which suggests that both nanosilver and microsilver ar e eliminated through biliary excretion. Although there was no significant differen ce between the amount of silver at the injection site or in excrements after administration of nanosilver or microsilver, the amount of silver in organs was significantly greater for nanosilv er. Animals injected with na nosilver were found to contain significantly more silver in the liver, kidney, spleen, brain, lung and blood than in animals injected with microsilver. Histopatholog ical observations found that nanos ilver-injected animals contained elemental silver spheres that were absent from the microsilver-treated animals. The elemental silver spheres were observed in different kinds of cells, such as renal tubul ar epithelial cells and hepatic cells. Moreover, these el emental silver spheres also in duced blood-brain barrier (BBB) destruction and astrocyte swelling, and caused neuronal degeneration. The serum kinetics, tissue distribu tion, and excretion of s ilver after sing le injections of 0.5 mg/kg and 5 mg/kg of citrate-coated na nosilver with average diameter of 7.9±0.95 nm was determined in SPF New Zealand White rabbits (n = 4) (Lee et al , 2012). There was no significant general toxicity reported in either the 0.5 or 5 mg/kg treatment gr oups. Accumulation of silver was observed in all the tested organs including liver, kidney, splee n, lung, brain, testes, and thymus, where the liver and spleen contained the greatest amount of silv er. As with the rat study above, the amount of silver in feces between 1 and 28 days after injec tion was significantly greater than in urine, which suggests biliary excretion of silver is the major route of eliminati on after injection of nanosilver. Blood kinetics, tissue distribution, and organ accumula tion of silver were determined after daily intravenous injections for 5 c onsecutive days of between 23.8 a nd 27.6 mg/L of nanosilver with average diameters of 20, 80, and 110 nm in six-w eek old male Wistar rats (n=21 in treatment groups; n= 2 in control) (Lankve ld et al., 2010). The concentrati on of silver in blood rapidly decreased during the initial 10 minut es following both single and re peated injection of nanosilver and then remained stable for up to one hour after injection. Silver was distributed to all organs evaluated, including the liver, lungs , spleen, brain, heart, kidneys and testes, regardless of the size of nanosilver injected. After injecting 20 nm diameter nanos ilver, silver was found to be distributed mainly to the liver, followed by the ki dneys and spleen, whereas after injection of the15 80 and 110 nm diameter nanosilver, silver was dist ributed mainly to the spleen, followed by the liver and lung. Thus, there was a size-dependent tissue distribution. Repe ated administration of nanosilver resulted in accumulation of silver in the liver, lung and spleen, indicating that these organs may be potential ta rget organs for toxicity after repeated exposure.

4.3.2 Oral Administration of Nanosilver The organ distribution and cellu lar localization of silver wa s determined following 28-day repeated oral administration at 9.0 mg/kg/day of PVP-stabilized na nosilver with average diameter of 14 ± 4 nm and silver acetate to four week ol d female Wistar Hannover Galas rats (n = 9 for nanosilver and n = 7 for silver acetate) (Loeschner et al., 2011). Although the distribution of silver in organs for animals treated with nanosilver and silver acetate was similar, the concentration of silver in the organs treated with silver aceta te was greater than it wa s for nanosilver-treated animals. This was in agreement with the higher fecal excretion of nanosilver as compared to silver acetate. Besides the intestinal syst em, the largest silver concentrations were detected in the liver and kidneys; however, silver was also found in th e lungs and brain. Remark ably, silve r-containing granules in the same size range as that of the ad ministered nanosilver were observed in the ileum and kidney tissues of rats exposed to nanosilver and silver acetate. Using transmission electron microscopy (TEM), sulfur and selenium containing gr anules were detected in the ileum of animals exposed to nanosilver and silver acetate, and were mainly located in the basal lamina of the ileal epithelium and in lysosome s of macrophages within the lamina propria. The results of the present study demonstrate that the organ di stribution and form of silver was similar when nanosilver or silver acetate were administered orally to rats.

The toxicokinetics and tissue distri bution of silver was determined following 28-day oral gavage at 90 mg/kg/day of nanosilver w ith average diameter of 17.7±3.3 nm by TEM, PVP-coated nanosilver with average diameter of 12.1±8.0 nm by TEM, and 9 mg/kg/day of silver nitrate to six-week-old male pathogen free Sprague-Dawley rats (n = 5 per group) (Van der Zande et al., 2012). Greater than 99% of the silver administered to the rats was ex creted in their feces indicating that only a small fraction (<1%) of silver from nanosilver and silver nitrate was absorbed. After normalizing for gavage dose, the concentration of s ilver in the blood of the silver nitrate treated animals was significantly higher than for the nano silver-treated animals at all timepoints during exposure. This clearly illustrates a much highe r uptake of silver wh en silver nitrate was administered as compared to nanosilver. One da y after the oral gavage, a significant reduction in blood silver concentration was obs erved. One week after the oral gavage, the concentration of silver in blood was reduced to nondetectable levels indicating rapi d clearance of silver from the blood for both nanosilver and silver nitrate treated anim als. After normalizing for gavage dose, silver was observed in all examined organs with the highest levels in th e liver and spleen where animals treated with silver nitrate had accumula ted significantly more silver than in animals treated with nanosilver. Silver was cleared from most organs eight weeks after the final gavage, but remarkably not from the brain and testes, wh ere between 94 and 100% of the silver was still present in the brain compared to the amount one da y after the last gavage of nanosilver and silver nitrate, respectively. Using si ngle-particle inductively coupl ed plasma mass spectrometry, nanosilver was detected one day after the fi nal gavage in the liver, spleen, lungs, and gastrointestinal contents of nanosilver gavaged rats. Nanosilver was also detected in the liver, spleen, lungs, and gastroin testinal contents one day after the final gavage from silver nitrate treated rats demonstrating the in vivo formation of nanosilver from silv er nitrate. Blood enzyme levels were not significantly differentfrom untreated animals indicating that there was no acute hepatotoxicity observed. Also, there was no indi cation that nanosilver caused nonspecific immune responses based on immunotoxic responses.16 4.4 Safety Factor for Infants and Children (FQPA Safety Factor) Under section 408(b)(2)(C) of the Federal Food, Drug and Cosmetic Act (FFDCA) (as amended in 1996 by the Food Quality Protection Act (FQPA), P.L. 104-170), EPA was direct ed to "ensure that there is a reasonable certainty that no harm w ill result to infants and children" from aggregate exposure to a pesticide chemical residue when establishing, modifying, l eaving in effect, or revoking a tolerance or tolerance ex emption. The law further states that for dietary exposures in the case of threshold effects, "an additional tenfold margin of safety for the pesticide chemical residue and other sources of exposur e shall be applied for infants and children to take into account potential pre- and post-natal toxi city and completeness of the data with respect to exposure and toxicity to infants and childre n. Notwithstanding such requirement for an additional margin of safety, the Administrator may use a different margin of safety for the pesticide residue only if, on the basis of reliable data, such margin will be safe for infants and children." There is no food use for NSPW-L30SS at this time, and therefore, no FQPA Safety Factor and no FFDCA aggregate exposure analysis addressing potential NSPW-L30SS residues is required.

4.5 Antimicrobial Resistance Silver is currently used as a broad-spectrum antibio tic in wound dressings. There is a concern that increasing use of silvers, includ ing nanosilver used for preservi ng plastics, may result in more bacteria developing resistance to silver and will limit its use as an antibiotic agent for wound care (Gupta and Silver, 1998). In the wound-care sett ing, a recent review by Chopra (2007) concluded that the threat of bacterial resi stance to silver in the clinical setting is low. However, Chopra (2007) cautioned against use of wound dressings that release sublethal levels of silver over a long period of time allowing bacter ia to develop resistance. In terms of the environment, a recent study i nvolved releasing 1 mg/L of nanosilver into microcosms containing estuary wa ter overlying estuarine sediment cores (Bradford et al., 2009). The study found no impact to the microbial commun ity over a 30-day monitoring period. Evidence for antibacterial resistance was also evaluate d during this study and no increase in antibiotic resistance to the bacterial popul ation in the sediment was found (Mühling et al. 2010). Wigginton et al. (2010) suggested that the l ack of antimicrobial effect in the microcosm was expected given that bacterial proteins effi ciently bind to nanosilver. EPA concludes that, while development of antibacte rial resistance due to th e use of nanosilver in NSPW-L30SS is possible, the likelihood is low that the levels used in products incorporating NSPW-L30SS will lead to the develo pment of silver-resistant microbes that will spread throughout the environment and result in widespread bacterial resistance to silver. 4.6 Point of Departure Selections and Target Margin of Exposure The currently available oral toxicity studies indicate that nanosilver causes liver and kidney toxicity in laboratory animals in which silver is distributed to all orga ns and tissues. Inhalation toxicity studies also identifie d liver toxicity as well as l ung effects including alveolar inflammation. There were potential neurotoxic effects identified with increases in neurotransmitter concentrations and decrease in spatial cognitiv e ability; however, these same effects were not observed in a follow-on study. Together, these studies indicate to EPA that, if sufficient quantitie s of nanosilver break away from NSPW-L30SS, and if such nanosilver displays toxici ty similar to the nanosilver used in the oral and inhalation route-specific studies, then route-specific exposure to NSPW-L30SS-derived nanosilver may result in a dverse health effects.17 4.6.1 Point of Departures for NSPW-L30SS The toxicological point of departure (POD) is the lowe r confidence bound on the lowest experimental dose from a dose-response study that showed an effect. This dose is determined from dose-response data and marks the beginning of extrapolation to dete rmine the risk associated with environmentally relevant human exposures. Comm only, this is a NOAEL from a laboratory animal toxicity study, which represents the dose at wh ich no adverse effects were observed in laboratory animals. Oral or dermal subchronic and chroni c toxicology studies are not available for NSPWL30SS or the nanosilver that might break away from articles incorporating NSPW-L30SS. In place of these studies, EPA is determining NOAELs a nd LOAELs from subchronic inhalation and oral toxicity studies found in th e scientific literature fo r nanosilvers to evaluate the effects that could occur from exposure to the nanos ilver present in NSPW-L30SS. As stated in Section 2.2, th e FIFRA SAP was unsupportive of bridging among silver-based materials with different propertie s. However, the SAP indicated th at bridging would be appropriate for materials of similar size and essentially identical physical prope rties and that bridging between silver ions released from nanosilv er and the existing database for s ilver ions is feasible (FIFRA SAP, 2009). The SAP cautioned about extrapolating from one nanosilver fo rmulation to another when assessing hazards because differences in particle formulation (e.g., coating and inert ingredients) are likely to affect biological act ivity, among other things. The oral toxicity study by Ha drup et al. (2012a) used PVP-co ated nanosilver with average diameter of 14 nm, which is similar to the diam eter and surface coating of the nanosilver in NSPW-L30SS. However, EPA cannot determine a NOAEL or LOAEL from this study because histopathological patterns indicativ e of distinct adverse effects at the highest nanosilver dose of 9 mg/kg/day were not identified. A LOAEL was not iden tified. The oral toxicity study by Park et al. (2010) used uncoated nanosilver with an average di ameter of 42 nm, which is different from the diameter and surface coating of the nanosilver in NSPW-L30SS. In 2011, EPA used the NOAEL reported in the Park et al. (2010) study as the POD for evaluating th e oral and dermal toxicity of the nanosilver in HeiQ AGS -20 (U.S. EPA, 2011a). EPA reviewed this study again as part of this assessment and now concludes that although the re sults of the Park et al. (2010) study showed evidence of changes in clinical chemistry, it lacks histological suppor t for the effects used as the basis for the study NOAEL/LOAELs. In the absence of histopathological findings, the clinical chemistry changes observed alone are insuffi cient evidence of an adverse effect. The oral toxicity studies by Kim et al. (2008) and Kim et al. (2 010) used CMC-coated nanosilver with average diameter of 56 and 60 nm, respectiv ely, which is different from the diameter and surface coating of the nanosilver in NSPW-L30SS. These studies were stated as being completed according to OECD guidelines and identified histopa thological patterns in the liver that were indicative of distinct adverse effects. EPA has determined that the NOAEL is 30 mg/kg/day from the 28-day Kim et al. (2008) study based on sign ificant increases in ALP and cholesterol, significant changes in hematology, and accompanie d by histopathological evidences of liver toxicity (bile-duct hyp erplasia around central vei n, infiltration of inflammatory cells, and dilation of the central vein) at the LOAEL dose of 300 mg/kg/day. A NOAEL for the 90-day Kim et al. (2010) cannot be established because of adverse hist ological patterns evident at the lowest dose of 30 mg/kg/day. EPA has determined that the LOA EL from the 90-day Kim et al. (2010) study is 30 mg/kg/day based on histopathological evidences of liver toxicity (b ile-duct hyperplasia with focal, multifocal, or lobular necrosis) in both males and females. The inhalation toxicity studies by Sung et al. (2009) and Song et al. (2012) used uncoated nanosilver with average diameter s of 18 to 19 nm and 14 to 15 nm , respectively, which is similar18 to the diameter but different than the surface co ating of the nanosilver in NSPW-L30SS. EPA has determined that the NOAEL is 133 μg/m 3 from the 90-day Sung et al . (2009) study based on the minimal bile-duct hyperplasia, peri vascular mixed cell infiltrate a nd chronic alveolar inflammation and macrophage accumulation in the lungs in both male and female rats, and on decreases in physiological measures of lung function (respir atory minute volume, tidal volume, and peak inspiratory flow) in the male rats. EPA has determined that the NOAEL is 49 μg/m 3 from the 12- week Song et al. (2012) study based on adverse histopathological patterns in lung tissue observed at the 117 μg/m 3 dose. In 2011, EPA used the NOAEL reporte d in the Sung et al. (2009) study as the POD for evaluating the inhalation toxicity of the nanosilver in He iQ AGS-20 (U.S. EPA, 2011a). EPA compared the Sung et al. (2009) study to the Song et al. (2012) study and now concludes that the newly availa ble Song et al. (2012) study is mo re appropriate for evaluating inhalation exposure to nanosilver, as this study in cluded a recovery period in addition to an exposure period. This recovery period indicated that some effects pe rsisted in male rats past the end of the exposure period, something not studied in Sung et al. (2009). Based on the above analysis, EPA has determined that the NOAEL of 30 mg/kg/day from 28-day oral toxicity study by Kim et al. (2008) is the POD for short-term oral exposures (< 30 days) to the nanosilver in NSPW-L30SS (Table 3). EPA has al so determined that the LOAEL of 30 mg/kg from the 90-day oral toxicity study by Kim et al . (2010) is the POD for intermediate-term oral exposures (1 to 6 months) to the nanosilver in NSPW-L30SS. EPA has determined that the NOAEL of 49 μg/m3 derived from the Song et al . (2012) study is the POD for short- and intermediate-term inhalation expos ures to the nanosilver in NSPW-L30SS. These studies were conducted using OECD guidelines, th erefore they are the most robust and the effects are consistent across studies. The NOAELs/LOAELs from these stud ies are therefore protec tive of the types of effects seen in other studies. Table 3 – Toxicity Endpoints for Use in the Risk Assessment of NSPW-L30SS Nanosilver

Exposure Scenario Point of Departure (POD) Uncertainty Factor Target MOE for Risk Assessment Study

Incidental Oral

(short-term) NOAEL = 30 mg/kg/day UF A = 10 UF H = 10 UF D = 10 1000 Kim et al. (2008)

Incidental Oral (Intermediate-term) LOAEL = 30 mg/kg/day UF A = 10 UF H = 10 UF D = 10 UF L = 3 3000 Kim et al. (2010)

Dermal (short -term) NOAEL = 30 mg/kg/day DA = 6.7% UF A = 10 UF H = 10 UF D = 10 1000 Kim et al. (2008)

Dermal (Intermediate-term) LOAEL = 30 mg/kg/day DA = 6.7% UF A = 10 UF H = 10 UF D = 10 UF L = 3 3000 Kim et al. (2010)

Inhalation (short- and

intermediate- term) NOAEL = 49 μg/m 3 UF A = 10 UF H = 10 UF D = 10 1000 Song et al. (2012)

Cancer (oral, dermal, inhalation) At this time there is inadequate information to assess carcinogenic potential of nanosilver due to differences in results between the in vitro studies and in vivo study, and the limitations of the only available in vivo study.19 There are no acceptable dermal toxicity studies on nanosilver available to EPA. In the absence of any such dermal toxicity studies , EPA normally uses extrapolation from another route of exposure (usually oral) and assumes a default de rmal absorption (DA) of 100% when no in vivo data are available (see Section 4. 2.3). The available human in vivo study indicating absorption of nanosilver is 6.7% and the in vitro data indicating absorption of na nosilver from intact and abraded human skin is substantially below 0.1% provide sc ientific support for set ting a conservative DA of 6.7% for the nanosilver that might break away from the NSPW-L30SS. EPA has determined that the dermal toxicity for the nanosilver in NSPW-L30SS will be evaluated using the oral POD of 30 mg/kg/day and a DA of 6.7%. In 2011, EPA used a DA of 0.1% that was based on a human clinical study by Moiemen et al., (2011) for evaluating the dermal toxicity of the nanosilver in HeiQ AGS-20 (U.S. EPA, 2011a). EPA reviewed this study again as part of this assessment and now concludes that the 0.1% DAF was an underest imate because it was based on the Moiemen et al. (2011) study which only reported silver concentrations in the blood of patients. The 6.7% DA is based on the observational study by Wan et al. (1991) which repor ted silver concentration in serum as well as that eliminated through urine. The estimated DA of 6.7% is based on applicati on of 500 mg of silver to damaged skin while significantly less silver and nanosilv er are used to preserve materials. For example, the maximum dermal exposure to nanosilver from HeiQ AGS- 20 treated textiles was 0.17 mg/day (EPA, 2011) and was 0.0165 mg/day for NSPW-L30SS-treate d textile (EPA, 2013). Therefore, the concentration of silver in the burn wound studies were from approximately 3,000 to 30,000 times greater than those for materials treated with nanosilver. There are currently no acceptabl e studies on the reproductive a nd developmental toxicity of nanosilver. However, there were dose-dependent incr eases in the concentration of silver in the testes of rats after oral inge stion, inhalation and injection of nanosilver and, in another study, nanosilver was distributed to major maternal organs and extra-embryonic tissues although no adverse morphological effects on the developing em bryos were observed. Therefore, EPA believes that the short-term incidental oral NOAEL and incidental-oral LOAEL of 30 mg/kg/day and inhalation NOAEL of 49 μg/m 3 are expected to be protective of developmental and reproductive effects of nanosilver. The study completed by Hadrup et al. (2012b) , which reported significant increases in neurotransmitter concentrations (e.g., dopamine) after oral administ ration of nanosilver to rats at concentrations of up to 9 mg/kg/day, lacks hi stological support for de termining NOAEL/LOAELs. The study by Liu et al. (2013) reported no effects on spatial cognition or hippocampal neurogenesis of mice after injecting nanosilver into the body cavity (i.e., intr aperitoneal injection) at concentrations of up to 50 mg /kg. Therefore, EPA believes that the short-term incidental oral NOAEL and intermediate-term LOAEL of 30 mg/ kg/day are expected to be protective of neurotoxic effects of nanosilver. The effects on spatial cognition and hippocampal syna ptic plasticity observ ed by Liu et al. (2012) after administering nasal drops c ontaining nanosilver at concentrati ons of 3 and 30 mg/kg to rats suggest possible neurotoxic effect s from the inhalation of nanosilv er. However, EPA believes that the doses used in the Liu et al. (2012) study we re greater than the maximum dose used in the inhalation toxicity study by Song et al. (2012). Therefore, EPA believes that the inhalation NOAEL of 49 μg/m 3 based on the Song et al. (2012) study is expected to be protective of neurotoxic effects of nanosilver.20 4.6.2 Target Margin of Exposure The target margin of exposure (MOE) is based on uncertainty factors. There are two standard uncertainty factors that account for potential inte rspecies extrapolation an d intraspecies variation. The first is a 10 fold uncertainty factor (UF A ) assigned to account for extrapolation of laboratory animal data to humans (interspecies). The second is a 10 fold uncertainty factor (UF H ) assigned to account for variations in suscepti bility within the human population (intraspecies). In addition to the two standard uncertainty f actors given above, there is a third uncertainty factor (UF D ) which accounts for the incomplete characterization of nanos ilver toxicity (databas e). In this case, EPA has determined that the inhalation and oral toxicity data for nanosilv er are sufficient to assess the potential health effects caused by nanosilver rele ased from NSPW-L30SS (Table 3). However, as the SAP concluded, there is uncertainty on how part icle parameters such as size, surface charge and coating affect the toxicity pot ential of nanosilver. In addition, the database is incomplete with respect to reproductive and developmental effects. Therefore, EPA is using the maximum 10 fold database uncertainty factor to cover the missi ng information on reproductive and developmental effects and the potential for differe nces between the nanosilver in th e available toxicity data (U.S. EPA, 2002). Thus, the target MOE is: Target MOE: 10 (UF A ) × 10 (UF H ) × 10 (UF D ) = 1,000 The target MOE of 1,000 is for ev aluating short-term (< 30 days) and intermediate-term (1 to 6 months) continuous daily inhalation exposures to NSPW-L30SS because the inhalation POD was based on a 90-day study by Song et al. (2012). Th e target MOE of 1,000 is for evaluating the short-term exposure (< 30 days) continuous daily oral exposures to NSPW-L30SS because the oral POD was based on a 28-day oral t oxicity study by Kim et al., 2008.

The oral POD that was based on the LOAEL from the 90-day oral toxici ty study by Kim et al. (2010) has an additional 3 fo ld uncertainty factor (UF L ) to account for the lack of NOAEL. The target MOE for evaluating intermediate-term e xposure (1 to 6 months) continuous daily oral exposures to NSPW-L30SS is: Target MOE (intermediate-term oral): 10 (UF A ) × 10 (UF H ) × 10 (UF D ) × 3(UF L ) = 3,000 The maximum recommended long-term target MO E is 3,000 (U.S. EPA, 2002), which has already been established for intermediate-term exposures. 4.6.3 Margin of Exposure The margin of exposure (MOE) is used to determin e if exposure to a chemical can be expected to cause an adverse effect. The MOE is calculated by dividing the POD by the estimated daily dose to which humans will be exposed as expressed by the following: MOE = POD / Daily Dose After calculating a MOE from the POD and daily dos e, EPA evaluates the risk from exposure to a pesticide by comparing the calculated MOE to a target MOE (U.S. EPA, 2002). If a calculated MOE is equal to or greater than a target MOE, EP A may conclude that expos ure to the pesticide is unlikely to pose a risk concern and, therefore, wi ll not cause unreasonable adverse effects for that specific exposure scenario. If a calculated MOE is less than a target MOE, then EPA may have a risk concern. However, the MOE analysis is not the only factor EPA uses when determining if there is a risk concern from expos ure to a pesticide. EPA also c onsiders other scientific evidence in a weight of evidence evaluation such as the se verity of toxic effects, the controls used to minimize exposures, and the population exposed to the pesticide. A risk concern is not the21 equivalent of a determination th at the potential risk constitute s an unreasonable adverse effect. Rather, where EPA finds a risk concern, EPA will generally: (1) require protective measures or use restrictions to mitigate the risk; (2) further re fine its risk assessment analysis, particularly if conservative assumptions were used to produce the initial assessment; or (3 ) explicitly analyze any potential benefits of the pestic ide to determine whether, on bala nce, those benefits outweigh the identified risk. EPA’s MOE approach for nanosilver uses mass-ba sed metrics, both for determining the POD and for calculating exposure. EPA is aw are of the ongoing debate within the scientific community that metrics other than mass (such as particle num ber or surface area) may be more suitable for assessing nanosilver risks and th erefore acknowledges the potentia l for limitations of mass-based risk estimates. V. Occupational and Consumer Risk Assessment Based on the use pattern of NSPW-L30SS in plas tics and textiles, EPA anticipates humans could be exposed to the following substances: 1) Silver ions released from NSPW-L30SS; 2) NSPW-L30SS complex; and/or 3) Nanosilver that might break away from NSPW-L30SS. Because the levels of silver in plastics and te xtiles incorporating NSPW-L 30SS are lower than for other currently registered products which use si lver ions and because EPA determined that unreasonable adverse effects from use of silver-c ontaining products are unlikely (U.S. EPA, 1993), EPA concluded that the risk from exposure to silver ions derived from plastic and textile products incorporating NSPW-L30SS is not of concern. Na nosilva LLC has submitted a number of studies and other information to support it s application for regist ration, including information relevant to assessing the toxicity of and exposure to NSPW-L 30SS. In addition, EPA has also reviewed data and information on nanosilver from the scientif ic literature. This section discusses EPA’s assessment of the potential risks to human health from exposure to nanosilver from the use of NSPW-L30SS. 5.1 Occupational Risk Asse ssment for Mixing and Loading the NSPW-L30SS Liquid Suspension EPA expects that occupational inhalation and dermal exposures to NSPW-L30SS, including nanoparticles that might break aw ay from NSPW-L30SS, are likel y to occur during the following use scenarios :
1. Mixing and loading of NSPW-L30SS duri ng preparation of a master batch 2. Mixing, loading, and applying the NSPW-L30SS containing master ba tch during treatment of plastics and textiles 3. Handling plastics and textiles incorporating NSPW-L30SS EPA evaluates the risk of occupational exposures from mixing, loading, and applying or handling pesticide products. Although exposure to NSPW -L30SS during subsequent work activities involving plastics and textiles incorporating NSPW-L30SS can o ccur in occupational settings (items 2 and 3), the risk to workers for these s cenarios is unlikely to ex ceed the risk during mixing and loading NSPW-L30SS during preparation of th e master batch (item 1). Therefore, only the mixing and loading of NSPW-L30SS during preparat ion of a master batch is evaluated in the subsequent sections.22 5.1.1 Occupational Exposure Preparing th e Master Batch Containing NSPW-L30SS A master batch consists of polymer pellets (i .e., plastic pellets) c ontaining NSPW-L30SS which are subsequently used in treating plastics (MRID 48652901). A master batch is prepared by blending a polymer and the NSPW-L30SS liquid su spension using a mixer and compounding this mixture using an extruder. Workers may be expo sed to NSPW-L30SS and/or nanosilver particles that break away from NSPW-L30SS while load ing the NSPW-L30SS liquid suspension into the master batch mixer. Nanosilva LLC did not submit data or information quantifying the amount of NSPW-L30SS that workers are exposed to during lo ading of the NSPW-L30SS liquid su spension into a master batch mixer. To calculate worker exposure, EPA is using the standard occupational handler unit exposure values for mixing and loading of liqui ds, as shown in Table 4, which are from the Antimicrobial Exposure Assessment Task For ce II (AEATF II) liquid pour human exposure monitoring study (U.S. EPA, 2012a) and the Pestic ide Handlers Exposure Database (PHED) (U.S. EPA, 1998). Table 4 – Unit Exposures for Mixing and Loading the NSPW-L30SS Liquid Suspension

Mitigation Level A Exposure Route Unit Exposure Data Source None (No respirator or Engineering Control) Inhalation 0.21 μg/m 3 /lb AEATF II Engineering Control – Closed System Loading Inhalation 0.016 μg/m 3 /lb PHED None (No gloves or engineering control) Dermal 10 mg/lb B AEATF II Gloves Dermal 1.2 mg/lb C AEATF II Engineering Control – Closed System Loading Dermal 0.0086 mg/lb PHED
A. It is assumed that all workers will wear long pants, lo ng sleeve shirts, shoes and socks at all mitigation levels. B. Arithmetic mean for conventional liquid pour scenario C. Estimated from the no gloves unit exposure using a glove protection factor of 10. The inhalation unit exposure for mi xing and loading of liquids usi ng engineering controls (closed system loading) as listed in PHED is 0.083 mg /lb. This value was calculated based on the air concentrations measured in workers’ breathing zo ne during the handling of a pesticide, the time that elapsed during the sampling, an assumed br eathing rate of 29 liters per minute, and the amount of the pesticide that was handled dur ing the sampling period. When the PHED unit exposures were initially calculate d, they were expressed in units of mg/lb for comparisons to PODs that were expressed in units of mg/kg/ day. Because the POD for inhalation exposure to nanosilver is expressed as a daily- average air concentration of μg/m 3 , the PHED unit exposure was converted to a daily-average air concentration with units of μg/m 3 per lb of pesticide handled per day. This was done by dividing the air concentrat ions listed in the PH ED by the amount of pesticide handled while accounting for the time el apsed during sampling and an eight hour work day. This yielded an inhalation unit exposure of 0.016 μg/m 3 per lb of pesticid e and it represents an eight hour time weighted average (Table 4). Th is unit exposure is the arithmetic mean of 27 PHED monitoring events (formerl y called replicates) that repres ent inhalation exposure during the mixing and loading of pesticide containing liquids while using closed-system loading. 5.1.2 Occupational Margins of Exposure As detailed in the following sections, EPA eval uated the risk to workers who load the NSPWL30SS liquid suspension into a master batch mixer.23 Occupational Inhalation Exposure The inhalation exposure to the na nosilver in the NSPW-L30SS li quid suspension was calculated using the following: Inhalation Daily Exposure = Amount of nanosilver in NSPW-L30SS handled per day × Inhalation Unit Exposure

Where:

 Amount of nanosilver in NSPW-L30SS handled per day is 1.2 lb/day. This value was calculated assuming that the NSPW-L30SS liquid suspension was mixed into a low density polyethylene plastic using an extruder with the maximum screw diameter of 8 inches (Chanda and Roy, 2007) which yields a plas tic production rate of 5,000 lb/hour (Flite Technology, 2012). Assuming an 8 hour work day yields a plastics pr oduction volume of 40,000 lb per day at each treatment facility. Th e maximum amount of nanosilver applied to plastics or textiles is 0.003% by weight. Thus , the amount of nanosilver that is handled per day is 40,000 lb/day × 0. 003% ÷ 100% = 1.2 lb/day.

 The unit exposure for inhalation is 0.21 μg/m 3 /lb when no respirator is worn and 0.016 μg/m 3 /lb when closed-system loading is used (see Table 4). The MOEs shown in Table 5 were calculated by dividing the inhalation ex posure for nanosilver by the POD of 49 μg/m 3 , which is the NOAEL from a 90-day in halation toxicity study for nanosilver (Song et al., 2012). This assumes that all the nanosilver in NSPW-L 30SS is freely available after inhalation and behaves like the nanosilver used in the Song et al. (2012) study even though the nanosilver in NSPW-L30SS is attached to a silica core particle through a thiolate bond. When no respirator is worn the MOE is 200, and is less th an the target MOE of 1,000 which means that the risk is of concern. The MOE is 2,500 when closed -system loading is used. This MOE is greater than the target MOE of 1,000 which means the risks are not of con cern. These MOEs indicate that the risk concern for inhalation exposures to th e nanosilver in NSPW-L30SS is mitigated when workers use closed-system loading during mi xing and loading of the NSPW-L30SS liquid suspension.

Table 5 – Occupational Handlers Inhalation MOEs for the Nanosilver in NSPW-L30SS

Mix/Load NSPWL30SS Liquid

Suspension (i.e. Liquid Pour) Amount of Plastics Treated per day Application Rate A Amount of Nanosilver

Handled per day Inhalation Unit Exposure (μg/m 3 /lb) Exposure B (μg/m 3 ) MOE C,D

Without Respirator 40,000 lb 0.003% By Weight 1.2 lb 0.21 0.25 200 Closed System Loading 0.016 0.02 2,500
A. Maximum amount of nanosilver in plastics incorporating NSPW-L30SS B. Daily Exposure = Amount nanosilver Handled × Unit Exposure C. MOE = POD / Daily Exposure D. Target MOE is 1000 Occupational Dermal Exposure The dermal dose from exposure to the nanosil ver in the NSPW-L30SS liquid suspension was calculated using the following:24 Dermal Daily Dose = (Amount of nanosilver in NSPW-L30SS handled per day × Dermal Unit Exposure × Dermal Absorption) / Body Weight

Where:

 Amount of nanosilver in NSPW-L30SS handled per day is 1.2 lb/day (Table 5).

 The dermal unit exposures are 10 mg/lb when no gloves are worn, 1.2 mg/lb when gloves are worn, and 0.0086 mg/lb when closed-s ystem loading is used (Table 4).

 The dermal absorption (DA) for nanosilver is 6.7 percent (see Section 4.2.3).

 The body weight of an adult is 80 kg (U.S. EPA, 2011b).

The MOEs shown in Table 6 were calculated by dividing the dermal dose by the POD of 30 mg/kg/day, which is the NOAEL from a 28-day or al toxicity study (Kim et al., 2008) and the LOAEL from a 90-day oral toxicity study (Kim et al., 2010). This assumes that all the nanosilver in NSPW-L30SS is freely available after dermal c ontact and behaves like the nanosilver used in the Kim et al. (2008 and 2010) studies even though the nanosilver in NSPW-L30SS is attached to a silica core particle through a thiolate bond. The MOEs range from 3,000 when no gloves are worn to 3,500,000 when closed-system loading is used. These MOEs are well above the target MOEs of 1,000 and 3,000 for short and intermedia te term exposures, respectively, which means that the risks are not of concern. Table 6 – Occupational Handlers Dermal MOEs for the Nanosilver in NSPW-L30SS

Mix/Load NSPW-L30SS Liquid Suspension (i.e. Liquid Pour) Amount of Nanosilver Added or Handled A Dermal Unit Exposure

(mg/lb) Exposure B (mg/day) Dose C (mg/kg/day) MOE D, E

Without Gloves

1.2 lb/day 10 12 0.010 3,000

With Gloves 1.2 1.4 0.0012 25,000

Closed System Loading 0.0086 0.01 0.0000086 3,500,000 A. Based on the same assumptions as those used for the inhalation MOE (see Table 5) B. Dermal Exposure = Amount of nanosilver Handled × Dermal Unit Exposure C. Dermal Dose = (Dermal Exposure × Dermal Absorption)/ Body Weight D. Dermal MOE = POD / Daily Dose E. Target MOE is 1000 for short-term exposures and 3000 for intermediate term exposures. 5.1.3 Conclusions for Occupational Risk The MOE for occupational inhalation exposure to the nanosilver in the NSPW-L30SS liquid suspension is 2,500 if workers use close-system load ing. This MOE is greater than the target MOE of 1,000 which means that the risk is not of concern. The MOE for dermal exposure to the nanosilver in the NSPW-L30SS liquid suspensi on is 3,500,000 if workers use closed-system loading. This MOE is well above the target MO Es of 1000 and 3,000 for short and intermediate term exposures, respectively, which means that the risk is not of concern. EPA is requiring that closed-system loading as defined in 40 CFR pa rt 170.240(d)(4) be used when mixing and loading the NSPW-L30SS liquid suspension to mitigate risk s with inhalation exposure. The closed-system loading requirement is based on unit exposure da ta from PHED. The MOEs for handling NSPWL30SS without closed system loading were calcu lated using unit exposure data from AEATF II. EPA does not typically consider lo ng-term occupational exposures to materials preservatives used to treat plastics or textiles b ecause application of these chemi cals does not typically occur on a daily basis for more than 6 months.25 There are several uncertainties in the occupationa l risk assessment. The exposure analysis assumes that all the nanosilver in NSPW -L30SS is freely available even though the nanosilver in NSPWL30SS is attached to a silica co re particle thr ough a thiolate bond. The PODs used were based on inhalation and oral toxicity studies completed using nanosil ver, not NSPW-L30SS or the nanosilver that might break away from NSPW-L30 SS, and thus these PODs may not equal PODs determined from testing conducted with NSPW -L30SS or the nanosilver found in NSPW-L30SS. Because the nanosilver in the toxicity database is different from the nanosilver in NSPW-L30SS and because the database is incomplete with re spect to reproductive and developmental effects, EPA used a maximum 10-fold database uncertain ty factor when evaluating the risk from occupational exposure to NSPW-L30SS. There are al so uncertainties in ex trapolating from effects observed after feeding test-animals nanosilver (i.e ., oral route) to the effects that might be observed after applying nanosilver to the skin of test animals (i.e., dermal route). 5.1.4 Occupational Exposure and Health Data Requirements EPA is not requiring that Nanosilva LLC conduct an indoor applicator stu dy to quantify the unit exposure values during mixing and loading of the NSPW-L30SS liquid suspension into the master batch mixer. This is because closed-system loadi ng, such as lock and load containers, where the NSPW-L30SS liquid suspension is transferred to a plastic extruder th rough piping minimizes occupational exposure to NSPW-L30SS to the greatest extent possible. Although there is no risk concern for workers w ho use closed system loading when mixing and loading the NSPW-L30SS liquid suspension, EPA is requiring an inhalation route-specific subchronic test (OCSPP GLN 870.3465) to account for the lack of inhalation data on the specific form of nanosilver in assessing the risks to workers who mix and load NSPW-L30SS. Also, because workers of child bearing age could handle NSPW-L30SS during mixing and loading, EPA is requiring a modified reproduction/developmenta l toxicity screening te st since data on the specific form of nanosilver are missing from the toxi city database. The current points of departure and additional 10x database uncerta inty factor is sufficient and h ealth protective until these data are submitted to the Agency. In summary, the following guideline studies are required for NSPW-L30SS:

 90-Day Inhalation Toxicity (Rat) (OCSPP GL N 870.3465) modified to include a functional observational battery, motor activ ity and detailed neuropathology

 Reproduction/Developmental Toxicity Screeni ng Test via the inhala tion route (Modified OCSPP GLN 870.3550/ OECD TG 421)

5.2 Consumer Risk Assessment for Pr oducts Incorporating NSPW-L30SS EPA expects consumer exposures to NSPW-L30SS, the silver ions releas ed from NSPW-L30SS, and the nanoparticles that br eak away from NSPW-L30SS coul d potentially occur during the following use scenarios:

1. Incidental oral exposure to plastics and textiles incorpor ating NSPW-L30SS 2. Dermal exposure to plastics and te xtiles incorporating NSPW-L30SS NSPW-L30SS is registered to be mixed into po lymer and polymer based products to suppress the growth of bacterial, algae, fungus, mold and m ildew, which cause odors, discoloration, stains, and deterioration of plastics and textiles. Because plastics inco rporating NSPW-L30SS could be subsequently used to manufacture children’s toys a nd textiles worn by children, it is assumed that children will be exposed to pr oducts containing NSPW-L30SS.26 While children younger than 6 months may potentia lly be exposed to plastics and textiles incorporating NSPW-L30SS, it is believed that exposure for children older than 6 months will be equivalent, if not greater, due to behavioral and anatomical/physio logical development; therefore, EPA assessed exposure to children older than 6 months. EPA recently revised its standard operating procedures (SOPs) for residential pe sticide exposure assessments (U.S. EPA, 2012b). Based on the combined quantitative and qualitative an alysis of the life stag es, EPA has determined that the 1 to < 2 year-old life st age represents the most appropriat e “index life stage” for children for most exposure scenarios. This analysis of lifestages is incl uded in Appendix A of the SOPs (U.S. EPA, 2012b) and it focused on four specifi c child lifestages as defined the Guidance on Selecting Age Groups for Monitoring and Asse ssing Childhood Exposures for Environmental Contaminants (U.S. EPA, 2005). These lifestages included children 6 < 12 m onths old, children 1< 2 years old, children 2 < 3 years old and children 3 < 6 years ol d. The quantitative life stage analysis, which included such as factors as body weight, exposure time and frequency of object and hand to mouth activities, indi cated that 6 to < 12 month-old children and 1 to < 2 year-old children have the greatest potentia l for exposure. When qualitative factors are considered in addition to the quantitative factors; however, the 1 to < 2 year-old children are considered to be the most appropriate. In 2011, EPA evaluated the exposure to textiles containing the nanosilver based pesticide HeiQ AGS-20 using children in the 2 to 3 year-old life stage based on EPA’s determ ination, at that time, that the 2 to 3 year-old child was likely the mo st exposed population from chewing on and wearing textiles treated with HeiQ AGS-20 (U.S. EPA, 2 011a). After issuing the registration decision for HeiQ AGS-20, questions were raised about the use of the 2 to 3 year-old lif e stage. In light of those questions and in light of th e newly revised SOPs, in this case, EPA is evaluating exposure to children in the 6 to < 12 month, the 1 to < 2 year, and 2 to <3 year-o ld age range from plastic toys and flooring and textiles incorporat ing NSPW-L30SS for transparency. 5.2.1 Consumer Exposures to Plastics and Textiles Incorporating NSPW-L30SS Leaching studies are required to determine the am ount and form of silver that consumers will be exposed to when in contact with plastics and te xtiles incorporating N SPW-L30SS. These studies typically involve immersing produc ts incorporating nanosilver inbiological fluids such as simulated saliva solutions for ex tended periods of time at physio logical temperatures (i.e., 98.6 degrees Fahrenheit or 37 degrees Celsius) and meas uring the amount and form of silver released to those fluids. Such “leaching studies” were submitted by Nanosilva LLC using food contact solutions for plastic coupons incorporating NSPW-L30SS and using la undry detergent and simulated saliva for shirts incorporating NSPW-L30SS. Plastic Coupon Leaching Study (MRID 478289-25) Nanosilva LLC submitted a plastics leaching study on the migration of silver in NSPW-L30SS from a linear low-density polyethylene (LLDPE ) plastic (MRID 478289-25). The plastics leaching study was based on Food and Drug Administration methodology to determine the migration of chemicals from plastics which contact food. The plastics leaching study involved 44 LLDPE coupons with 14 being ashed to determine silver c ontent and 28 used in the leaching tests. The 28 plastic coupons were prepared with four NSPW -L30SS master batch concentrations where seven coupons each contained 0, 2.5, 5, and 10%, respectiv ely. These master batch concentrations corresponded to nanosilver concentrations of 0, 5, 10, and 20 mg/kg (supplementary information provided on 17 December 2010). The coupons each had a surface area of approximately 112 cm 2

, a volume of approximately 16.3 mL and an a pproximate mass of 15g. Each coupon was exposed27 to 50 mL of the food simulation fluids, which included aqueous solutions of low and high pH fluids, 10% alcohol, 10% sodium phosphate monbasic, 10% oliv e oil and 10% sodium phosphate and sucrose at temperatures of 40 and 100 degr ees Celsius for periods of up to 240 hours (10 days). The amount of nanosilver present in the coupons was 0, 75, 150, and 300 μg, respectively. Given that coupons were in contact with 50 mL of fluid, the maximum silver concentrations that could be obtained from each coupon wa s 0, 1500, 3000, and 6000 μg/L, respectively. Since EPA is only evaluating ri sk from non-dietary human cont act with non-food use plastics incorporating NSPW-L30SS, results for low and hi gh pH fluids, which are more representative of biological fluids, are included in th is assessment (other fluids woul d be used if dietary exposures from food use plastics were anticipated). Given that EPA is ev aluating human exposure to plastics incorporating NSPW-L30SS which occur at phys iological temperatures (i.e., 98.6 degrees Fahrenheit or 37 degrees Celsius), the leaching results for the trials conducted at 100 degrees Celsius are not addressed here. The 100 degrees Celsius conditions are more representative of microwaving plastics than of contact with humans. The concentration of silver in the low and high pH fluids contacting plas tic incorporating NSPWL30SS was below the analytical LOD. The analys is method, inductively coupled plasma atomic emission spectroscopy (ICP-AES), employed an acid di gestion and thus the s ilver concentrations are for total-silver content. The plastics leach ing study reported method detection limits that ranged from 0.8 to 2 μg/L. However, because th ere were no validation data provided to support these LODs, EPA is using the silver LOD of 5 μg /L, which is the value reported as the estimated LOD in U.S. EPA Method 6010C (U.S. EPA, 2007a) and as the reporting limit in the plastics leaching study (see Table 1, Page 30 of the study report).

Table 7 –Silver Concentrations in Fluid Co ntacting NSPW-L30SS Treated Plastic Coupons

Fluid (40 degrees C) Maximum Silver Concentration Detected ( μg/L) Silver Matrix Spike Recovery (%) Low pH, pH 2.0 using 10% nitric acid in
water 15 of 15 samples < 5 μg/L 93 to 110
High pH, pH 8.0 using 0.1N sodium hydroxide in water 15 of 15 samples < 5 μg/L 100

Because the concentration of silv er in the low and high pH fluids were all below the analytical LOD of 5 μg/L, the EPA used 2.5 μg/L as the con centration of silver released from plastics incorporating NSPW-L30SS. This was based on the assumption that all results reported as being below the LOD had a value of one-half the LOD (i .e., 2.5 μg/L), which is consistent with how EPA assigns values to non-detect results when evaluating pesticide re sidues in food (US EPA, 2000). Since this value is below the LOD, the fo rm of any potentially re leased silver is unknown and was assumed to be in the form of nanosilv er as found in NSPW-L30SS. Using the assumed concentration of 2.5 μg/L e quates to nanosilver releases of 0.17, 0.08, and 0.042% from the coupons containing 5, 10, and 20 mg/kg of nanosilver , respectively. Plastic products incorporating NSPW-L30SS are to contain a maximum of 0.0 03% nanosilver by weight or 30 mg/kg of nanosilver. Assuming that a leaching study condu cted using a coupon containing 30 mg/kg of nanosilver would also result in na nosilver releases below the L OD would result in a nanosilver release rate of 0.028% for a coupon c ontaining 30 mg/kg of nanosilver. The plastic coupon leaching study was initially de termined to be unacceptable by EPA because less than 0.8% of the si lver in the plastic coupons was rec overed during the ashing process, the wide range in silver matrix-spike recoveries for some of the f ood simulants (particularly alcohol28 and oil), and the lack of validation data for the silver analysis. Na nosilva LLC subsequently completed an additional study to determine the si lver content of identically prepared plastic coupons (MRID 48652901). As per EPA recomm endation, Nanosilva LLC employed the nondestructive Neutron Activation An alysis (NAA) to determine the silver content of the NSPWL30SS liquid suspension, a master batch co ntaining 2% NSPW-L30SS, and plastic coupons containing 5% of the NSPW-L30SS master batc h. Using NAA, Nanosilva LLC determined the amount of silver in coupons was within 99% of the amount added. In addition, EPA decided that the plastics leaching study could be used to ev aluate the release of silver from NSPW-L30SS treated plastics into physiologi cal fluids because the recoveries for the most relevant food simulants, low and high pH fluids , were close to 100 percent. The plastics leaching study was c onducted using a protocol that wa s not reviewed by EPA prior to conducting the study. The plastics leaching study did not employ phys iological fluids such as artificial saliva; however, the study was complete d at the physiological te mperature of 40 degrees Celsius. The coupons tested contained nanosilver at concentrations of 5, 10, and 20 mg/kg, which are less than the maximum nanosilver concen tration of 30 mg/kg that on the NSPW-L30SS pesticide label. Even though the study was not conducted using physiolo gical fluids, the study does provide a reasonable estimate for the amount of silver transferred to the mouth while mouthing and transferred to skin while cont acting plastics incor porating NSPW-L30SS. Based upon this study, the transferab le surface residue is 0.0011 ug/cm 2 . This value represents the concentration of silver detected in low and hi gh pH fluids, which are more representative of biological fluids, contacting NSPW-L30SS contai ning coupons that is transferable from the surface of the toy into the child’s mouth. The concentr ation of silver in the low and high pH fluids were all below the analytical LOD of 5 μg/L for coupons containing 5, 10, and 20 mg/kg of NSPW-L30SS. EPA is assuming that non-detectable concentrations of silver would be found in a similar test conducted with coupons containing 30 mg/kg of NSPW-L30SS. EPA is using 2.5 μg/L, which is one-half the LOD of 5 μg/L, as the concentration of silver released from plastic incorporating NSPW-L30SS. Using the concentra tion of silver, the volume of the leachate (50 mL), and the surface area of the test coupons, the amount of transferable nanosilver at the coupon surface is:

Transferable Surface residue: ଶ.ହ	௨௚

௅ ൈ ହ଴	௠௅ ൈ ௅

ଵ଴଴଴	௠௅ ൈ ଵଵଶ	௖௠ మ ൌ ଴.଴଴ଵଵ௨௚

௖௠ మ Textile Leaching Study Nanosilva LLC prepared and submitted a draft protocol titled “The Quantification and Characterization of Silver Released from Tex tiles Treated with NSPW-L30SS (NSPW-L30) as a Results of Washing” with the stated purpose to quantify and characterize silver released from textiles incorporating NSPW-L 30SS during laundering. The prot ocol was based on methods developed by Geranio et al. (2009) and Lorenz et al. (2012) who m odified the ISO Colour Fastness test (ISO, 1997) to determine the amount and form of silver released to the environment when washing textiles containing silver and nanosilver. B ecause EPA must also evaluate the release of nanosilver when children chew and mouth textiles incorporating NSPW-L30SS, EPA recommended including an additional test using simulated human saliva. EPA does not anticipate dermal exposure will result in a risk concer n for textiles incorporating NSPW-L30SS, and therefore a separate leaching study involving simulated human sweat was not recommended. EPA will use the results of the ISO Colour Fastness test completed with simulated human saliva to evaluate the dermal exposure to nanosilver from textiles incorporating NSPW-L30SS.29 Nanosilva LLC completed and submitted the m odified ISO Colour Fastness test (MRID 49045301) using shirts incorporati ng NSPW-L30SS. The washing tests were conducted on shirts composed of polyethylene terephthalate (PET), which incorporated NSPW-L30SS into the PET yarn for a final nanosilver content of 0.00262% or 26.2 mg/kg per shirt. For the detergent wash test, a section was cut from each of three shirts a nd washed separately in 150 mL of distilled water with commercial laundry dete rgent and 10 hard rubber balls with diameter of 10 mm for 30 minutes at 40 degrees Celsius. The detergent wash was followed by two rinse cycles with deionized water. The concentrations of silver in the wash and ri nse water were determined after filtering samples through a 0.45 μm pore size f ilter using inductively coupled plasma mass spectrometry (ICP-MS). The concentr ation of silver retained on the filter was also determined by ICP-MS. These samples were acid digested prior to analysis and thus the silver concentrations are for total-silver content and do not distinguish between silver ions, nanosilver, or NSPW-L30SS. Although the NSPW-L30SS wash test study reported an ICP-MS LOD of 0.0094 μg/L for aqueous samples and 0.94 μg/kg for filter samples, EPA determined that the LOD for aqueous samples was 10 μg/L and 1,000 μg/kg for solids samples. EPA determined these LODs based on a statistical analysis of seven ICP-MS calibrati on curves and the analysis resu lts from 100 μg/L quality control samples. The concentration of silver in the wash and rinse water from the detergent wash test for three shirt sections was below the analytical L OD of 10 μg/L (Table 8). The concentration of silver retained on 0.45 μm pore size filters was al so less than the analyt ical LOD of 1,000 μg/kg.

Table 8 – Concentration of Silver Released from Shirts Incorporating NSPW-L30SS

Number of Shirt Sections Wash Medium Concentration of Silver

Wash/Rinse Water 0.45 μm Pore Size Filter (Particles with Diameters >0.45 μm)

3 Distilled Water with Detergent < 10 ug/liter < 1,000 ug/kg

9 Simulated Human Saliva < 10 ug/liter < 1,000 ug/kg

A separate study was conducted using three secti ons from each of three shirts and washed separately in 150 mL of simulated human saliva w ith 10 hard rubber balls with diameter of 10 mm for 45 minutes at 40 degrees Celsius. The concentr ation of silver in the simulated saliva was determined after filtering thr ough a 0.45 μm pore size filter using IC P-MS where the concentration of silver in the saliva for nine shirt sections was below the analytical LOD of 10 μg/L (Table 8). The concentration of silver retained on filters was less than the analytical LOD of 1,000 μg/kg.

Given that none of the silver co ncentrations in detergent or sa liva solutions were above 10 μg/L and none of the filters containe d silver at a concen tration above 1000 μg/kg, EPA evaluated the amount of silver released from textiles incorp orating NSPW-L30SS by replacing the non-detect values with half the LOD, 5 μg/L for liquid sa mples and 500 μg/kg for solids samples (U.S. EPA, 2000). The amount of silver released from te xtiles was calculated based on the volume of detergent and rinse water, and saliva along with the mass of the 0.45 μm filters used for each test (Table 9).30 Table 9 – Amount of Silver Released from Shirts Incorporating NSPW-L30SS

Wash Medium Volume/Mass Concentration Amount of Silver (μg) Total-silver Potentially Released (μg) % Distilled Water with Detergent 190 mL wash/rinse water 5 μg/L B 0.95 1.05 1.6 0.2 g filters A 500 μg/kg B 0.1 Simulated Human Saliva 150 mL Saliva 5 μg/L 0.75 0.81 0.9 0.12 g filter 500 μg/kg 0.06 A. There was a 0.1 g filter used for the detergent wash and another 0.1 g filter for the rinse. B. Set to half the LOD.

The initial amount of silver in textiles washed with distille d water and detergent was 66.54 μg, therefore the amount of silver releas ed during the detergent wash was:

Silver released in detergent wash = ଵ.଴ହஜ௚ ଺଺.ହସ	ஜ௚ ൈ 100% ൌ 1.6%

The initial amount of silver in textiles washed with simulated human saliva was 94.9 μg, therefore the amount of silver released during the saliva wash was:

Silver released in saliva wash = ଴.଼ଵஜ௚ ଽସ.ଽ	ஜ௚ ൈ 100% ൌ 0.9%

The value of 1.6% is used in evaluating releases to the environment from wash water and the value of 0.9% is used in calculating oral and dermal exposures to textiles in corporating NSPW-L30SS. Since these releases were determined using concen trations that were below the ICP-MS LOD, the form of silver is unknown. EPA assumes the form of silver is identical to the nanosilver present in NSPW-L30SS in the absence of further information.

The results of these studies de monstrate that PET shirts whic h incorporate NSPW-L30SS at 26.2 μg/kg of nanosilver do not release silver at concentrations above the analytical LOD. The ISO Colour Fastness test is thought to represent aggressive washing conditions with one wash cycle representing up to five domestic or commerci al laundering cycles when the multiple test is employed. The amount of silver released during one IS O Colour Fastness test is believed to exceed the daily exposure to nanosilver from a treated textile because the ISO Colour Fastness test involves immersing the textile in water containing detergents or simulated human saliva and hard rubber balls followed by mechanical agitation for 30 to 45 minutes. Thus, results from studies which are based on the ISO Colour Fastness test will be used to determine the daily dose of nanosilver for children who chew and mouth, adults who wear, and workers who manufacture items from nanosilver-treated textiles even though this likely overestimates the daily dose of nanosilver. 5.2.2 Consumer Margins of Exposure to Plastics Incorporating NSPW-L30SS EPA expects that consumers will be exposed to plastics incorporating NSPW-L30SS by the routes of incidental oral and dermal exposures. As de tailed in the following sections, EPA evaluated the risk to children, as the likely most vulnerabl e subpopulation, from mouthing toys, and mouthing and crawling on flooring incorporating NSPW-L30SS. Incidental Oral Exposures for Toys Incorporating NSPW-L30SS Incidental oral exposures to t oys incorporating NSPW-L30SS were calculated using the following: Incidental Oral Exposure = Transferable Surface Residue × Surface Area of Toy Mouthed31 Where:

 The transferable surface residue is 0.0011 ug/cm 2 based on the Plastics Leaching Study

(MRID 478289-25). This value represents the con centration of silver detected in low and high pH fluids, which are more representativ e of biological fluids, contacting NSPW-L30SS containing coupons that is transferable from th e surface of the toy into the child’s mouth.

 The surface area of a toy mouthed per day is 800, 560 and 396 cm 2 for 6 to <12 month, 1

to < 2 year and 2 to < 3 year-old childre n, respectively. These values were calculated assuming that a child mouths an area of 10 cm 2 each time they contact a toy, that 6 to < 12 month-old children contact objects 20 times pe r hour, 1 to < 2 year-old children contact objects 14 times per hour, and 2 to < 3 year-o ld children contact objects 9.9 times per hour (U.S. EPA, 2011a) for 4 hours per day.

The incidental oral dose was calculated from the incidental oral exposure using the following:

Incidental Oral Dose = Exposure / Body Weight

Where:

 Exposure is determined in the calculations above.

 The body weight is 9.2, 11.4 and 13.8 kg for children 6 to < 12 months old, 1 to <2 years old and 2 to < 3 years old, respectively (U.S. EPA, 2011b).

The MOEs in Table 10 are for incidental oral ex posures and were calculated from the incidental oral dose using the POD of 30 mg/kg/day, which is the NOAEL from a 28-day oral toxicity study (Kim et al., 2008) and the LOAEL from a 90-day or al toxicity study (Kim et al., 2010). The MOEs range from 310,000 to 940,000, depending on the age of the child. These MOES are well above the target MOEs of 1,000 and 3,000, for short an d intermediate terms exposures, respectively, which means that the risk for s hort- and intermediate-term exposur e to children who mouth plastic toys containing NSPW-L30SS is not of concern.

Table 10 – Incidental Oral MOEs for Plastic Toys Incorporating NSPW-L30SS

Age of Child Body Weight

(kg) Transferable Surface Residue (ug/cm 2 ) Surface Area Mouthed per

Day (cm 2 ) Exposure A (ug/day) Dose B (ug/kg/day) MOE C,D

6 to < 12 months 9.2

0.0011 800 0.88 0.096 310,000

1 to < 2 years 11.4 560 0.62 0.054 560,000

2 to < 3 years 13.8 396 0.44 0.032 940,000 A. Exposure = Transferab le Surface Residue (ug/cm 2 ) × Surface Area of Toy Mouthed (cm 2 ) B. Dose = Exposure (ug/day) / Body Weight (9.2, 11.4 or 13.8 kg) C. MOE = [POD (30 mg/kg/day) x 1000 ug/mg] / Daily Dose (ug/kg/day) D. Target MOE is 1000 for short-term exposures and 3000 for intermediate term exposures. Dermal Exposure to Flooring Incorporating NSPW-L30SS The dermal exposure to flooring incorporating NSPW-L30SS was calculated using the following: Dermal Exposure = Transferable Surface Residue (mg/cm 2 ) × Body surface area in contact with floor (cm 2 /day)32 Where:

 The transferable surface residue is 0.0011 ug/cm 2 based on the Plastics Leaching Study (MRID 478289-25).

 The body surface area contacti ng flooring is 4,500, 5300 and 6,100 cm 2 /day for children 6 to < 12 months old, 1 to < 2 years old, and 2 to < 3 years old, respectively (U.S. EPA, 2011b). These are the total surface area of children and do not account for the portion of child covered with clothing.

The dermal dose was calculated from the dermal exposure using the following:

Dermal Dose = [Exposure × Dermal Absorption]/ Body Weight

Where:

 Exposure is determined in the calculations above.

 The dermal absorption is 6.7 percent (see Section 4.2.3).

 The body weight is 9.2, 11.4 and 13.8 kg for children 6 to < 12 months old, 1 to <2 years old and 2 to < 3 years old, respectively (U.S. EPA, 2011b). The MOEs in Table 11 are for dermal exposures to flooring incorporati ng NSPW-L30SS and were calculated from the dermal dose using the POD of 30 mg/kg/day, which is the NOAEL from a 28- day oral toxicity study (Kim et al., 2008) and the LOAEL from a 90-day oral toxicity study (Kim et al., 2010). The MOEs range from 820,000 to 910,000, depending on the age of the child. These MOES are well above the target MOEs of 1,00 0 and 3,000 for short and intermediate terms exposures, respectively, which mean s that the risk for short- and intermediate-term dermal exposure to children who contac t flooring incorporating NSPW -L30SS is not of concern. Table 11 – Dermal MOEs for Flooring Incorporating NSPW-L30SS

Age of Child Body Weight

(kg) Transferable Surface Residue (ug/cm 2 ) Exposed Body Surface Area (cm 2 ) Exposure A (ug/day) Dose B (ug/kg/day) MOE C,D

6 to < 12 months 9.2

0.0011 4,500 5.0 0.036 830,000

1 to < 2 years 11.4 5,300 5.8 0.034 880,000

2 to < 3 years 13.8 6,100 6.7 0.033 910,000 A. Exposure = Transferab le Surface Residue (ug/cm 2 ) × Exposed Body Surface Area (cm 2 ) B. Dose = [Exposure (ug/day) × Dermal absorption (6.7%)] / Body Weight (9.2, 11.4 or 13.8 kg) C. MOE = [POD (30 mg/kg/day) x 1000 ug/mg] / Daily Dose (ug/kg/day) D. Target MOE is 1000 for short-term exposures and 3000 for intermediate term exposures. Incidental Oral Exposures to Fl ooring Incorporating NSPW-L30SS The incidental oral exposure to nanosilver in flooring incorporating NS PW-L30SS was calculated using the following: Incidental Oral Exposure = Transferable Surface Residue (mg/cm 2 ) × Surface area of hand that contacts the floor and the child’s mouth (cm 2 /event) × Frequency of hand-to-mouth contacts (events/hr) × Exposure time (h r/day) x Saliva Extraction Efficiency

Where:

 The transferable surface residue is 0.0011 ug/cm 2 based on the Plastics Leaching Study (MRID 478289-25).33  The hand to mouth (HTM) surface area (i.e. th e surface area of the ha nd that contacts both the floor and the child’s mouth) is 16 cm 2 /event for 6 to < 12 month-old children, 20

cm 2 /event for 1 to < 2 year-old children and 21 cm 2 / event for 2 to < 3 year-old children. The HTM value of 20 cm 2 for 1 to < 2 year children is listed in the SOPS (US EPA, 2012b) and is based on the mean surface area of one hand (150 cm 2 ) for children 1< 2 years old as listed in Table 7-2 of the Exposure Factor s Handbook (US EPA, 2011b) and the fraction of the hand mouthed per event (0.13) from an anal ysis included in Zart arian et al. (2005). Table 7-2 also lists mean hand surface areas of 120 cm 2 for 6 to < 12 month-old children, 140 cm 2 for 2 to < 3 year-old children and 185 cm 2 for 3 to < 6 year-o ld children. The hand surface area of 120 cm 2 for 6 to < 12 month-old children was used in conjunction with the fraction of the hand mouthed per event ( 0.13) to derive the HTM value of 16 cm 2 /event for 6 to < 12 month-old children. The average of the hand surface areas values of 140 cm 2 (2 to < 3 old children) and 185 cm 2 (3 to < 6 year-old childre n) along with the fraction hand mouthed value (0.13) was used to derive the hand to mouth surface of 21 cm 2 for 2 to < 3 old children.

 Frequency of hand-to-mouth events is 19 even ts/hr for 6 to < 12 month-old children, 20 events/hr for 1 to < 2 year-old children, and 13 events/h r for 2 to <3 year-old children (U.S. EPA, 2011b).

 Exposure time is 101 minutes ( 1.68 hours) per day for 6 to < 12 month-old children, 126 minutes (2.10 hours) per day for 1 to < 2 y ear-old children, and 108 minutes/day (1.80 hours) for 2 to <3 year-old children. These ar e the mean times for “D oers” in kitchens and bathrooms (Table 16-15, U.S. EPA, 2011b)

 Saliva extraction efficiency from hand to mouth is 50% (U.S. EPA, 2012b)

The incidental oral dose was calculated from the incidental oral exposure using the following:

Incidental Oral Dose = Exposure / Body Weight

Where:

 Exposure is determined in the calculations above.

 The body weight is 9.2 kg, 11.4 kg and 13.8 kg for ch ildren 6 to < 12 months old, 1 to <2 years old and 2 to < 3 years ol d, respectively (U.S. EPA, 2011b).

The MOEs in Table 12 are for incidental oral exposures to flooring incorporating NSPW-L30SS and were calculated from the incidental oral dose using the POD of 30 mg/kg/day, which is the NOAEL from a 28-day oral toxic ity study (Kim et al., 2008) and the LOAEL from a 90-day oral toxicity study (Kim et al., 2010). The MO Es range from 970,000 to 1,500,000 depending upon the age of the child. These MOES are well above th e target MOEs of 1000 and 3,000 for short and intermediate terms exposures, resp ectively. This means that the ri sk for short- and intermediateterm exposure to childre n who mouth their hands after cont acting flooring incorporating NSPWL30SS is not of concern.34 Table 12 – Incidental Oral MOEs fo r Flooring Incorporating NSPW-L30SS

Age of Child Transferable Surface Residue (ug/cm 2 ) Frequency of Hand to Mouth Contacts (Events/ Hour) Daily Exposure Time (Hours) Exposure A (ug/day) Dose B (ug/kg/day) MOE C,D

6 to < 12 months

0.0011 19 1.68 0.28 0.031 970,000

1 to < 2 years 20 2.10 0.46 0.041 730,000

2 to < 3 years 13 1.80 0.27 0.020 1,500,000 A. Exposure = Transferab le Surface Residue (ug/cm 2 ) × Surface area of hand that contacts the floor and the child’s mouth (16, 20 and 21 cm 2 /event) × Frequency of Hand to Mouth Contacts (Events/hour) × Daily Exposure Time (hours) × Saliva extraction efficiency (50 %) B. Dose = [Exposure (ug/day)] / Body Weight (9.2, 11.4 or 13.8 kg) C. MOE = [POD (30 mg/kg/day) × 1000 ug/mg] / Daily Dose (ug/kg/day) D. Target MOE is 1000 for short-term exposures and 3000 for intermediate term exposures. 5.2.3 Consumer Margins of Exposure to Textiles Incorporating NSPW-L30SS EPA expects that consumers will be exposed to textiles incorporating NSPW-L30SS by the routes of inhalation, dermal, and in cidental oral exposures. Consumer Inhalation Exposures EPA recognizes the potential for inhalation expo sure to nanosilver during laundry drying of textiles incorporating NSPW-L30S S. However, EPA lacks information on the release rate of nanosilver from textiles incorporating NSPW-L 30SS during laundry dryin g. While exposure may occur during laundry drying, EPA believes that wh en compared to exposure through dermal and oral contact with textiles in corporating NSPW-L30SS, exposure during laundry drying will likely be of lower significance. To suppor t this, an estimate of the inhala tion dose that might occur from exposure to textiles incorporat ing NSPW-L30SS during laundry dr ying was calculated using the following:

Inhalation Dose = Amount of NSPW-L30SS in clothi ng handled per day × Unit Exposure

Where:

 Amount of NSPW-L30SS in clothing handled pe r day assumed that one t-shirt containing NSPW-L30SS was laundered where the amount of na nosilver in a t-shirt is equal to 150 g × 30 mg/kg × kg ÷ 1000 g = 4.5 mg.

 The unit exposure for inhalation of wettable powder is 7.8 μg/m 3 /lb when no respirator is worn (U.S. EPA, 2011a). The MOE shown in Table 13 was calculated by dividing the inhalation dose by the POD of 49 μg/m3, which is the NOAEL from a 90-day inhala tion toxicity study for nanosilver (Song et al., 2012). This assumes that NSPW-L30SS released from textiles during drying behaves like nanosilver after inhalation. This anal ysis further assumes that all of the nanosilver from the textile incorporating NSPW-L30SS becomes airborne during a single laundry drying event and this exposure would be similar to mixing and loadin g of wettable powders (U.S. EPA, 2011a). The MOE of 640,000 is well above the target MOE of 1000 which means that the risks are not of concern. Even if 100 t-shirts incorporating N SPW-L30SS were laundered per day, the MOE would be 6,400 which is well above the target MOE of 1,000.35 Table 13 – Inhalation MOE for Drying Textiles Incorporating NSPW-L30SS

Scenario Number of Nanosilver Treated T-shirts Laundered per day Amount of Nanosilver Laundered per day A Unit Exposure B (μg/m 3 /lb AI) Dose C (μg/m 3 ) MOE D,E

Unload Clothes Dryer ( Without

Respirator) 1 4.5 mg (0.0000099 lb) 7.8 0.000077 640,000

100 450 mg (0.00099 lb) 0.0077 6,400
A. Amount of Nanosilver laundered per day = Amount of NSPW-L30SS in t-shirt × Mass of t-shirt B. PHED unit exposure value converted to air concentration units based on mean 8 hour TWA C. Dose = Amount Nanosilver Handled × Unit Exposure D. MOE = POD / Daily Dose E. The target MOE is 1000. Consumer Dermal Exposures to Textiles The dermal exposure to textiles incorporating NSPW-L30SS was calculated using the following: Dermal Exposure = Amount of NSPW-L30SS in Textile × Cloth Density × Surface Area Exposed × Transfer Efficiency

Where:

 The textile incorporating NSPW-L30 SS contains 30 mg/kg nanosilver.

 The cloth density is 10 mg/cm 2 based on the density of mixed cotton and synthetics. This value is a standard assumption used in O PP risk assessments and was taken from the HERA Guidance Document Met hodology (AISE/CEFIC, 2005).

 The total surface area for a 6 to < 12 month-old is 4,500 cm 2 /day, for a 1 to < 2 year-old is 5,300 cm 2 /day, and for a 2 to < 3 year-old is 6,100 cm 2 /day (U.S. EPA, 2011b).

 The cloth-to-skin transfer efficiency was based on the amount of silver released during the leaching study which was 0.9% based on one-half the LOD (see Table 9).

The dermal dose was calculated from the dermal exposure using the following:

Dermal Dose = Exposure × Dermal Absorption / Body Weight

Where:

 Exposure is determined in the calculation above.

 The dermal absorption (DA) is 6.7% (see Section 4.2.3).

 The body weight of a child with age between 6 and < 12 months is 9.2 kg, between 1 and < 2 years is 11.4 kg, between 2 and < 3 years is 13.8 (U.S. EPA, 2011b). The MOEs in Table 14 for dermal exposures were calculated from the dermal dose using the POD of 30 mg/kg/day, which is the NOAEL from a 28-day oral toxicity study (K im et al., 2008) and the LOAEL from a 90-day oral toxi city study (Kim et al., 2010). The MOEs range from 340,000 to 370,000 depending upon the age of the child. These MOES are well above th e target MOEs of 1000 and 3,000 for short and intermediate term expos ures, respectively, which means that the risks are not of concern.36 Table 14 – Dermal MOEs for Textiles Incorporating NSPW-L30SS

Age of Child Application Rate (mg/kg) Cloth Density (mg/cm 2 ) Surface Area Exposed (cm 2 /day) Cloth-to-Skin Transfer Efficiency Exposure A (mg/day) Dose B (ug/kg/day) MOE C,D

6 to <12 months

30 10 4,500

0.9% 0.012 0.087 340,000

1 to < 2

years 5,300 0.014 0.082 370,000

2 to < 3

years 6,100 0.017 0.083 360,000
A. Exposure = Application Rate × Cloth Density × Surf ace Area Exposed × Cloth-to-Skin Transfer Efficiency B. Dose = [Exposure (mg/day) × 1000 ug/mg × DA (6.7%)] / Body Weight (9.2, 11.4 or 13.8 kg) C. MOE = [POD (30 mg/kg/day) x1000 ug/mg] / Daily Dose (ug/kg/day) D. Target MOE is 1000 for short-term exposures and 3000 for intermediate term exposures. Consumer Incidental Oral Exposures to Textiles Incidental oral exposures were calculated using the following: Incidental Oral Exposure = Amount of NSPW-L30SS in Text ile × Cloth Density × Surface Area Mouthed × Saliva Extraction Efficiency

Where:

 The textile incorporating NSPW-L 30SS contains 30 mg/kg nanosilver.

 The cloth density is 10 mg/cm 2 based on the density of mixed cotton and synthetics. This value is a standard assumption used in O PP risk assessments and was taken from the HERA Guidance Document Met hodology (AISE/CEFIC, 2005)

 The surface area of fabric that is mouthed by a toddler per day is assumed to be 100 cm 2 (~16 in. 2 ) which represents an estimate, for example, of the area of blanket or shirt sleeve.

 The nanosilver saliva extraction efficiencies fo r mouthing fabric are based on the results of the leaching study which was 0.9% based on one-half the LOD (see Table 9).

The incidental oral dose was calculated from the incidental oral exposure using the following:

Incidental Oral Dose = Exposure / Body Weight

Where:

 Exposure is determined in the calculation above.

 The body weight of a child with age between 6 and < 12 mont hs is 9.2 kg, between 1 and <2 years is 11.4 kg, between 2 and < 3 year s is 13.8, which is for children who mouth textiles (U.S. EPA, 2011b). The MOEs in Table 15 for incidental oral exposures were calculated from the incidental oral dose using the POD of 30 mg/kg/day, which is the NOA EL from a 28-day oral t oxicity study (Kim et al., 2008) and the LOAEL from a 90- day oral toxicity study (Kim et al., 2010). The MOEs range from 1,000,000 to 1,500,000 depending upon the age of th e child. These MOEs are well above the target MOEs of 1000 and 3,000 for short and inte rmediate terms exposures, respectively, which means that the risk for short- and intermediate -term exposure to childr en who mouth textiles incorporating NSPW-L30SS is not of concern.37 Table 15 – Incidental Oral MOES fo r Textiles Incorporating NSPW-L30SS
Application Rate (mg/kg) Cloth Density (mg/cm 2 ) Surface Area Mouthed (cm 2 /day) Saliva Extraction Efficiency Exposure A (mg/day) Dose B (ug/kg/day) MOE C, D

30 10 100 0.9% 0.00027 0.029 1,000,000 0.024 1,200,000 0.020 1,500,000 A. Exposure = Application Rate × Cloth Density × Surface Area Mouthed × Saliva Extraction Efficiency B. Dose = [Exposure (mg/day) x 1000 ug/mg] / Body Weight (9.2, 11.4 and 13.8 kg) C. MOE = [POD (30 mg/kg/day) x 1000 ug/mg] / Daily Dose (ug/kg/day) D. Target MOE is 1000 for short-term exposures and 3000 for intermediate term exposures. 5.2.4 Aggregate MOES for Plastics and Textiles Incorporating NSPW-L30SS In the Federal Food, Drug, and Cosmetic Act (FFDCA ), Congress specified th at, to establish an acceptable level of a given pesticide’s chemical re sidue that could be found in or on food products, EPA must determine that “there is a reasonable ce rtainty that no harm will result from aggregate exposures to the pesticid e chemical residue, including all an ticipated dietary exposures and all other exposures for which there is reliable in formation.” Furthermore, when enacting this provision of the FFDCA, Congress also amende d FIFRA’s definition of unreasonable adverse effects. Specifically, Congress redefined unreasonab le adverse effects to include “a human dietary risk from residues that result from a use of a pe sticide in or on any food inconsistent with the standard under” the FFDCA. In other words, C ongress explicitly required the consideration of aggregate exposures for registration decisions und er FIFRA for food-use pesticides, but chose not to similarly alter the statutory requ irements for nonfood-use pesticides. The following analysis is for children who are simultaneously exposed to nanosilver via the incidental oral and dermal routes of exposur e while mouthing toys, contacting flooring, and mouthing textiles inco rporating NSPW-L30SS. Inhalation exposure to children during laundry drying of textiles incorp orating NSPW-L30SS is not anticipated and is not considered as part of the aggregate exposure. The aggregate daily dos e was calculated by adding the daily oral and dermal doses using the following: Aggregate Dose = Incidental Oral Dose to Toys In corporating NSPW-L30SS + Dermal Dose to Flooring Incorporating NSPW-L30SS + Incide ntal Oral Dose to Flooring Incorporating NSPW-L30SS + Dermal Dose to Textiles Incorp orating NSPW-L30SS + Incidental Oral Dose to Textiles Incorporating NSPW-L30SS

Where:

 Incidental Oral Dose to Toys Incorp orating NSPW-L30SS is from Table 10;

 Dermal Dose to Flooring Incorporat ing NSPW-L30SS is from Table 11;

 Incidental Oral Dose to Flooring Incorporating NSPW -L30SS is from Table 12.

 Dermal Dose to Textiles Incorporati ng NSPW-L30SS is from Table 14; and

 Incidental Oral Dose to Textiles Inco rporating NSPW-L30SS is from Table 15. The oral and dermal daily doses can be summed because they are evaluated using the same POD of 30 mg/kg/day. The aggregate MOEs in Table 16 range from 110,000 to 160,000 depending on the age of the child. These MOEs are all well above the target MOEs of 1,000 and 3,000 for short and intermediate terms exposures, respectively, which means that the risk for short- and intermediate-term aggregate exposure to children is not of concern.38 Table 16 – Aggregate MOEs for Toys, Floor ing and Textiles Incorporating NSPW-L30SS

Age of Child Toys Flooring Textile Aggregate Incidental Oral Dose Dermal Dose Incidental Oral Dose Dermal Dose Incidental Oral Dose Dose A (ug/kg/day) MOE B,C

6 to < 12 months 0.096 0.036 0.031 0.087 0.029 0.28 110,000

1 to < 2 years 0.054 0.034 0.041 0.082 0.024 0.24 120,000

2 to < 3 years 0.032 0.033 0.020 0.083 0.020 0.19 160,000 A. Aggregate Dose = Sum of the Incidental Oral and Dermal Doses (ug/kg/day) for Toys, Flooring and Textiles B. Aggregate MOE = [POD (30 mg/kg/day) × 1000 ug/mg] / Dose (ug/kg/day) C. The target MOE is 1000 for short-term exposure and 3000 for intermediate term exposures.

The assessment above is for the concurrent e xposure to the nanosilver in NSPW-L30SS via the incidental oral and dermal routes of exposure while mouthing toys and textiles, and contacting flooring incorporating NSPW-L30SS. Aggregate assessments can al so include other sources of exposure such as to NSPW-L30SS in food and dri nking water, and to other nanosilvers in the market place that are identical to the nanosil ver in NSPW-L30SS. Ther e are no anticipated food exposures to NSPW-L30SS since Nanosilva LLC ha s not supplied the necessary information to support a food contact application a nd the pesticide label for NSPW-L 30SS states that it is only for non-food contact use. Neither the nanosilver that might break away fromNSPW-L30SS or NSPWL30SS is anticipated to enter dr inking water because any particulates released to surface water would be removed by gravitational sediment ation and adsorption (see Section 5.1.1). The only other pesticide product registered as co ntaining nanosilver is HeiQ AGS-20 (U.S. EPA, 2011a). The nanosilver in HeiQ AGS-20 has differe nt size range and surface coatings than the nanosilver in NSPW-L30SS, and EPA has not determined that AGS-20 and NSPW-L30SS contain the same active ingredient or present exposur es that should necessarily be aggregated. As to exposure to other nanosilvers in the market place, an aggregate risk assessment involves the analysis of exposure to a single chemical by multipl e pathways and routes of exposure that share a common toxic effect. For EPA to conduct such an assessment requires that EPA possess sufficient information such as:

 identification of toxicological endpoints for each exposure route and duration;

 identification of potential exposures for each pathway (food, water, and/or residential);

 reconciliation of dura tions and pathways of exposure with durations and pathways of health effects;

 determination of which possible residential expos ure scenarios are likely to occur together within a given time frame (co-occurrence);

 determination of magnitude and duration of exposure for all expos ure combinations;

 determination of the appropria te technique (deterministic or probabilistic) for exposure assessment; and

 determination of the appropriate risk metric to estimate aggregate risk.

 When EPA has the above information, or as in the case of tric losan where populationbased biological monitoring data were availa ble to assess the co-occurrence of uses, EPA develops an aggregate exposur e assessment for residential uses of a single chemical.

 However, in the case of nanos ilver, with the exce ption of the HeiQ products, EPA does not yet have reliable information on the compositi on of the nanosilver or potential nanosilver in other silver based pe sticides to determine if all nanos ilvers should be treated as a single39 chemical. These limitations include understandi ng how size, charge and coating effect the toxicity of nanosilvers.

5.2.5 Conclusions for Consumer Risk The MOEs for the children’s expos ure scenarios (toys, flooring and textiles) routes of exposure (dermal and incidental oral) and life stages (6 months to <1 year, 1 to <2 years and 2 to <3 years) range from 310,000 to 1,500,000 depending upon the scen ario, exposure route and life stage. All of these MOE are well above the target MOEs of 1000 and 3,000 for short and intermediate term exposure, respectively, which means that there is no risk concern for children’s short- and intermediate-term exposure to toys, flooring and textiles incorporating NSPW-L30SS. The aggregate MOEs for children who are, within the same short time frame, mouthing a toy, flooring, and a textile while wearing a textile an d crawling on flooring all incorporating NSPWL30SS are greater than 100,000 for all life stages, which indicates that the risk for short- and intermediate-term simultaneous exposure to plastics and textiles incorporating NSPW-L30SS is
not a concern. Although children’s short- and inte rmediate-term exposure durations to plastic toys and textiles treated with NSPW-L30SS are expe cted, children are not anticipate d to play with NSPW-L30SStreated toys or contact NSPW-L30SS-treated textil es every day for a period of greater than six months, thus long-term exposures are unlikely. B ecause of the potential for NSPW-L30SS to be used in flooring, long-term dermal and incidental oral exposures to children contacting flooring are possible. However, EPA does not believe that child ren will have continuous and daily exposure to flooring for a period of greater th an 6 months. Even if these unlik ely long-term exposures were to occur, the calculated MOEs for children’s dermal and incidental oral exposure to plastics and textiles incorporating NSPW-L30S S are greater than 100,000 indica ting that daily exposure to plastics and textiles incorporat ing NSPW-L30SS for greater than 6 months are not of concern.

There are several uncertainties in the cons umer risk assessment. Because Nanosilva LLC completed leaching tests using plastic coupons c ontaining nanosilver at concentrations of 5, 10, and 20 mg/kg, EPA assumed that non-detectable concen trations of silver w ould also be found if a leaching test was conducted with coupons contai ning nanosilver at 30 mg/kg. This assumption was reasonable because the concentra tion of silver in the textile leaching studies, which were conducted on textiles incorporati ng nanosilver at 30 mg/kg, was also below the analytical LOD, making it unlikely that NSPW-L30SS incorporated into plastic at 30 mg/kg will release nanosilver at detectable concentrations. The PODs used we re based on inhalation and oral toxicity studies completed using nanosilver, not NSPW-L30SS or the nanosilver that might break away from NSPW-L30SS, and thus these PODs may not equal PODs determined from testing conducted with NSPW-L30SS or the nanosilver found in NSPW-L30SS. Because the nanosilver in the toxicity database is different from the nanosilver in N SPW-L30SS and because the database is incomplete with respect to reproductive a nd developmental effects, EPA used a maximum 10-fold database uncertainty factor when evaluating the risk from exposure to NSPW-L30SS and the nanosilver that might be released from products incorporating NSPW-L30SS. There are also uncertainties in extrapolating from effects observe d after feeding test-animals nanos ilver (i.e., oral route) to the effects that might be observed af ter applying nanosilver to the skin of test animals (i.e., dermal route). Taking the above into account, EPA conclude s that use of NSPW-L30SS will not cause unreasonable adverse effects on the environment dur ing the period when newly required data are being developed.40 5.2.6 Consumer Exposure and Health Data Requirements EPA has determined that an additional leaching study is required for plastics incorporating NSPWL30SS using biological fluids and physiological temperatures, which includes methods to determine the form of silver released. EPA ha s determined that the textile leaching study submitted by Nanosilva LLC is acceptable and no additional studies on textiles incorporating NSPW-L30SS are required. EPA is requiring that Nanosilva LLC conduct plastic leaching tests as part of Tier I studies because the available study was completed using a protocol that was not reviewed by EPA prior to conducting the study, the coupons tested had less th an the maximum nanosilver concentration of 30 mg/kg, and the study did not use physiologi cal fluids. The newly required leaching study should determine the nature and quantity of silv er released from plastics incorporating NSPWL30SS under conditions of use. EPA will evaluate the results from the plastic leaching test along with results from Tier I toxici ty studies discussed in Section 5.1.4 using a weight of evidence approach based on physical and chemical propertie s, toxicity, and exposur e. If EPA identifies a concern, then EPA may require add itional toxicity data to assess potential adverse health outcomes in young children resulting from incidental oral exposure to NSPW-L30SS- treated plastic toys (i.e., children chewing on toys). If additional toxic ity data are needed, the study should evaluate peri- and post-natal exposure to NSPW-L30SS. A more detailed descri ption of these data requirements is provided in Appendix B.

VI. Environmental Risk Assessment EPA anticipates the following substances could en ter the environment through leaching of plastics and textiles incorporating NSPW-L30SS: 1) silver ions released from NSPW-L30SS; 2) NSPW-L30SS; and/or 3) nanosilver that might break away from NSPW-L30SS.

There are no studies available to characterize the environmental fate or ecotoxicity of NSPWL30SS. However, there are studie s available in the scientific li terature for nanosilver. Since nanosilver may be released from NSPW-L30SS, EP A has considered the scientific literature studies on nanosilver fate and ecotoxicity releva nt to NSPW-L30SS. The following sections cover the environmental fate of nanosilver, the enviro nmental hazards posed by silver and nanosilver, and the potential risk to aquatic sp ecies from nanosilver. Because it recognizes that differences in formulations from one nanosilver to another are lik ely to affect biological activity and fate, EPA has attempted to be cautious in how it has us ed information about ot her nanosilvers from the literature in assessing risks from NSPW-L30SS. As part of these di scussions, additional data that EPA is requiring in order to confirm its assessm ent of the environmental risks of NSPW-L30SS are also identified in Section 7.3.1.

6.1 Environmental Fate Nanosilva LLC has not conducted any studies to ch aracterize the environmental fate of NSPWL30SS or the other particles that could be rel eased during leaching or di sposal of plastics and textiles incorporating NS PW-L30SS. In lieu of this info rmation, EPA is relying on studies available in the scientific literature, discussed in the following section, as the basis for determining the fate of nanosilver in the environment.41 6.1.1 Nanosilver

The rate at which nanosilver transforms into ioni c silver determines the length of time that these particles will reside in the envi ronment. Although there are studies reporting that nanosilver will completely transform into ionic silver within si x days after being disper sed into deionized water (Liu and Hurt, 2010), these results are only for on e form of nanosilver and under conditions which are not representative of the environment. In the environment, nanosilver is likely to complex with naturally occurring anions such as chloride and sulfide or natura l organic matter such as humic acids, which will significantly delay the rate at which nanosilver transforms into ionic silver. For example, Choi et al., (2009) pr ovided spectroscopic evidence show ing that nanosilver reacts with sulfide to produce stable silver-sulfide comple xes, which were shown by Liu et al. (2010) and Levard et al. (2011) to have unde tectable rates of nanosilver to ionic silver transformation. These stabilized nanosilver complexes are likely to partiti on to sediments, rather than remain suspended in water, due to gravitationa l settling and coagulation pro cesses (FIFRA SAP, 2009, p. 19). Likewise, nanosilver is anticipated to partition to biosolids during wastew ater treatment but may also be released in the effluent. Thus, there is the potential for nanos ilver to reside or persist in the environment for a significant period of time where th ese particles are most likely to be associated with sediments.

6.1.2 Silver Ions Ionic silver typically has low concentrations in natural waters, in the nanogram per liter range, due to its reactivity with chloride , sulfides, and natural organic matter (Andren and Armstrong, 1999). As with nanosilver, ionic silver is found in sedime nts and associated with biosolids in wastewater treatment plants. 6.1.3 Impacts to Wastewater Treatment/Septic Systems There is the potential for nanosilver that might be released from plastics and textiles incorporating NSPW-L30SS to reach publicly owned wastewater treatment and privately owned septic systems where they will most likely complex with sulfid e and partition to biosolids (Kaegi et al., 2011). Once entrained in the biosolids, the nanosilver could serve as a l ong-term source of ionic silver and could potentially adversely a ffect microorganisms that are vital to the wastewater treatment process. There are contradictory reports in the scientific literature regarding the impact of nanosilver on wastewater treatment systems. Fo r example, nanosilver was reported to inhibit nitrification in the range of 50% (Choi and Hu, 2009a) to 84% (Choi and Hu, 2009b) based on a reduction in oxygen uptake rate in simulated wastew ater sludge. However, Burkhardt et al. (2010) found no impact to nitrification at nanosilver dosag es of 1 mg/L, the same dosage that Choi and Hu (2009a and 2009b) reported as in hibitory in municipal wastewat er sludge. These two research groups are reporting different find ings with the Burkhardt group suggesting little impact of nanosilver to nitrification and the Hu group sugges ting that an impact to wastewater treatment systems from nanosilver is expected. A third gro up independently determined that nanosilver at concentrations from 0.5 to 1.5 mg/L had no detect able effect on the ability of the wastewater bacteria to biodegrade organic material, as measured by chemical oxygen demand (COD) (Wang et al., 2012a). More recent work by the Hu group re ported that nanosilver at concentrations of up to 40 mg/L had negligible imp act on anaerobic digestion and me thanogenic organisms (Yang et al., 2012). While there are reports suggesti ng the potential for nanosilver to impact wastewater treatment operations, EPA does not anticipate that registeri ng NSPW-L30SS will lead to negative impacts to wastewater treatment systems. This conclusion is based on the limited amount of silver released42 from plastics and textiles incorporating NSPW -L30SS (see Section 5.2.1) and the small volume of nanosilver (i.e., < 1,693 kg/yr as estimated in S ection 6.3.1) expected to be introduced into commerce from plastics and textile s incorporating NSPW-L30SS. 6.2 Environmental Toxicity Nanosilva LLC has not conducted any studies to ch aracterize the ecotoxic ity of NSPW-L30SS or the other particles that could be released during leaching or disposal of plastics incorporating NSPW-L30SS. In lieu of this information, EPA is relying on studies availa ble in the scientific literature, discussed in the fo llowing section, as the basis for determining the ecotoxicity of nanosilver.

6.2.1 Silver Ions EPA has considerable data on the environmental hazards posed by the release of silver ions from conventional silver-based products. The precious metal silver is a trace element found in the Earth's crust and is generally natu rally present in surface waters in relatively low concentrations as compared to metals such as copper and zinc. Ho wever, it may become toxic to aquatic life at elevated concentrations. Thus, silver concentrat ions in natural environments, and its biological availability, are important. Naturally occurring concentrations of s ilver have been reported from about 0.0002 to just over 1 μg/L in freshw ater systems (Campbell et al., 2002). Elevated concentrations of silver in surface waters have generally been associated with wastewater treatment plant effluent disc harges (Bell and Kramer, 1999). Based on the most recent Agency assessment of silver, EPA does not expect unreasona ble adverse effects on the environment from registered uses, including the materials pr eservative use patter n (U.S. EPA, 1993). 6.2.2 Aquatic Toxicity of Nanosilver Although no tests with NSPW-L30SS and aquatic or ganisms were submitted, there are studies in the scientific literature covering the toxicity of nanosilver to aquatic organisms (Table 17). These studies indicate to EPA that, if sufficient quantities of nanosilver break away from NSPW-L30SS and reach surface water, and if such nanoparticles display toxicity sim ilar to the nanosilver used in these studies, then exposure of NSPW-L30SS derive d nanosilver may result in adverse effects to aquatic species. These re sults also indicate that, of the organisms tested, Daphnia magna is the most acutely sensitive to nanosilver . As with the toxicity for silver ions, the toxic ity of nanosilver may depend on the ligands or counter ions (e.g., Ca 2+ ) present in the test media. For example, the LC 50 reported by Laban et al. (2010) was up to 27 times greater than the LC 50 reported by Kennedy et al. (2010) for the same organism where this difference may have been caused by the greater amount of calcium carbonate present in th e work by Laban et al. (2010). Additionally, the D. magna results for the nanosilvers on a total-silver ba sis in Table 17 may be lower than expected when food or organic matter is pres ent in the environment. In sim ilar tests with silver salts, the acute LC 50 is 3 to 40 times higher than acute tests where the daphnids are not fed during the test (EPA 1980, 1987; Ratte 1999). Standa rd acute test guidance for D. magna call for the daphnids to not be fed during testing and all of the D. magna studies listed in Table 17 were conducted in such

a manner.43 Table 17 – Nanosilver Toxicity to Aquatic Species

Aquatic Organisms

(Study Citation) Toxicity Silver Nitrate Nanosilver

Totalsilver ( μ g/L) Totalsilver ( μ g/L) Dissolved Silver ( μ g/L) Water Characteristics

Freshwater Algae, Raphidocelis subcapitata (Wang et al., 2012b) 4.5-hr EC 50 A 31.3 929- 120,000 G Not reported Dutch Standard Water (demineralized water with sodium bicarbonate, potassium bicarbonate, calcium chloride, and magnesium sulfate and then aerated for 24 h at 27 °C) Newborn Neonates of Freshwater Zooplankton, Chydorus sphaericus (Wang et al., 2012b) 48-hr EC 50 4.3 36.7- 128.4 G Not reported

Freshwater Fish Embryos, Danio rerio (Wang et al., 2012b) 96-hr EC 50 78.8 88.4- 210.3 G Not reported

D. magna (Kennedy et al., 2010) 96-hr LC 50 B 1.2±0.5 1.8 to 97.0 C 0.3 to 1.9 C,D Moderately hard reconstituted water (MHRW), 80 mg/L as calcium carbonate Freshwater Fish Larval, Pimephales promelas (Kennedy et al., 2010) 96-hr LC 50 6.1±0.6 9.0 to 125.6 C 1.5 to 5.6 C,D

D. magna (Hoheisel et al., 2012) 48-hr LC 50 0.98 4.3 to 30.4 E Not reported Moderate ionic strength Lake Superior water D. magna (Asghari et al., 2012) 48-hr LC 50 0.23 2 to 187 E Not reported OECD 202 Media

D. magna (Hoheisel et al., 2012) 24-hr LC 50 0.81 4.3 to 10.5 E 3.5 to 8.1 MHRW, 96 mg/L as sodium bicarbonate Natural Bacterioplankton (Das et al., 2012) 48-hr EC 50 Not reported 15 to 276 No reported EC 50 values did not correlate with any of the basic limnological parameters Freshwater Fish Embryo, P. promelas (Laban et al., 2010) 96-hr LC 50 15 9,400 to 10,600 40 215 to 240 mg/L as calcium carbonate; pH 8.3 to 8.5
Freshwater Fish Embryo, D. rerio (Wang et al., 2012b) 96-hr LC 50 77 to 82 72 to 223 G 0.7 to 22.3 H Not reported

A. EC 50 (medium effect concentration): Concentration at which the response is halfway between the baseline and maximum after the specified exposure time. B. LC 50 (median lethal concentration): Concentration required to kill half the tested population after the specified
exposure. C. Range of eight different nanosilver formulations (see Table 18 ). D. Expressed as the fraction of suspended nanosilver. E. For nanosilvers with diameters from 10 to 50 nm (see Table 18 ). F. Range of three nanosilver formulations (see Table 18). G. Range of three different nanosilver formulations (see Table 18). H. Estimated44 Table 18 – Reported Characteristics of Nanosilv er Materials in the Aqua tic Species Toxicity Studies

Study Source Nano Material Shape Primary Size (nm) ζ -Potential (mV) Asghari et al., 2012 ABC Nanotech Co., LTD SARPU 200 KW A (citrate capping agent), suspension spherical 7.32 Not reported

Nano Nasb Pars Co., Ltd Nanocid L2000, suspension spherical 6.47 Not reported

Xuzhou Hongwu Nanometer Material Co., Ltd Dry powder spherical 17.97 Not reported

Das et al., 2012 Vive Nano, Toronto PN 3010L (20% Ag w/w, carboxy-functionalized polyacylate-capped), aqueous suspension Not reported 1 to 10 Not reported

Hoheisel et al., 2012 NanoComposix Aqueous suspensions in 2 mM phosphate buffer: 10 nm spherical 10.2 Not reported 20 nm spherical 20.3 Not reported 30 nm spherical 34.4 Not reported 50 nm spherical 53.1 Not reported Kennedy et al., 2010 NanoComposix MQ; MHRW5; MHRW24 D NC10 spherical 10 (cv = 53%) -21 + 2; 5 + 4; -8 + 3 NC20 Mix of spheres and rods 20 (cv = 80%) -23 + 1; -1.7 + 0.1; -11 + 6

NC50 Mix of spheres

rods 49 (cv = 46%) B -34 + 1; -0.9 + 0.7; -21 + 1

NC80 spherical 50 (cv = 35%) -20 + 1; -0.9 + 0.1; -24 + 2 Luna Innovations PVP C -coated spherical -15 + 1; -0.7 +0.1; -6 + 4 Virginia Tech Citrate dispersion Mix of spherical and rods 29 (cv = 57%); 23 + 8 x 150 + 47 -46 + 3; -27 + 1; -17 + 3

EDTA dispersion spherical 36 (cv = 35%) -33 + 1; -22 + 2; 25 + 1 American Biotech Laboratories Colloidal silver drink spherical 31 (cv = 63%) Not reported

Laban et al., 2010 NanoAmor powder spherical 35 Not reported

Sigma-Aldrich powder spherical 21 to >300 (>50% in 21 to 60) Not measured45 Study Source Nano Material Shape Primary Size (nm) ζ -Potential (mV) Wang et al., 2012b Nanostructure & Amorphous Materials Powder, bare, 99.5% Ag spherical 35 1000 and 10 μM at pH 7.0: -23.2 + 0.6, - 21.3 + 0.3 Powder, PVP-coated ~0.3% PVP spherical 15 (90% <20) -24.1 + 1.2, - 20.6 + 1.5 Joint Research Centre of the European Union monodispersed with dispersant (4% each of polyoxyethylene glycerol trioleate and polyoxyethylene sorbitan monolaurate) spherical 80 -10.3 + 1.0, - 13.9 + 1.5

Wu and Zhou, 2013 Synthesized in the lab powder spherical 29.9 (25.9-36.7) -43.4

A. Reference material in OECD WPMN sponsorship program. B. Particles less than 5 nm were not included in primary diameter size; if included m ean diameter is 12 nm (CV = 96%). C. polyvinylpyrrolidone D. MQ = milli-Q water; MHRW5 = moderately hard reconstituted water after 5 minutes of media interaction; MHRW24 = moderately hard reconstituted wa ter after 24 hours of media interaction Based on the studies listed in Table 17, the most sensitive aquatic speci es to nanosilver is Daphnia

magna . The lowest 96-hr EC 50 was 1.8 μg/L (Kennedy et al., 2010), which is 1.5 μg/L after subtracting the fraction of ionic silver present. This test was conducted in MHRW, which is a test medium representative of surface water in the United States. Although these studies are useful fo r indicating the concentration of nanosilver that might lead to effects in aquatic species, and to identify for th is assessment an effect level for evaluating the ecotoxicity of nanosilver that could be released from plastics or textil es incorporating NSPWL30SS, the data represent short-term (e.g., 96 h r) acute exposures and do not characterize the effects from longer-term exposur e to nanosilver. There is one l ong-term study on the effects of nanosilver on a freshwater fish ( Oryzias latipes ) that reported a no obser ved effect concentration (NOEC) of 100 μg/L based on the lethal effect ob served at the concentration of 250 μg/L during a 14-day subchronic exposure period (Wu and Zhou, 2013). Non-lethal effects (biomarker effects, histological lesions in the fish tissues along with silver accumulation in the liver) were reported for all nanosilver concentrations te sted (50 to 500 μg/L). The study did not include any measurements of non-lethal apical endpoints (e .g., growth—length and/or dry weight ) and there is no established model or relationship to predict from such data to apical non-lethal endpoi nts used in the risk assessment. While this is a longer-term study, it do es not provide a lower apical endpoint for use in the risk assessment as the 14-d NOEC for lethality of 100 μg/L, on a total-silver basis, is about 55 times higher than the lowest acute nanosilver endpoint (48-h EC 50 of 1.8 μg/L) for the most acutely sensitive species, D. magna . In such a case use of the 14- d lethality NOEC for estimating chronic risks would underestimate the chronic risk to aquatic sp ecies. Non-apical endpoints are typically used in risk characterizat ion, but again since this is not the most sensitive species, this information only provides evidence that there is potential for non-lethal apical endpoints in chronic studies to be affected at lower concentrations than where increased mortality is observed.46 6.3 Aquatic Risk Assessment EPA expects that NSPW-L30SS, silver ions re leased from NSPW-L30SS, and nanosilver that might break away from NSPW-L30SS could enter the environment primar ily through leaching of plastics and textiles in corporating NSPW-L30SS. Leaching of plastics and textiles incorporating NS PW-L30SS is anticipated to be the primary route by which NSPW-L30SS, silver ions, and nanosilver reach the environment. The silver released during leaching of plastics and textiles incorporating NSPW-L30SS could be discharged to the sanitary sewer system leading to publically ow ned wastewater treatment and privately owned septic systems, also known as the down-the-dra in discharge scenario. Once NSPW-L30SS, silver ions, or nanosilver reach wastewater treatment and septic systems they will most likely complex with sulfide and partition to bi osolids. However, some fraction of the silver compounds will reach surface water and may potentially im pact aquatic organisms. Leaching of building material such as plastic siding and decks, and outdoor furniture and carpeting incorpora ting NSPW-L30SS will be discharged directly into the environment. As stated in Section 5.2.1, silv er was not found above the analyt ical LOD leaching from plastic coupons and shirts incorporating NSPW-L30SS. EPA does not expect unreasonable adverse effects on the environment from registered uses of silver ions, including the materials preservative use pattern (U.S. EPA, 1993). To evaluate the impacts on surface water from leaching of NSPWL30SS from plastics and textiles, EPA is assumi ng that nanosilver as found in NSPW-L30SS is the only silver compound released. 6.3.1 Aquatic Risk Quotient EPA uses a Risk Quotient (RQ) approach to asse ss impacts to surface water, which is similar to the MOE used for the human health risk assessmen t. The RQ is used to compare toxicity from environmental exposure by dividing a point estimate of exposure by a point estimate of effects. This ratio is a simple, screening-level estimate that identifies high- or low-risk situations. In this method, the estimated environmental concentration is compared to an effect level, such as an LC 50

. After the RQ is calculated, it is compared to EP A's Level of Concern (LOC). An LOC is a policy tool that EPA uses to interpret the RQ and to anal yze potential risk to non-target organisms and the need to consider regulatory action (U.S. EPA, 2011c ). Risks to aquatic organisms associated with two scenarios of in-service use of preserved text iles and plastics (an i ndoor textile use and an outdoor plastic lumber use) were used to screen for aquatic envi ronmental loading of nanosilver from NSPW-L30SS and risks from all in-service uses of these materials listed on the label. Based on the results of the screening ri sk assessment, no further refinement of the environmental loading and risks by in-service use were conducted.

Textile Use

 Mass of silver release per year: 1123 kg/year. The amount of silver released from textiles incorporating NSPW-L30SS was derived assuming that 300 million people (U.S. population) purchase one t-shirt incorporat ing NSPW-L30SS each year. Each t-shirt weighs 150 grams and contains 0.003% nanosil ver by weight silver and releases 1.6% silver per wash (see Section 5.2.1).

 Release Weeks: Each t-shirt is wa shed once per week for 52 weeks/yr.

 Wastewater Removal Efficiency: Ranged from 85 to 99% based on the range provided by Blaser et al. (2008). Wang et al. (2012a) reported nanosilver removal of 88% with biomass present.47  Speciation, Agglomeration and Sedimentation in the Surface Water Column: All of the silver released to surface water from a wast ewater facility was assumed to be in the nanosilver form and it was assumed that all of the nanosilver was retained in the water column (i.e., no removal of released silver fr om the water column). The model used does not include these fate mechanisms and theref ore surface water concentrations are expected be overestimated).

Plastic lumber use

 Mass of silver release per year: 570 kg/year. The amount of silver released from plastics incorporating NSPW-L30SS was derived assuming that 1% of the yearly plastic lumber production contains NSPW-L30SS at 0.003%. Y early plastic lumber production for 2006 was projected to be approximately 1900 million kg/yr (Stutz et al., 2003). This assumes that all the nanosilver in plastic incorporati ng NSPW-L30SS is released during a year even though results from the plastic leaching study indi cate that little silver is released from plastic incorporating NSPW-L30SS (i.e., <0.042 % in 10 days, see Section 5.2.1) and that this release is likely even lower in less porous plastics than the LLDPE plastic used in the study.

 Release Days: 365 days per year. This assumes that each plastic incorporating NSPWL30SS releases all its silver as nanosilver ove r the course of one y ear (i.e., 0.274% leaches out per day) even though not all in-service pl astic lumber will be in contact with water every day.

 Wastewater Removal Efficiency: 0% to represen t direct release into aquatic environments from the plastic lumber use of plastics in corporating NSPW-L30SS (i.e., no removal of nanosilver in the run-off due to adsorption to soil).

 Speciation, Agglomeration and Sedimentation in the Surface Water Column: All of the silver released to surface water from a wast ewater facility was assumed to be in the nanosilver form and it was assumed that all of the nanosilver was retained in the water column (i.e., no removal of released silver fr om the water column). The model used does not include these fate mechanisms and theref ore surface water concentrations are expected be overestimated).

Down the Drain Model

The concentration of nanosilver in surface wate r resulting from the use of NSPW-L30SS in plastics was calculated using the Down the Dr ain Module of the Exposure and Fate Assessment Screening Tool (E-FAST model, version 2) . The following input values were used:

 Stream Dilution Factor: 1.0 or 20.1. These values are the 10 th and 50 th percentile values for the dilution that occurs during one day of lowest stream flow over a ten year period (1Q10) (U.S. EPA, 2007b).

 The toxicity value for nanosilver : 1.5 μg/L based on the 96-hr EC 50 value for Daphnia

magna

.

 Level of Concern for the RQ: The presumptiv e acute level of concern (LOC) is 0.05 for listed (i.e., endangered or threatened) aqua tic animals and 0.5 for non-listed animals.48 Table 19 – Risk Quotients for Nanosilver in Surface Water

Use Scenario Wastewater Treatment Removal A Stream Dilution Factor Surface Water Silver Concentration (μg/L) Acute Risk Quotient D Acute RQ Exceeds LOC?* Textiles (t-shirts) 85% 1.0 B 0.004 0.003 No 20.1 C 0.0002 0.0001 No Plastics (lumber) 0% E 1.0 0.014 0.009 No 20.1 0.001 0.0007 No *The presumptive acute LOC is 0.05 for listed anim al species and 0.5 for n on-listed animal species. A. Silver removed from wastewater during treatment befo re discharge to a water body (e.g., lake, river etc.). B. 10 th Percentile dilution factor for 1Q10 stream flow. C. 50 th Percentile dilution factor for 1Q10 stream flow. D. Acute RQ = Surface Wate r Concentration / 96-h EC 50 for D. magna (1.5 μg/liter) E. Assumes direct discharge to surface wate r with no rete ntion in soil or sediment. The down-the-drain modeling results are shown in Table 19 and these results were divided by the 96-hr EC 50 of 1.5 μg/L for D. magna to obtain acute risk quotients (RQs ). The effect level used to calculate the acute RQs (i.e., 1.5 ug/L) was chos en to represent the most sensitive aquatic organism D. magna and to represent conditions that are representative of su rface water in the United States. The acute RQ of 0.009 at the worst- case stream dilution factor of 1.0 indicates that it is unlikely that the registration of NSPW-L30SS as a plastics and textiles preservative will lead to adverse effects for listed or non-listed aqua tic organisms during the four year period of conditional registration. However, this assessmen t does not consider long-term exposures or exposure to estuarine and marine species. 6.3.2 Conclusions for Aquatic Risk The acute RQs for D. magna exposed to nanosilver ranged from 0.0001 to 0.009, which are not of concern for listed or non-listed organisms because they were less than the acute LOC of 0.5 and 0.05, respectively. The effect level used to calculate the acute RQ s (i.e., 1.5 μg/L) was chosen to represent the most sensitive aquatic organism D. magna and to represent conditions of MHRW, which is a test medium representative of surface water in the United States. This analysis assumes that 1% of the projected yearly production of plastic lumber in the U.S. would contain NSPW-L30SS at 0.003% resultin g in a nanosilver production-volume of 570 kg/year. This is likely an overest imate given that the total mass of silver distributed as a material preservative in the U.S. during 2009 was less th an 6,800 kg, based on EPA records. This analysis also assumes that all the nanosilver in plas tic lumber incorporating NSPW-L30SS would be released into the environment during a one year period (i.e., 0.274% leaches out per day) when in fact the silver in the leaching study for plastic coupons was below the an alytical LOD indicating that significantly less silver would be released from plastic lumber incorporating NSPW-L30SS (see Section 5.2.1; <0.042% would leach out in 10 days or <0.0042% daily). This assumption also requires nanosilver to migrate from the interior of the plastic at the same or higher rate daily from the interior to the exterior of the plastic lumber to replenish nanosilver for leaching availability the next day. There are several uncertainties regarding the ex posure portion of this evaluation. The production volume of NSPW-L30SS is unknown si nce this product is not curren tly available in the U.S. Because of this, EPA made the assumption that 1% of all plastic lumber produced every year in the U.S. would contain 0.003% NSPW-L30SS. The na nosilver that could be released from the lumber incorporating NSPW-L30SS was assumed to directly enter the aquatic environment without any removal in soil or sediments. The rate at which nanosilver ente red the sanitary sewer49 systems was assumed to be constant in the dow n-the-drain model, which does not account for variability in the distribution of plastic produc ts incorporating NSPW-L 30SS among other factors. The ecotoxicity values shown in Table 17 are for acute effects and mortality, and there is only one study on the long-term exposure to nanosilver. Th e longer-term exposure study is not with a sensitive aquatic species and does not provide a chronic apical endpoi nt lower than the acute data used in the risk assessment for the most acute ly sensitive species. Exposure models did not account for reduction in exposure due to removal a nd sorption of nanosilver particles to organic matter in the water column. The acute daphnid results in Table 17 are from tests where the daphnids were not fed during the study per standard test guidance for this species (e.g., OPPTS 850.1010, OECD 202). In tests with silver salts, chronic NOEC endpoints on a total-s ilver basis for daphnid have been found to be higher than acute endpoints, this has been attributed to the pr esence of food in the long-term exposure studies as compared to the acute (USE PA Ambient Water Quality Criteria for Copper 1980, 1987). When daphnids are fed during the silver salt acute tests, acute toxicity has been observed to be reduced (2.9 to 40 times less toxi c) (USEPA 1980). Similar decreases in acute toxicity have been observed for ot her sensitive cladoceran species ( Ceriodaphnia reticulata, Daphnia pulex ) when fed (USEPA 1987). While EPA has not reviewed more current public literature on acute toxicity for silver salts for acceptability for use in a risk assessment, open literature reviews of available silver toxicity data based on total recoverable silver demonstrate this pattern has also been observed in later studies with D. magna . (Ratte 1999). This difference has been attributed to the presence of food providi ng a mitigating effect (i.e., food binds silver, rendering it less bioavaila ble). Organic matter in the water column may potentially reduce the acute toxicity, on a total recovera ble silver basis, of nanosilver. Given that all calculated RQs were below th e presumptive LOC for listed species and the conservative assumptions used in calculating these RQs, EPA conclude s, with a level of confidence acceptable for the period of conditional registration, that there is a low probability of adverse risk to the environment from plas tics and textiles inco rporating NSPW-L30SS. 6.4 Ecological Effects and Environm ental Fate Data Requirements. Even though EPA does not anticipate adverse eco logical effects from plastics and textiles incorporating NSPW-L30SS during the period of conditional registrati on, EPA is requesting additional testing to confirm its conclusions. The testing is base d on a tiered approach. Tier I studies will determine the nature and quantity of silver released from plastics incorporating NSPW-L30SS and confirm that the nanosilver in NS PW-L30SS is consistent with the risk from Table 19. The Tier I environmental fate testing categories include:

 NSPW-L30SS Characterization Testing

 Release Characteristics/Exposure which includes Silver Release from Plastics: Form, Rate and Characteristics The Tier I effects testing categories to confirm assumptions and include:

 Health Effects (Inhalation Studies)

 Ecological Effects (acute daphnid study). Completion of these Tier I studies will pr ovide NSPW-L30SS product characteristics and determine the substance or substances releasedduring leaching of plastics incorporating NSPWL30SS. Information from these studies will be us ed to substantiate NSPW-L30SS’s claim that no nanosilver is released from plastics incorporati ng NSPW-L30SS during use. However, if50 nanosilver is found to be released during Tier I studies, and the RQ calculated using this release rate results in a risk concern or is within one order of magnitude of the LOC, then Tier II studies will be required. If necessary, the Tier II studies will provide quantitative toxicity data on the amounts and forms of silver released, which will th en be used by EPA to re-run the environmental risk assessment for NSPW-L30SS. The Tier II envi ronmental fate and ecological effects testing categories include:

 Product Characterization

 Sorption/Desorption Characteristics

 Bioaccumulation Characteristics

 Impacts to Wastewater Treatment

 Wastewater Treatment Removal Efficiency

 Aquatic Plant testing

 Acute Effects to Freshwater Fish

 Chronic Effects to Freshwat er Fish and Invertebrates

 Acute and Chronic Effects to Estuarine/Marine Animals

 Chronic Effects to Sedi ment Dwelling Organisms

 Terrestrial Plant Toxicity A more detailed description of these data requirements is provided in Appendix B. VII. Regulatory Action A registration for a new active ingredient may be granted under either sec tion 3(c)(5) or section 3(c)(7)(C) of FIFRA. A FIFRA section 3(c)(7)(C) re gistration is appropriat e where, as here, the data for registration of the new active ingr edient are newly required or identified as needing adaptation to fit nanosilver. As discussed more thoroughly below, because EPA had not reached a final decision with regard to which types of da ta would be further requi red, the data requirements for this registration are c onsidered newly required. In addition, we believe granting the conditiona l registration with the terms and conditions identified in Appendix B is particularly appr opriate given EPA suspects that some alreadyregistered like-situated products on the market c ontain nanosilver as the active ingredient. While EPA approved these registrations without knowledge that these products may contain nanoscale silver and without specifically assessing any poten tial risks that might be associated with any nanosilver contained in those pr oducts, they are nonetheless on the market. To avoid disparate treatment and consistent with the analytic decisi on framework in this document, EPA is seeking similar data along with comparable terms on alre ady registered products that are identified to contain nanosilver. As part of this effort, EPA opened the Nanosilver Regi stration Review (docket EPA-HQ-OPP-2011-0370) on July 6, 2012, which is the fi rst step towards issu ing a Data Call-In for products containing nanosilver. 7.1 Legal Framework The Federal Insecticide, Fungici de, and Rodenticide Act (FIFRA) section 3(c)(7)(C) provides that: The Administrator may conditionally register a pesticide contai ning an active ingredient not contained in any currently re gistered pesticide for a peri od reasonably sufficient for the generation and submission of required data (whi ch are lacking because a period reasonably sufficient for generation of the data has not el apsed since the Administ rator first imposed the data requirement) on the condition that by the en d of such period the Administrator receives such data and the data do not meet or exceed ri sk criteria enumerated in regulations issued51 under this Act, and on such other conditions as the Administrator may prescribe. A conditional registration under this su bparagraph shall be granted only if the Administrator determines that use of the pesticide during such period will not cause any unreasonable adverse effect on the environment and that use of the pe sticide is in the public interest. Therefore, EPA must make four findings in or der to grant a section 3(c)(7)(C) conditional registration for a pesticide product containing a new active ingredient:

1. NSPW-L30SS contains nanosilver as an activ e ingredient and the nanosilver in NSPWL30SS is not in any currently registered pesticide; 2. Use of NSPW-L30SS will not cause unreasonable adverse effects on the environment during the period when newly require d data are being developed; 3. Insufficient time has elapsed for the newly required data to be generated and submitted; and 4. Use of NSPW-L30SS is in the public interest. 7.2 Findings and Registration Decision EPA is granting a conditional, four year regist ration for NSPW-L30SS in accordance with FIFRA section 3(c)(7)(C) and is requiring as a condition of registration speci fied use conditions as well as data from a number of studies th at will allow EPA to refine its current assessment of the risks. Specific data requirements are outlined in A ppendix B. Although these data requirements are specific to NSPW-L30SS, they form a starting po int for identifying the types of data EPA will require for other nanomaterial-based products on a case-by-case basis.

Consistent with Nanosilva LLC’s application, label language is required to reduce potential exposures. The pesticide label must include the following:

1. The application rate must be less than 30 pp m (mg/kg) or 0.003% silv er on a weight basis for plastics and textiles whic h incorporate NSPW-L30SS. 2. NSPW-L30SS may only be incorporated into linear low-density polyethylene (LLDPE) plastic and polyethylene terephthalate (PET) based textiles consistent with the materials used in the leaching studies. 3. NSPW-L30SS may only be used to preserve plas tics and textiles and may not be used for drinking water or food contact uses. 4. It is required that the following mitigation measures be employed when mixing and loading NSPW-L30SS during treatme nt of articles:

 Closed system loading of NSPW -L30SS containing suspension.

 NIOSH certified full-face respirators with P100 or equivalent filter cartridges immediately available for use in an emergency.

 Gloves which are chemically resistant to all of the components of the NSPWL30SS liquid suspension.

 A long-sleeve shirt, long pants, shoes plus socks.

5. The label must have an environmental haza rd statement consisting of the following: “This pesticide is toxic to fish, aquatic i nvertebrates, and birds. Do not discharge effluent containing this product into lake s, streams, ponds, estuaries, oceans, or other waters unless in accordance with the requirements of a National Pollutant Discharge Elimination System (NPDES) permit and the permitting authority has been notified in writing prior to discharge. Do not discha rge effluent containing this product to sewer systems without previously notifying the local sewage treatment52 plant authority. For guidance contact your St ate Water Board or Regional Office of the EPA.” 7.3 Basis for Conditional Registration

EPA’s basis for the conditional registration is as follows:

7.3.1 Data Generation Nanosilva LLC submitted its registration application thinking its product was a new pesticide ingredient not contained in any currently registered silver-based pesticide product and should be given a registration under FIFRA sec tion 3(c)(5). At the time of application for a new chemical, Nanosilva LLC was required to submit all the app licable information and studies under 40 CFR 161. Although Nanosilva LLC submitted some of the required studies using guidelines not modified to be applicable for testing nanosilver, th ey also submitted a request to waive some of the data requirements. In addition, Nanosilva LLC co mpleted a leaching study using a protocol that was modified to be applicable for nanosilver . Although Nanosilva LLC provided EPA with some data in an effort to address Agency questions and concerns, EPA had not reached a final decision with regard to which types of data would be fu rther required for NSPW-L30SS. This was due in large part to the need to understand and appl y the advice provided in the report from the consultation with the FIFRA Scientific Advisory Panel. As a result, EPA has determined that insufficient time has elapsed from the point at which EPA determined and informed Nanosilva LLC of the data requirements needed to assess Nanosilva LLC’s application for Nanosilva LLC to have generated the data. EPA is requiring the tests listed below to account for potential physiochemical differences of the nanosilver and because there are currently no acceptable studies on the reproductive and developmental toxicity for nanosilver. Because th is list of data requirements are only being imposed with today’s action, Nanosilva LLC has not yet been able to conduct these studies. Therefore, EPA is requiring these studies as a co ndition of registration, a llowing sufficient time for the study protocols to be agreed upon, the studies to be conducte d, and for EPA to review them. The required studies are:

 90-Day Inhalation Toxicity (Rat) (OCSPP GL N 870.3465) modified to include a functional observational battery, motor activ ity and detailed neuropathology

 Reproduction/Developmental Toxicity Screening Test via the inhalati on route. (Modified OCSPP GLN 870.3550/ OECD TG 421)

 Acute Freshwater Invertebrate Testing using Daphnia magna

 Product Characterization and Testing

 Dissolution Kinetics

 Silver Release from Plastics: Form, Rate and Characteristics

EPA will evaluate the results from the above stud ies using a weight of evidence approach based on physical and chemical properties, toxicity, and ex posure. If EPA identifies a possible concern, then one or a combination of the fo llowing tests will be triggered:

 Peri- and post-natal exposure to NSPW-L30SS

 Product Characterization

 Sorption/Desorption Characteristics

 Bioaccumulation Characteristics

 Impacts to Wastewater Treatment

 Wastewater Treatment Removal Efficiency53  Aquatic Plant testing

 Acute Effects to Freshwater Fish

 Chronic Effects to Freshwat er Fish and Invertebrates

 Acute and Chronic Effects to Estuarine/Marine Animals

 Chronic Effects to Sedi ment Dwelling Organisms

 Terrestrial Plant Toxicity As discussed above, a listing of the studies that are required as terms and conditions of this registration of NSPW-L30SS are in Appendix B. If Nanosilva LLC does not meet the conditions set forth in this Decision Document, EPA will is sue a notice of intent to cancel Nanosilva LLC’s registration under section 6(e). In addition, Nano silva LLC’s conditional registration for NSPWL30SS will automatically expire four years afte r being issued. Nanosilva LLC should request an amendment to remove the expiration date once they have submitted the required data if they wish to continue to sell and distribute NSPW-L30SS in the United States.

7.3.2 Public Interest As required under FIFRA section 3(c)(7)(C), fo r the reasons summarized below, EPA believes granting a conditional registration with the terms a nd conditions specified is in the public interest. Concluding that granting the regi stration is in the public interest is not an endorsement of the product. Under its authority set forth in FIFRA section 3(c)(7)(C), EPA is making only a public interest finding, which is provided for informati onal purposes only and not to be inferred as any endorsement of a pesticide product. EPA and its employees do not endorse any particular product, service or enterprise. If any re gistrant were to include "any st atement directly or indirectly implying that the pesticide or device is recomm ended or endorsed by any agency of the Federal Government," then such statement, pursuant to 40 CFR 156.10(a)(5)(v), would be false or misleading. EPA believes that use of NSPW-L30SS offers the potential for conservation of the environment which contribute to the public interest . These points are discussed in more detail below:

Conservation of the Environment EPA has already registered a number of silver-b ased pesticide products for use as materials preservatives. All silver-based pesticide products act via the release of a low concentration of silver ions that then interact with bacteria. Co mmonly, regardless of the s ilver additive type, upon contact with moisture, silver ions are released from the object incorporating the additive. The antimicrobial potency of a silver additive is theref ore directly related to th e potential for releasing silver ions. The release potential differs between various silver ma terials. As the size of silver particles decreases (e.g., from micr o-size silver to nano-size silve r), the potentia l for releasing silver ions increases, due to the increasing unit of su rface area (i.e., availability of ions for release) per unit mass of silver. Specifically, most silver-based pe sticide products curren tly contain a silver salt, [e.g., AgCl or AgNO 3 ]. Compared to the amount of silver in Nanos ilva LLC’s product, most currently registered silver-based materials preservatives incorporate larger amounts of silver (i.e., NSPW-L30SS is applied at a lower rate). Furthermore, the nanosilver in NSPW-L30SS is co mplexed to a larger particle that is then embedded into a polymer. The submitted leaching studies show that nanosilver is unlikely to be released in quantities above the detection limit from this polymer. Absent release from the polymer, the mobility of nanosilver in NSPW-L30SS is greatly reduced. Taking into consideration54 the low application rates, low mobility, and the exp ected gradual and controlled release of silver ions from nanosilver as opposed to the rapid releas e of silver ions from a zeolite structure or the immediate dissolution of a silver salt, EPA believes there is potential for reduced environmental loading and likely reduced environmental risk.

7.3.3 No Unreasonable Adverse Effects In assessing the potential risks to human health and the envi ronment associated with the distribution and use of NSPW-L30SS as a material s preservative, EPA relied on data submitted by Nanosilva LLC and data in the public literature on nanosilver, and use of uncertainty factors and conservative assumptions. As a result of this analysis and taking into account the terms and conditions on this conditional re gistration, EPA believes that the likely risks from the use of NSPW-L30SS during the period of the conditional re gistration are small. Moreover, EPA expects the overall quantity of silver used in plastics and textiles as a result of th is conditional registration will be lower than the overall qua ntity of silver used in othe r materials preservative products containing conventional silver resul ting in expected reductions in e nvironmental loadings of silver and to humans. EPA concludes th at the registration would not cau se unreasonable adverse effects on the environment during the conditional period . This conclusion is based on the findings discussed in the following sections.

Risks to Human Health As discussed above, humans could be exposed to silver ions, to NSPW-L30SS, and to nanosilver released from NSPW-L30SS. With respect to silv er ions, EPA completed a risk assessment (U.S. EPA, 1993) for silver salts and determined that the risks from registered uses, including the materials preservative use pattern, were acceptable. EPA concludes that hu man exposure to silver ions from the use of NSPW-L30SS and consumer exposures to plastics a nd textiles incorporating NSPW-L30SS is not unreasonable. With respect to NSPW-L30SS, the acute toxici ty of NSPW-L30SS is low by all routes of exposure. Because there are no subc hronic or chronic oral or dermal toxicity studies available for NSPW-L30SS or on the nanosilver that might br eak away from products incorporating NSPWL30SS, the subchronic or chronic toxicities were estimated using studies on nanosilvers reported in the literature. Because of the potential difference between the nanosilver in the available toxicity data and the nanosilver in NSPW -L30SS along with the lack of an acceptable developmental and reproductive toxicity study for nanosilver, EPA used a maximum 10-fold database uncertainty factor when evaluating the risk from exposure to NSPW-L30SS and the nanosilver released from produc ts incorporating NSPW-L30SS. To evaluate occupational exposure to the nanosilv er that might break away from NSPW-L30SS, EPA calculated the daily-dose to workers assuming that all the silver in NSPW-L30SS was freely available as nanosilver. EPA be lieves that this assumption ove restimates the daily-dose of nanosilver that a person coul d potentially receive when working with NSPW-L30SS. To assess consumer exposures to treated textil es and plastics, EPA re lied on the results from studies submitted by Nanosilva LLC where the liqui ds in contact with plastic and shirts incorporating NSPW-L30SS were analyzed for silv er content to estimate exposure the amount of NSPW-L30SS and nanosilver that mi ght be released from plastic s and textiles. EPA recognized the uncertainties and incorporated multiple uncertainty factors and conservative assumptions when calculating the Margin of Exposures (MOEs) for es timating the risk to children from plastics and textiles incorporat ing NSPW-L30SS.55 Based on all of the foregoing, EPA concludes that use of NSPW-L30SS will not cause unreasonable adverse effects on the environment dur ing the period when newly required data are being developed.

Risks to the Environment The environment could be exposed to silver ions, to NSPW-L30SS, and to nanosilver released from NSPW-L30SS. With respect to silver ions , EPA completed a risk assessment (U.S. EPA, 1993) for silver salts and determined that the ri sks from registered uses , including the materials preservative use pattern, were accep table. EPA concludes that environmental exposure to silver ions from the use of NSPW-L30SS and plastic s and textiles incorporating NSPW-L30SS is acceptable. With respect to the fate and eco toxicity of NSPW-L30SS, because there are no fate or ecotoxicity studies available for NSPW-L30SS or the nanosilver that might break away from plastics or textiles incorporating NS PW-L30SS, the fate and ecotoxicity were estimated using studies on nanosilvers reported in the scientific literature. Direct release from leaching of plastics and laundering of textiles incorporating NSPW-L30SS were anticipated to be the primary route by which NSPW-L30SS, silver ions, and nanosilver re ach the environment. EPA assessed impacts to surface waters assuming that nanosilver was the only compound released. Environmental exposure from indoor use was assessed assuming that 300 million people (U.S. population) each purchased one t-shirt containing 0.003% nanos ilver where 1.6% of the silver in the t-shirt incorporating NSPW-L30SS was released during each weekly washing over a one year period. Environmental exposure from plastic lumber use was assessed assuming that 1% of the projected yearly production of plastic lumber in the U.S. w ould contain NSPW-L30SS at 0.003% where all the silver would be released into the environment as nanosilver during a one year period even though silver was released below the LOD in the leach ing study for plastic coupons incorporating NSPWL30SS. EPA estimates, derived from modeling, of the concentrations of nanosilver resulting from the use of NSPW-L30SS do not exceed EPA’s estimat e of the highest concen tration of nanosilver in surface water to which an aquatic community can be expo sed without resulting in an unacceptable effect. Thus, EPA concludes that use of NSPW-L30SS will not cause unreasonable adverse effects on the environm ent during the period when newly required data are being developed.56 

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Critical Observati ons on the Neurotoxicity of Silver. Critical Reviews in58 Toxicology 37:237-250. Lankveld, D.P., Oomen, A.G., Krystek, P., Nei gh A, Troost-de Jong A, Noorlander CW, Van Eijkeren JC, Geertsma RE, De Jong WH. 2010. Th e kinetics of the ti ssue distribution of silver nanoparticles of different sizes. Biomaterials 31:8350-8361. Larese, F.F., D'Agostin, F., Crosera, M., et al., 2009. Human Skin Penetration of Silver Nanoparticles through Intact and Damaged Skin. Toxicology 255(1-2):33-7. Lee, J.M., Kim, D.W., Kim, T. H., Oh, S.G. 2007. Facile route fo r preparation of silica-silver heterogeneous nanocomposite partic les using alcohol reduction method. Materials Letters 61:558-1562. (MRID 48379902 and 48379904) Lee, J.M., Kim, D.W., Jun, Y.D., Oh, S.G. 2006. Preparation of silica-silver heterogeneous nanocomposite particles by one-pot prepara tion strategy using pol yol process: Sizecontrolled immobilization of silver nanoparticles. Materials Research Bulletin 41:1407- 1416. (MRID 48379901 and 48379903) Levard, C., Reinsch, B.C., Michel, F.M., et al. 2011. Sulfidation Processes of PVP-Coated Silver Nanoparticles in Aqueous Solution: Impact on Dissolution Rate. Environmental Science and Technology 45:5260-5266. Li, Y., Chen, D.H., Yan, J., Chen, Y., et al. 2012. Genotoxicity of silver nanoparticles evaluated using the Ames test and in vitro micronucleus assay. Mutation Research 745:4-10. Li, Y., Bhalli, J.A., Ding, W., et al. (2013) Cyto toxicity and genotoxicity assessment of silver nanoparticles in mouse. National Center fo r Toxicological Research, USFDA, MRID 49291901. Nanotoxicology, early online Liu P, Huang Z, and Gu N 2013. Exposure to silver nanoparticles does not affect cognitive outcome or hippocampal neurogenesis in adu lt mice. Ecotoxicol Environ Saf. Jan;87:124- 30. doi: 10.1016/j.ecoenv.2012.10.014. Epub 2012 Nov 10. Liu Y, Guan W, and Ren G, et al. 2012. The possi ble mechanism of silver nanoparticle impact on hippocampal synaptic plasticity and spatial cognition in rats. T oxicology Letters, 209: 227- 231. Liu, J., Hurt, R.H. 2010. Ion Releas e Kinetics and Particle Persis tence in Aqueous Nano-silver Colloids. Environmental Science Technology 44:2169-2175. Liu, J., Sonshine, D.A., Shervani, S., Hurt, R. H. 2010. Controlled Release of Biologically Active Silver from Nanosilver Surfaces. ACS Nano 4:6903-6913. Liu, Z., Ren, G., Zhang, T., Yang, Z. 2009. Action Potential Changes Associated with the Inhibitory Effects on Voltage-Gated Sodi um Current of Hippocampal CA1 Neurons by Silver Nanoparticles. Toxicology 264(3):179-84. Loeschner et al. 2011. Distribution of silver in rats following 28 days of repeated oral exposure to silver nanoparticles or silver acetate. Particle and Fibre Toxicology , 8:18 Lorenz, C., Windler, L., von Goetz, N. et al. 2012. Characterization of silver release from commercially available functional (nano) textiles. Chemosphere 89:817-824. Mahabady, M.K. 2012. The evaluation of teratogeni city of nanosilver on skeletal system and placenta of rat fetuse s in prenatal period. African Journal of Pharmacy and Pharmacology 6: 419-424. Moiemen, N.S., Shale, E., Drysdale, K.J., et al . 2011. Acticoat dressing an d major burns: Systemic silver absorption. Burns 37:27-35. Mei et al. 2012. Silver Nanoparticle-Induced Muta tions and Oxidative Stre ss in Mouse Lymphoma Cells. Environmental and Mo lecular Mutagenesis 53: 409-419. Mühling, M., Bradford, A., Readman, J.W., Some rfield, P.J., Handy, R.D. 2009. An Investigation into the Effects of Silver Nanoparticles on Antibiotic Resistance of Naturally Occurring Bacteria in an Estuarine Sediment. Marine Environmental Research 68:278-283.59 NIOSH. 2009. Approaches to Safe Nanotechnology: Managing the Safety and Health Concerns Associated with Engineered Nanomater ials. DHHS (NIOSH) Publication No. 2009-125 Park, E.J., Bae, E., Yi, J., et al. 2010. Repeated -Dose Toxicity and Inflammatory Responses in Mice by Oral Administration of Silver Nanoparticles. Environmental Toxicology and Pharmacology 30: 162-168. Ratte, H. 1999. Bioaccumulation and toxicity of silver compounds: a review. Environmental Toxicology and Chemistry 18(1): 89-108. Song et al. 2012. Recovery from silver-nanopartic le-exposure-induced lung inflammation and lung function changes in Sprague-Dawley rats. Nanotoxicology doi:10.3109/17435390.2011.648223 Stutz, J., Donahu, S., Mintzer, E., et al. 2003. Pl astic Lumber in Landscape Applications. Tellus Institute, Boston, MA. Available at: www.epa.gov/osw/conserve/rrr/g reenscapes/pubs/lumber.pdf Sung, J.H., Ji, J.H., Park, J. D., et al. 2009. Subchr onic Inhalation Toxicity of Silver Nanoparticles. Toxicological Sciences 108:452-461. (MRID 477575-05) Tang, J., Xiong, L., Wang, S. et al. 2009. Distributi on, translocation, and accumulation of silver nanoparticles in rats. J. Nanoscience Nanotechnology 9(8):4924-32. The Minimum Information for Nanomaterial Characterization (MIN Char) Initiative, 2008. Available at : http://characteriza tionmatters.files.wordpr ess.com/2008/11/mincharparameters-list.pdf Trop, M., Novak, M., Rodl, S. et al., 2006. Silver-C oated Dressing Acticoat Caused Raised Liver Enzymes and Argyria-like Sy mptoms in Burn Patient. Journal of Trauma- Injury Infection and Critical Care 60:648-652. Van der Zande et al. 2012. Distribution, Elimina tion, and Toxicity of Silver Nanoparticles and Silver Ions in Rats af ter 28-Day Oral Exposure. ACS Nano DOI: 10.1021/nn302649p. U.S. EPA, 1980. Ambient Water Quality Criteria for Silver. Office of Water, Regulations and Standards, Criteria and Standards Division, Washington, DC. EPA 440/5-80-071. U.S. EPA, 1987. Ambient Aquatic Life Water Qualit y Criteria for Silver. Office of Research and Development, Environmental Research Laborat ories, Duluth, MN and Narragansett, RI. EPA 440/5-87-011. U.S. EPA, 1993. RED Fact Sheet for Silver , EPA-738-F-93-005. EPA Office of Pesticide Programs, June 1993. U.S. EPA, 1998. PHED Surrogate Exposure Guid e, EPA Office of Pesticide Programs, August 1998, available at: http://www.epa.gov/pesticides /science/phedsur-expo-guide.pdf U.S. EPA, 2000. Assigning Values to Non-Detect ed/Non-Quantified Pesticide Residues in Human Health Food Exposure A ssessments. Available at: http://www.epa.gov/pesticid es/trac/science/trac3b012.pdf U.S. EPA, 2001. General Principles for Perfor ming Aggregate Exposure and Risk Assessments. November 28, 2001. Available at: http://www.epa.gov/opp00001/trac/science/aggregate.pdf U.S. EPA, 2002. A review of the Reference Do se and Reference Concentration Processes. EPA/630/P-02/002F. December. U.S. EPA. 2005. Guidance on Selecting Age Groups for Monitoring and Assessing Childhood Exposures to Environmental Contam inants. EPA/630/P-03/003F. November, 2005. U.S. EPA, 2007a. Method 6010C, Inductively Coupl ed Plasma-Atomic Emission Spectrometry. In EPA SW846: Test Methods for Evaluating Solid Waste, Physical/Chemical Methods. U.S. EPA, 2007b. Exposure and Fate Assessm ent Screening Tool (E-FAST), Version 2.0, Documentation Manual. Oct ober, 2007. Available at:60 http://www.epa.gov/oppt/exposure/pubs/efast2man.pdf U.S. EPA. 2008. Child-Specific Exposure Fact ors Handbook. National Center for Environmental Assessment. Washington, DC; EP A/600/R-06/096F. September. U.S. EPA. 2011a. Decision Document, Conditional Registration of HeiQ AGS-20 as a Materials Preservative in Textiles. December 1, 2011. U.S. EPA. 2011b. Exposure Factors Handbook: 2011 Edition. EPA/600/R-090/052F. September, 2011. U.S. EPA, 2011c. Technical Overview of Ecolog ical Risk Assessment, Risk Characterization. Risk Presumptions for Aquatic Animals. Available at: http://www.epa.gov/oppefed1/ ecorisk_ders/toera_risk.htm U.S. EPA, 2012a. Science Review of the AE ATF II Liquid Pour Hu man Exposure Monitoring Study. October 9. Available at: http://www. epa.gov/hsrb/files/meeting-materials/nov-1- 2012-public-meeting/AEATF-Liquid-Pour-Study.pdf U.S. EPA, 2012b. Standard Operating Procedures for Residential Pesticide Exposure Assessment. Health Effects Division, Office of Pesticide Programs, Office of Chemical Safety and Pollution Prevention. U.S. EPA, 2013. Part 158 Toxicology Data Requi rements: Guidance for Neurotoxicity Battery, Subchronic Inhalation, Subchronic Dermal and Immunotoxicity Studies. May 1 st . Available
at: http://www.epa.gov/pesticides/regulat ing/part158-tox-da ta-requirement.pdf Wan, A.T., Conyers, R.A.J., Coombs, C.J., et al. 1991. Determination of Silver in Blood, Urine, and Tissues of Volunteers and Burn Patients. Clinical Chemistry 37:1681-1687. Wang, Z., Liu, S.J., Ma, J. et al. 2013. Silver Nanoparticles Induced RNA Polymerase-Silver Binding and RNA Transcription Inhib ition in Erythroid Progenitor Cells. ACS Nano 7: 4171-4186. Wang, Y., Westerhoff, P., Hristovs ki, K.D. 2012a. Fate and biological effects of silver, titanium dioxide, and C60 (fullerene) nanomaterials during simulated wastewater treatment
processes. Journal of Hazardous Materials 201-202:16-22 Wang, Z, Chen, J., Li, J. et al. 2012b. Aquatic To xicity of Nanosilver Colloids to Different Trophic Organisms: Contributions of Particles and Free Silver Ion. Environmental Toxicology and Chemistry 31: 2408-2413. Wigginton, N.S., De Titta, A., Piccapietra, F., et al. 2010. Binding of Silver Nanoparticles to Bacterial Proteins Depends on Surface Modifi cations and Inhibits Enzymatic Activity. Environmental Science Technology 44:2163. Wu, Y., Zhou, Q. 2012. Silver Nanoparticles Caus e Oxidative Damage and Histological Changes in Medaka ( Oryzias latipes ) after 14 days of Exposure. Environmental Toxicology and Chemistry , 32:165-173. Yang, Y., Chen, Q., Wall, J.D., Hu, Z. 2012. Potentia l nanosilver impact on anaerobic digestion at moderate silver concentrations. Water Research 46: 1176-1187. Yu, Wook-Joon, Son, Jung-Mo, Lee, Jinsoo, et al . (2013) Effects of silver nanoparticles on pregnant dams and embryo-fetal development in rats. Korea Institute of Toxicology, MRID 49291902. Nanotoxicology early online: 1-7. Zartarian V.G., J. Xue, H.A. Ozkaynak, W. Dan g, G. Glen, L. Smith, and C. Stallings. (2005). A Probabilistic Exposure Assessment for Childre n Who Contact CCA-tr eated Playsets and Decks Using the Stochastic Human Exposure and Dose Simulation Model for the Wood Preservative Scenario (SHEDS-WOOD). Fi nal Report. U.S. EPA, Washington, DC, EPA/600/X-05/009.61 

### Appendix A – Evaluation of Nanos ilva LLC’s Data Waiver Requests

I. Introduction On August 10, 2009, Nanosilva LLC submitted an app lication to register NSPW-L30SS with a new nanosilver active ingredient (PRIA A420). In 2009, as an applicant for registration of a new active ingredient, Nanosilva LLC wa s required to submit all the applicable information and studies under 40 CFR part 161. EPA published a Notice of Proposed Rulemaking on October 8, 2008 proposing to revise and update the existi ng data requirements under 40 CFR part 161 for antimicrobial pesticides. These data requirement s were made final as 40 CFR part 158W on May 8, 2013 (78 FR 26936). In 2009, the Nanosilva LLC app lication included a request to waive the requirement to generate data for NSPW-L 30SS using the followi ng test guidelines:

1. Hydrolysis (OCSPP GLN 835.2120) 2. Aquatic Invertebrate Acute Toxicity Test , Freshwater Daphnids (OCSPP GLN 850.1010) 3. Fish Acute Toxicity Test, Freshw ater and Marine (OCSPP GLN 850.1075) 4. Avian Acute Oral Toxicity Test (OCSPP GLN 850.2100) 5. 90-Day Dermal Toxicity (OCSPP GLN 870.3250) 6. Prenatal Developmental Toxi city Study ( OCSPP GLN 870.3700) 7. Bacterial Reverse Mutati on Test (OCSPP GLN 870.5100) 8. Detection of Gene Mutations in Soma tic Cells in Culture (OCSPP GLN 870.5300) 9. in vitro Mammalian Cytogeneti cs (OCSPP GLN 870.5375) 10. Mammalian Bone Marrow Chromosome Aberration Test (OCSPP GLN 870.5385) 11. Immunotoxicity (OCSPP GLN 870.7800) The test guidelines in items 1 through 4 were required in 40 CFR part 161 at the time Nanosilva LLC applied to register NSPW-L30SS. Guidelines in items 5 through 11 were proposed in 2008 to be included in 40 CFR part 158W, which was finalized in 2013 and therefore, the test guidelines in items 5 through 11 were not requir ed at the time Nanosilva LLC applied to register NSPW-L30SS. Even though these guidelines were not required at the time of application, EPA evaluated the request to waive each of these data requirements.

Waivers for the above studies were requested because of the lack of acute toxicity noted in the six acute toxicity studies (M RID 47828918 through 47828923), because NSPW-L30SS has a specific gravity greater than water, because NSPW-L30SS ha s low solubility in water as determined by the water column generator method (MRID 47828917), a nd due to the non-leaching characteristic of NSPW-L30SS because silver leached below the L OD in leaching studies conducted with a plastic incorporating NSPW-L30SS (MRID 47828925). The data requirements listed above in item 1 and in items 3 through 11 are either waived or satisfied. In general, the data requireme nt is waived or satisfied because:

 certain guideline studies are not appropriate for nanosilver an d where appropriate are being modified and required as terms a nd conditions on the registration

 certain environmental data requirements are satisfied by using literature studies

 additional dermal studies are not needed base d on calculated margins of exposure (MOEs)

 in vitro and in vivo mutagenicity study requirements ar e satisfied and there is no concern for mutagenicity or genotoxicity

Each of the above studies is a ddressed in the Environmental Fate , Wildlife and Aquatic Organism, and Toxicology data requirements listed in 40 CFR part 161 for antimicrobial pesticides. The following sections contain an eval uation of each waiver request.62 A. Environmental Fate Data Requirements The following test for environmental fate is required for antimicrobials under 40 CFR 161.202 to assess the exposure to pesticides after application: 1. Hydrolysis, OCSPP GLN 835.2120 This test is waived because nanosilver undergoes dissolution with the gene ration of ionic silver rather than undergoing h ydrolytic transformation. B. Wildlife and Aquatic Organism Data Requirements The acute ecotoxicity testing waiver request for the following tests required under 40 CFR 161 was based on the non-leaching characteristic of NSPW-L30SS: 2. Aquatic Invertebrate Acute Toxicity Te st, Freshwater Daphni ds (OCSPP GLN 850.1010) 3. Fish Acute Toxicity Test, Fres hwater and Marine (OCSPP GLN 850.1075) 4. Avian Acute Oral Toxici ty Test (OCSPP GLN 850.2100) The results of these tests are required for use in determining the appropriat e precautionary labeling on packages containing NSPW-L30SS prior to inco rporating into a product. As summarized in Table 17, EPA has information on the acute toxicity of nanosilver for Daphnids and Freshwater Fish (items 2 and 3 above). Based on that information, because the EC 50 values were less than 1 mg/L, as indicated in Chapter 8 of the Pestic ide Label Review Manual, EPA is requiring the following environmental hazard statement on the NSPW-L30SS label: This pesticide is toxic to fi sh and aquatic invertebrates. Given that there are no test results for avian speci es available for nanosilver , EPA requires that the environmental hazard statement for the NSPW-L30SS label be expanded to include birds: This pesticide is toxic to fish, aquatic invertebrates, and birds. However, if Nanosilva LLC wishes to remove birds from this statement they can submit results from an Avian Acute Oral Toxicity Test (O CSPP GLN 850.2100) showing that the nanosilver in NSPW-L30SS is non-toxic to birds with an LD 50 of greater than 100 mg/kg. In addition, Nanosilva LLC is required to pla ce the following statement on the NSPW-L30SS label: “Do not discharge effluent containing this product into lakes, streams, ponds, estuaries, oceans, or other waters unless in accord ance with the requirements of a National Pollutant Discharge Elimination System (NPDES) permit and the pe rmitting authority has been notified in writing prior to discharge. Do not disc harge effluent containing this product to sewer systems without previously notifying the local sewage treatm ent plant authority. For guidance contact your State Water Board or Regional Office of the EPA”. EPA has waived the acute toxicity studies for fi sh and birds. The acute invertebrate study is required as a condition of registration to confirm the risk conclusions. C. Toxicology Data Requirements The following tests are required for antimicr obials under 40 CFR part 158.2230 (i.e., part 158W): 5. 90-Day Dermal Toxicity (OCSPP GLN 870.3250 6. Prenatal Developmental Toxicity Study (OCSPP GLN 870.3700)63 The requirement for the 90-Day Dermal Toxicity study is waived because the calculated MOEs for dermal and incidental exposure to products incorporating NSPW-L30 SS were greater than 1,000,000 as compared to the highest target MOE of 3,000. However, if nanosilver is determined to be released during the leaching test of pl astics incorporating NSPW-L30SS in amounts well above those found in the plastics leaching study already submitted such that the MOE calculated using the newly determined nanosilver release rate results in a possible risk concern, then the following route-specific subchroni c tests is required to confirm the risk assessment for the nanosilver in NSPW-L30SS:

 Dermal penetration study in rats (OCSPP GLN 870.7600) The requirement for the Prenatal Developmental Toxicity Study is waived because the following study has been determined to be more appropriate for NSPW-L30SS:

 Modified 870.3550/ OECD TG 421: Reproduction/ Developmental Toxicity Screening Test The following tests are required for pestic ides under 40 CFR part 158.2230 (e.g., 158W):

7. Bacterial Reverse Mutati on Test (OCSPP GLN 870.5100) 8. Detection of Gene Mutations in Soma tic Cells in Culture (OCSPP GLN 870.5300) 9. In Vitro Mammalian Cytogeneti cs (OCSPP GLN 870.5375) 10. Mammalian Bone Marrow Chromosome Aberration Test (OCSPP GLN 870.5385) 11. Immunotoxicity (OCSPP GLN 870.7800) The mutagenicity studies [Bact erial Reverse Mutation Test (OCSPP GLN 870.5100), Detection of Gene Mutations in Somatic Cell s in Culture (OCSPP GLN 870.5300), In Vitro Mammalian Cytogenetics (OCSPP GLN 870.5375), and Mamma lian Bone Marrow Chromosome Aberration Test (OCSPP GLN 870.5385)] are satisfied because there are in vitro studies available for nanosilver in the scientific literature which address/ satisfy these data needs. There are also several well-conducted in vivo literature studies, showing cytotoxici ty and evidence th at the nanoparticles reached the target tissue, suggesting that the in vitro mutagenic potential observed may be intrinsic to nanoparticles, but is not expressed in whole an imals. There is evidence that silver nanoparticles are genotoxic, expressing DNA damage in cultured human lung cells and in the lungs and liver of intact animals (comet assay) via intracellula r generation of reactive oxygen species (ROS). However, since initiation of ROS-induced oxidative DNA damage can be blocked by ROS scavengers, there is not a concern for mutagenici ty or genotoxicity and additional data are not needed. The immunotoxicity (OCSPP GLN 870.7800) is wa ived at this time. OPP has published a document (U.S. EPA, 2013) recommending that imm une related endpoints be evaluated during the inhalation toxicity test, required as a term and condition on the regi stration, before determining if the immunotoxicity test is required. The currently available oral toxicity studies indicate that nanosilver causes liver and kidney to xicity in laboratory animals where silver is distributed to all organs and tissues with accumulation of silver in the brain and male an imal testes. Inhalation toxicity studies also identified liver toxicity as well as lung e ffects including chronic alveolar inflammation. The toxicology database for nanosilv er does not reveal any evidence of treatmentrelated effects on the immune system, which suggest s that nanosilver does not directly target the immune system.64 

### Appendix B – Data Requirements, Enforcea ble Schedule, and Conditions of the Conditional Registration for NSPW-L30SS

I. Introduction NSPW-L30SS is a liquid suspension containing 1% nanosilver by weight where the nanosilver active-ingredient is bonded to amorphous silica via a thiolate bond. The diameter of the spherical silica core is 320 nm on av erage (Lee et al., 2006) and the nanosilv er particles that are attached to the silica core have mean diameters between 6.9 and 10.6 nm (Lee et al., 2007). The complex is formed by reacting spherical silica particles modified with thiol groups with silver nitrate and polyvinylpyrrolidone (PVP) in ethanol (Lee et al. 2007). II. Exposure Pathways The human and environmental exposures resulting from the use of NSPW-L30SS, and use and disposal of plastics and textiles incorporati ng NSPW-L30SS will largely be a function of what materials are available for inhalation or dermal exposure during treatment of plastics and textiles or what materials leach or break away from the plastics and textiles incorporating Nanosilva during use and disposal. EPA antic ipates that humans and the e nvironmental will potentially be exposed to the following materials:

1. Silver ions released from NSPW-L30SS; 2. NSPW-L30SS complex; and/or 3. Nanosilver that might break away from NSPW-L30SS. EPA expects that occupational inhalation a nd dermal exposures to NSPW-L30SS and the nanoparticles that might break aw ay from NSPW-L30SS are likely to occur during the following use scenarios :
4. Mixing and loading of Nanosilver duri ng preparation of a master batch 5. Mixing, loading, and applying the NSPW-L30SS containing master ba tch during treatment of plastics and textiles 6. Handling plastics and textil es incorporating NSPW-L30SS EPA expects consumer exposures to NSPW-L30SS, the silver ions derived from NSPW-L30SS, and the nanoparticles that br eak away from NSPW-L30SS coul d potentially occur during the following use scenarios :
1. Incidental oral exposure to plastics and textiles incorpor ating NSPW-L30SS 2. Dermal exposure to plastics and te xtiles incorporating NSPW-L30SS Leaching of plastics and textiles incorporating NS PW-L30SS is anticipated to be the primary route by which NSPW-L30SS, silver ions, and nanosilver reach the environment. The silver released during leaching of plastics and textiles incorporating NSPW-L30SS could be discharged to the sanitary sewer system leading to publically ow ned wastewater treatment and privately owned septic systems, also known as the down-the-d rain discharge scenari o. Leaching of building material such as plastic siding and decks and outdoor furniture incorporating NSPW-L30SS will be discharged directly into th e environment. Once NSPW-L30SS, s ilver ions, or nanosilver reach wastewater treatment and septic systems they w ill most likely complex with sulfide and partition to biosolids. However, some fraction of the silver compounds will reach surface water and may potentially impact aquatic organisms.65 III. Data Needed to Confirm the Estimate s for Risks of Exposure to NSPW-L30SS As a condition of the registration, EPA is requiri ng the Nanosilva LLC to conduct a number of studies, based on a tiered approach, which will allow EPA to confirm the findings of the risk assessment completed for the conditional regist ration and discussed in the decision document. These tests include Tier I studies to determine th e nature and quantity of silver released from plastics incorporating NSPW-L30S S under conditions of use, produc t characterization testing, and silver release characteristics from the NSPW-L30SS complex. In addition, the Tier I studies include inhalation toxicity studies and an acute daphnid toxicity st udy. If nanosilver is determined to be released from plastics during the leaching te sts as part of Tier I testing and the weight of evidence based on physical and chemical properties, toxicity, and exposure re sult in a risk concern then route-specific toxicity and environmental fate tests as part of Tier II studies to confirm the nanosilver risk assessment will be triggered. Data must be submitted within four years and according to the schedule provided in Table B3 to avoid cancellation of the conditional registration. The duration of four years was chos en to allow time for protocol re views prior to initiation of the studies, completion of the studies, and Agency re view of the studies following completion. EPA will evaluate these data as they are submitted du ring the period of the conditional registration to confirm EPA’s determination th at the product is not expected to cause unreasonable adverse effects to human health and the environment. If EPA determines that Nanosilva LLC has failed to take appropriate steps to initiate the required studie s, or failed to submit the protocols or studies, as required pursuant to this Appendix, EPA will issue a notice of intent to cancel Nanosilva LLC’s registration under FIFR A section 6(e). The following factors were considered in deve loping the data requirements for NSPW-L30SS:

 Submitted as a new active ingredient application, NSPW-L30SS was subject to the data requirements for the registration of antimicrobi al pesticides that are detailed in 40 CFR Part 161. These requirements include studies on physical and chemical characteristics, residue chemistry, environmental fate, toxicolo gy, reentry protection, spray drift, wildlife and aquatic organisms, plant protection, nont arget insects, and pr oduct performance.

 Although some studies, such as those dealing wi th physical and chemical characteristics, are required for all use patterns, many of the data requirements are conditional based on the use pattern. Information provided by Nanosilva LLC and information from the literature was used to tailor the data requirements to the use pattern.

 Additional studies in the area of physical and chemical ch aracterization that are not specifically included in 40 CFR Part 161 ar e needed because NSPW-L30SS contains nanosilver. These studies are needed because nanosized materials may have unique and new characteristics which are not present in the bulk or conventional materials. These characteristics have been recognized in th e FIFRA SAP Report (FIFRA SAP, 2009) and by the MINChar In itiative (2008). In addition, the following recomm endations from the FIFRA SAP re port were considered in the development of the data requirements as terms and conditions on the conditional registration of NSPW-L30SS:

 Both nano-sized particles of silver (Ag) as well as ionic silver (Ag + ) can contribute to toxic effects of nanosilver. The rate of silver i on production, as well as the distribution of nanosilver in tissues and the environment, ma y differ substantially between nanosilver and other forms of silver, as nanosilver can potentia lly deliver silver ions directly to specific66 tissues, cell membranes or inside cells – pla ces where other forms of silver compounds cannot reach. Therefore, the haza rd profile of nanosilver ma y differ from other forms of silver.

 Particle size can substantially impact particle properties, such as rate and concentration of silver ion release, reactivity and catalytic efficiency, plasmon resonance, and quantum effects. Smaller sized-particles are more eas ily taken up by organisms and are distributed more widely. Other physicochemical properties, such as shape, surface area, surface charge or coating, are also likely to impact bi ological response and environmental fate.

 The Panel “disagreed that nanosilver applied to a substrate will permanently bind with the substrate.” It is “especially challenging to determine that there is no release of nanomaterials from a substrate” under current state of science and available measurement standards. The Panel suggested that EPA require tests that simulate realistic use of products and potential nanosilver release along with quantitative life-cycle analysis and risk
assessment. A listing of the studies that are needed for the registration of NSPW-L30SS is included in Tables B1 and B2. The studies included in Table B1 are consid ered to be Tier I, meaning that their need is not based on the results of other st udies. The studies listed in Table B2 are considered to be Tier II, because they may or may not be required depend ing upon the results of the Tier I studies. NSPWL30SS and plastics/textiles incorporating NSPW-L 30SS will be the test material during Tier I studies and the test material for Tier II studies will depend on the results of the Tier I leaching and dissolution studies. Figure B1 contains a conceptual diagram outlining the Tier I and Tier II testing approach.67 Tier I – Testing

NSPW-L30SS Complex Characterization, Stability, and Release Characteristics Particle Size Distribution Particle Surface Area Particle Stability Dissolution Kinetics Occupation Inhalation Exposure 90-day Inhalation Toxicity with Neuro Repro/Developmental Screening by Inhalation Route

Silver Nanoparticle Ecological Effects

Figure B1 - Test material and tiered approac h for NSPW-L30SS and plastic incorporating NSPW-L30SS. See Tables B1 and B2 for the specific tests required for each material. Tier II

NSPW-L30SS Complex Ecological Effects Environmental Fate Tier I – Testing

NSPW-L30SS Treated Plastic

Weight o f evidence is based on physical and chemical properties, toxicity, and exposure. Lo w risk potential, high confidence in available data, p otential for no further testing.

Additional characterization o f toxicity needed.68 IV. Enforceable Schedule EPA has prepared an enforceable schedule (Table B3) that is an estimation of the time required for developing and submitting protocols for revi ew, conducting the studies, and submitting the resulting data, as well as EPA’s review of the submitted data. Technical issues may arise due to the unique nature of NSPW-L30SS, which may cause a delay in testing. In such a case, Nanosilva LLC shall submit a written request justifying the nature of the delay. In addition to technical delays, there may be delays by EPA in reviewing protocols and data submitted by Nanosilva LLC. In such a case, the EPA sha ll submit a written statement to Nanosilva LLC outlining the nature of the delay. In either case, if EPA determines a delay in the enforceable schedule is appropriate, it will amend the terms and conditions on the conditional registration. Notwithstanding technical delays or delays in review ing data, if Nanosilva LLC fails to take appropriate steps to initiate the required studies, or fails to submit the protocols or data, as required pursuant to this Appendix, EPA will issue a notice of intent to cancel Nanosilva LLC’s registration under FIFRA section 6(e) . Specifically, Nanosilva LLC shall: 1. Submit protocols modified for NSPW-L30 SS to EPA’s satisfaction for each of the data requirements listed in Tables B1 and B2. 2. Perform each test and submit the results as described in Tables B1 and B2. These items shall be submitted according to th e schedule provided in Table B3. If EPA determines that Nanosilva LLC has failed to initia te or to submit the required studies by the dates indicated in Table B3, then EPA will issue notice of intent to cancel Nanosilva LLC’s registration under FIFRA section 6(e). In addi tion, the conditional registration for NSPW-L30SS will automatically expire four years after being issued. All tests shall be conducted using laboratories that comply with the Good Laboratory Practice (GLP) standards of 40 CFR Part 160. In accordance with those regulations, the pesticide testing facility and the sponsor should comply with GLPs in the following areas:
1. Organization and personnel 2. Facilities 3. Equipment 4. Testing facilities operation 5. Test, control, and reference substances 6. Protocol for and conduct of a study 7. Records and reports EPA expects that all studies performed to s upport, among other things, the registration or continued registration of a pesticide will address those requirements. However, studies deviating from those requirements may be considered if, as set forth in 40 CFR 160 , a statement describing in detail all differences between the practices used in the study and those required by 40 CFR Part 160 is provided [see 40 CFR Part 160.12(b)]. As part of its review of the subject study, EPA will evaluate any such differences in determinin g if the study is acceptable for use in supporting, among other things, product regist ration or continued registration. V. Time-Limited Conditional Registration The conditional registration for NSPW-L30SS will automatically expire four years after being issued. Nanosilva LLC should request an amendm ent to remove the expi ration date once they have submitted the required data if they wish to continue to sell and distribute NSPW-L30SS in the United States.69Table B1 - Summary of Tier I Required Data for NSPW-L30SSOCSPP Data Requirement1Guideline Number: Study TitleReason for StudyTestMaterialCommentsNSPW-L30SS CharacterizationNon-Guideline: Particle Size and Diameter(size) DistributionRequired to characterizeproduct.NSPWL30SSDetermine both the size of the silica core particle and thenanosilver particles.Non-Guideline: Surface AreaDeterminationRequired to characterizeproduct.NSPWL30SSDetermine both the surface area of the silica core particleand the nanosilver particles.Non-Guideline: Particulate StabilityRequired to assess stability ofNSPW-L30SS.NSPW-L30SSNanosilva LLC claims that NSPW-L30SS is a stablecomplex that does not release nanosilver. This test shouldbe designed to demonstrate stability to mechanical stress(normal and shear) and common reactants and solvents.EPA will use this information to evaluate Nanosilva LLCclaims of stability.Release Characteristics/ExposureDissolution KineticsRequired to quantify the rate ofdissolution of the nanosilver inNSPW-L30SS.NSPWL30SSDissolution under environmental relevant conditions isneeded: moderate hard water at pH 5, 7, 9; soft water atpH 6.5; and an estuarine condition of 15 parts perthousand salinity.Non-Guideline: Plastic Leaching Study(Confirm Nanosilva LLC study results)Required to confirm assessmenton human and environmentalexposure. This test willdetermine what test substancewill be used for Tier II studies.NSPWL30SSTreatedPlasticsA leaching study is needed todetermine the quantity andform of silver that is released from NSPW-L30SS treatedplastics under conditions ofuse. Nanosilva LLC submitteda leaching study using food simulants and determined thetotal-silver concentration.EPA requires an additional,follow-up study using physiological fluids and methods todetermine the form of silver released from NSPW-L30SStreated plastic. The exposure data will be used in toconfirm EPA’s evaluation of the risk of exposure toNSPW-L30SS.Health Effects870.3465: 90-Day Inhalation Toxicity (Rat)(Replace Song et al., 2012 study results)Modified to include a functionalobservational battery, motor activity anddetailed neuropathologyRequired to account for the lackof inhalation data on thespecific form of nanosilverNSPWL30SSThe inhalation study is a route-specific study required toevaluate the effects of inhaling nanosilver when mixingand loading NSPW-L30SS. The guideline is modified toinclude a functional observational battery, motor activity,and detailed neuropathology to examine potentialneurotoxicological effects.70Table B1 - Summary of Tier I Required Data for NSPW-L30SSOCSPP Data Requirement1Guideline Number: Study TitleReason for StudyTestMaterialCommentsModified 870.3550/ OECD TG 421:Reproduction/Developmental ToxicityScreening Test*Required to account for the lackof reproductive anddevelopmental data on thespecific form of nanosilverNSPWL30SSThe combined repeated-dosetoxicity study with thereproduction/developmental toxicity screening test willprovide additional information on possible effects onreproduction and/or development. In addition, theinhalation study may also provide a toxicity endpointapplicable to a risk assessment for oral incidentalexposure.Ecological Effects850.1010: Aquatic Invertebrate AcuteToxicity, Freshwater DaphnidsRequired to confirmfindings/conclusions of theecological risk assessment.Nanosilverin NSPWL30SSThe plastic lumber scenario has an RQ within a magnitudeof the LOC for endangered aquatic animals. This study isrequired to confirm the conclusion that the nanosilver inNSPW-L30SS is not significantly more acutely toxic thannanosilvers from the open literature used to conduct therisk assessment.1These guidelines only provide general guidance. Protocols shall be submitted prior to conducting these studies.*The use of a combined study that utilizes the two-generation reproduction study in rodents as a basic protocol for the addition of other endpoints or functionalassessments in the immature animal is encouraged.71Table B2 - Summary of Tier IIRequired Data for NSPW-L30SSOCSPP Data Requirement1Guideline Number: Study TitleReason for StudyTest MaterialCommentsProduct ChemistryNon-Guideline: Plasmonresonance (UV-Visible Light)These studies are required tocharacterize the nanosilver ifreleased during Tier I stability,dissolution or leaching studies.Nanosilver in NSPWL30SSNon-Guideline: Particle Size andDiameter (size) DistributionNon-Guideline: Surface AreaDetermination830.7840: SolubilityNon-Guideline: Zeta Potential andSurface Charge DeterminationHuman Exposure –No Studies are Required in Tier IIHealth Effects870.7600: Dermal PenetrationChildren’s exposure tonanosilver while contactingNSPW-L30SS treated plastics(flooring).**Based on the results of the plastics leaching study,EPA may require additional toxicity data to assesspotential adverse health outcomes in young childrenresulting from dermal exposure to NSPW-L30SStreated plastics (flooring).Non-Guideline: peri- and postnatal exposure to NSPW-L30SSChildren’s exposure tonanosilver while contactingNSPW-L30SS treated plasticsand textiles.**Based on the results of the plastics leaching study,EPA may require additional toxicity data to assesspotential adverse health outcomes in young childrenresulting from incidental oral exposure to NSPWL30SS-treated plastic toys (i.e., children chewing ontoys).Ecological Effects850.1850: Modified Aquatic FoodChain TransferRequired to determinebioavailability andbiomagnification.**Traditionally use Fish and Oyster BCF to estimatebioaccumulation for chemicals, however, mesocosmtests more likely to yield useful information fornanomaterials. If nanosilver particles are not found tobe attached to or within aquatic plant cells in theaquatic plant, Tier II studies, and are not found withininvertebrates in the chronic whole sediment studiesthis study may potentially be waived.72Table B2 - Summary of Tier IIRequired Data for NSPW-L30SSOCSPP Data Requirement1Guideline Number: Study TitleReason for StudyTest MaterialComments850.4100: Terrestrial PlantToxicityRequired to determine effectsto plants during early criticalstages in their development.**If green algae (Selenastrum capricornutum) sensitivityis the same or less sensitive to other nanosilvers orsilver, EPA would consider a waiver request for thisstudy.850.4400: Aquatic Plant Toxicity,Tier II (Definitive Test)Required to determine thetoxicity to freshwater andaquatic plants.**Aquatic plants are the primary source of cellularcarbon and chemical energy for aquatic environments.Impacts to these primary producers would have broadimplications for the aquatic food chain.850.4500: Algal Toxicity, Tier II(Definitive Test)Required to determine thephytotoxicity to freshwater andmarine algae.**Algae are the primary source of cellular carbon andchemical energy for aquatic environments. Impacts tothese primary producers would have broadimplications for the aquatic food chain. Testing withthe green algae (S. capricornutum) is required toconfirm the findings/conclusions of the riskassessment. If theS. capricornutumresult is less toxicor of similar toxicity to the value used for aquaticplants in the risk assessmentor for silver for thisspecies then EPA would consider a waiver request forstudies with the other three functional species (afreshwater diatom (Navicula pelliculosa), a saltwaterdiatom (Skeletonema costatum), and a cyanobacteria(Anabaena flos-aqua) test).850.4550: Cyanobacteria Toxicity,Tier II (Definitive Test)Required to determine thetoxicity to an importantfunctional aquatic plant group.**Non-Guideline: Measuring theChronic Effects of FreshwaterSediment-AssociatedContaminants onChironomusdilutesRequired to determine chronicimpact to freshwater sedimentdwelling organisms.** If more than one testmaterial is identified fortesting, only the moretoxic material based ontheHyalella aztecaresultsis required for testing withC. dilutes.Silver nanoparticles if released into aquaticenvironments are likely to partition to sediment.Chronic tests on a freshwater benthic emergent insect(Chironomus dilutes,formerlyChironomus tentans)with epibenthic ecological niche will be used toestimate potential risks to freshwater benthicorganisms.73Table B2 - Summary of Tier IIRequired Data for NSPW-L30SSOCSPP Data Requirement1Guideline Number: Study TitleReason for StudyTest MaterialCommentsNon-Guideline: Measuring theChronic Effects of FreshwaterSediment-AssociatedContaminants onHyalella aztecaRequired to determine chronicimpact to freshwater sedimentdwelling organisms.**Silver nanoparticles if released into aquaticenvironments are likely to partition to sediment.Chronic tests on a freshwater benthic amphipod(Hyalella azteca) with infaunal ecological niche willbe used to estimate potential risks to freshwaterbenthic organisms.Non-Guideline: Measuring theChronic Effects of Marine andEstuarine Sediment-AssociatedContaminants onLeptocheirusplumulosusRequired to determine chronicimpact to marine sedimentdwelling organisms.** If more than one testmaterial is identified fortesting, only the moretoxic material based ontheHyalella aztecaresultsis required for testing withL. plumulosus.Silver nanoparticles if released into aquaticenvironments are likely to partition to sediment.Chronic tests on an estuarine/marine benthic amphipod(Leptocheirus plumulosus) will be used to estimatepotential risks to marine benthic organisms.850.1075: Acute Toxicity Testwith Freshwater and Marine FishRequired to confirm the acuteeffects of nanosilver.**Acute toxicity test with fishspecies is to help in theassessment of possible risk to similar species in naturalenvironments, as an aid in determination of possiblewater quality criteria for regulatory purposes, and foruse in correlation with acute testing of other speciesfor comparative purposes. Because fish have beendemonstrated to be less sensitive to variousnanosilvers than daphnia, if the acute freshwaterdaphnid results demonstrate that the NSPW-L30SSand its nanosilver are not significantly more toxic thanthe other nanosilvers used to support the riskassessment, testing with a freshwater and marine fishmay potentially be waived.850.1300: Daphnid ChronicToxicity TestRequired to determine thechronic effects of nanosilver.**This guideline prescribes a chronic toxicity test inwhich daphnids are exposed to a chemical either in astatic-renewal or a flow through system.74Table B2 - Summary of Tier IIRequired Data for NSPW-L30SSOCSPP Data Requirement1Guideline Number: Study TitleReason for StudyTest MaterialComments850.1400: Early-Life StageToxicity Test for Freshwater,Estuarine, and Marine FishRequired to determine thechronic effects of nanosilver.**Tests with the early-life stages of fish are intended todefine the lethal and sublethal effects of chemicals onthe stages and species tested. They yield informationof value for the estimation of the chronic lethal andsublethal effects of the substance on other fish species.Environmental FateNon-Guideline: Rate ofDeposition/AggregationThe rates of aggregation andsedimentation are required forconfirming estimates on theenvironmental fate andpotential ecological impacts ofthese materials.**Knowing the rate of aggregation and sedimentation ofthe products released from textiles would give primaryinformation on the behavior of these compounds in theaquatic environment.835.1100: Activated SludgeSorption IsothermRequired to determinewastewater treatment removalefficiency.**The EPA uses Guideline 835.1110 test results toestimate the removal efficiency of a chemical as itpasses through a wastewater treatment plant.835.1230: Adsorption/Desorption(Batch Equilibrium) modified toalso measure leaching (835.1240)Required to determinepartitioning to solids and todetermine the mobility insubsurface environment.**If the material is removed during wastewatertreatment, it may be deposited on land through thedeposition of sludge (i.e. land farming). If the materialis not removed during wastewater treatment it may bereleased into aquatic environments and may bind tosediment. Adsorption/desorption equilibrium studiesrequired to determine the mobility of NSPW-L30SS ornanosilver in the environment.The distance that nanoparticles move in soil andgroundwater is thought to depend on interaction withsoil grains. Attachment of nanoparticles to soil grainsdepends on the physical processes of sedimentation,interception, and diffusion rather than partitioning tonatural organic matter. Nanoparticle to soil graininteraction depends on nanoparticle diameter, aqueouschemistry, and the arrangement of soil grains and mustbe measured using soil column tests.75Table B2 - Summary of Tier IIRequired Data for NSPW-L30SSOCSPP Data Requirement1Guideline Number: Study TitleReason for StudyTest MaterialComments850.3300 or OECD 209: ModifiedActivated Sludge, RespirationInhibition Test for SparinglySoluble ChemicalsRequired to determine impactto wastewater treatmentsystems.**Silver from industrial processes (e.g., film processing)has been shown to reduce microbial activity inwastewater treatment systems. The purpose of thestudy is to assess the impact of NSPW-L30SS ornanosilver on microbial activity during wastewatertreatment.1These guidelines only provide general guidance. Protocolsshall be submitted prior to conducting these studies.**The test material shall include materials that are releasedduring the stability and plastics leaching studies. These materialsinclude nanosilverreleased from NSPW-L30SS or plastics treated with NSPW-L30SS and or NSPW-L30SS released from plastics treated with NSPW-L30SS.76 Table B3 – Enforceable Schedule NSPW-L30SS and plastics treated with NSPW-L30 SS will be the test material during Tier I (Phases 1 – 4) studies. The test material for Ti er II studies (Phases 5 – 9) will depend on the results of the Tier I leaching a nd dissolution studies (nanosilver and /or NSPW-L30SS released).

Tier I Guideline Protocols* Results† Phase 1 – NSPW-L30SS Characterization 3 12 N GL Particle Size Distribution N GL Surface Area

Phase 2 – Product Testing 3 12 N GL Particulate Stability

Phase 3 – Release Characteristics/Exposure 3 15 N GL Dissolution Kinetics N GL Leaching Test of Plastic

Phase 4 – Health Effects 6 32

870.3250 90-Day Inhalation modified to include a functional observational battery, motor activity and detailed neuropathology

870.3550 Modified Reproduction/Developmental Toxicity Screening Test via the inhalation route

Phase 5 – Ecological Effects 6 18 850.1010 Aquatic Invertebrate Acute Toxicity, Freshwater Daphnids

Tier II Guideline Protocols* Results† Phase 6 – Characterization 18 24 N GL Plasmon Resonance N GL Particle Size Distribution N GL Surface Area 830.7840 / 830.7860 Solubility

N GL Zeta-potential

Phase 7 - Health Effects 18 45 N GL p eri- and post-natal exposure to NSPW-L30SS 870.7600 Dermal Penetration

Phase 8 – Ecological Effects 18 45 850.1850 Modified Aquatic Food Chain Transfer 850.4100 Terrestrial Plant Toxicity, Seedling Emergence 850.4400 Aquatic Plant Toxicity, Tier II (Definitive) Test 850.4500 Algal Toxicity, Tier II (Definitive) Test 850.4550 Cyanobacteria Toxicity, Tier II (Definitive) Test77 Guideline Protocols* Results† Phase 8 – Ecological Effects (continued) 18 45

N GL Measuring the Chronic Effects of Freshwater SedimentAssociated Contaminants on Chironomus dilutes

N GL Measuring the Chronic Effects of Freshwater SedimentAssociated Contaminants on Hyalella azteca

N GL Measuring the Chronic Effects of Marine and Estuarine Sediment-Associate d Contaminants on Leptocheirus

p lumulosus 850.1075 Acute Toxicity Test with Freshwater and Marine Fish 850.1300 Daphnid Chronic Toxicity Test

850.1400 Early-Life Stage Toxicity Test for Freshwater, Estuarine, and Marine Fish

Phase 9 – Environmental Fate 18 45 N GL Rate of Deposition/Aggregation 850.1100 Activated Sludge Sorption Isotherm

835.1230 Adsorption/Desorption (Soil/Sediment) modified to include leaching component 850.3300 or OECD 209 Activated Sludge, Respiration Inhibition Test for Sparingly Soluble Chemicals

*Number of months after conditional registration is issued to prepare and submit protocols. †Number of months after conditional registration is issued to perform study and submit results. NGL = Non-Guideline