## Draft Decision Document

Proposed Conditional Registration of Nanosilva as a Materials
Preservative in Textiles and Plastics

August 27, 2013

Environmental Protection Agency Office of Pesticide Programs
Antimicrobials Divisionii TABLE OF CONTENTS

EXECUTIVE SUMMARY ............................................................................................................. IV

I. REGULATORY DECISION SUMMARY ................................................................................1

II. BACKGROUND ................................................................................................................ .........2

2.1 R EGULATORY H ISTORY

............................................................................................................................... ........ 2
2.2 FIFRA S CIENTIFIC A DVISORY P ANEL (SAP) M EETING

....................................................................................... 2
III. PRODUCT DESCRIPTION, TESTING, AND USE .............................................................4

3.1 P RODUCT D ESCRIPTION

............................................................................................................................... ........ 4
3.2 P RODUCT U SE

............................................................................................................................... ....................... 4
3.3 P RODUCT T ESTING AND W AIVERS

....................................................................................................................... 4

IV. HAZARD CHARACTERIZATION OF NANOSILVER .....................................................8

4.1 A CUTE H UMAN T OXICOLOGY OF N ANOSILVA

..................................................................................................... 8
4.2 S UBCHRONIC AND C HRONIC T OXICOLOGICAL S TUDIES A VAILABLE FOR N ANOSILVER

....................................... 9
4.2.1 Oral .................................................................................................................... ......................................... 9
4.2.2 Inha lati on .............................................................................................................. .................................... 10
4.2.3 Dermal .................................................................................................................. .................................... 12 4.2.4 Reproductive and Developmen tal Toxicity of Nanosilver ................................................................... ..... 14 4.2.5 Mutagenicity of Nanosilver............................................................................................... ........................ 14
4.2.6 Silver Ions ............................................................................................................. .................................... 16
4.3 A BSORPTION , D ISTRIBUTION , M ETABOLISM , AND E XCRETION (ADME) OF N ANOSILVER

................................ 17 4.3.1 Injection of Nanosilver ................................................................................................. ............................. 17
4.3.2 Oral Administra tion of Nanosilver ....................................................................................... ..................... 18
4.4 S AFETY F ACTOR FOR I NFANTS AND C HILDREN (FQPA S AFETY F ACTOR )

......................................................... 19
4.5 A NTIMICROBIAL R ESISTANCE

............................................................................................................................ 20
4.6 P OINT OF D EPARTURE S ELECTIONS AND T ARGET M ARGIN OF E XPOSURE

......................................................... 20 4.6.1 Point of Departures for Nanosilva ....................................................................................... ...................... 21 4.6.2 Target Margin of Exposure ............................................................................................... ........................ 24 4.6.3 Margin of Exposure ...................................................................................................... ............................ 25

V. OCCUPATIONAL AND CONSUM ER RISK ASSESSMENT ..............................................27

5.1 O CCUPATIONAL R ISK A SSESSMENT FOR M IXING AND L OADING THE N ANOSILVA L IQUID S USPENSION

............ 27 5.1.1 Occupational Exposure when Preparing the Master Batch Cont aining Nanosilva ................................... 28 5.1.2 Occupational Margins of Exposure ........................................................................................ ................... 29
5.1.3 Conclusions for Occupationa l Risk ....................................................................................... .................... 32 5.1.4 Occupational Exposure and Health Data Re quirements ...................................................................... ..... 32
5.2 C ONSUMER R ISK A SSESSMENT FOR P RODUCTS I NCORPORATING N ANOSILVA

.................................................. 33 5.2.1 Consumer Exposures to Plastics an d Textiles Incorpora ting Nanos ilva ................................................... 3 4 5.2.2 Consumer Margins of Exposure to Plastics Incorporating Nanosilva ....................................................... 40 5.2.3 Consumer Margins of Exposure to Textiles Incorporat ing Nanosilva ...................................................... 4 5 5.2.4 Consumer Aggregate Margins of Exposure to Plastics and Textile s Incorporating Nanosilver ............... 50
5.2.5 Conclusions for Consumer Risk ........................................................................................... ..................... 52 5.2.6 Consumer Exposure and Health Data Requirements .......................................................................... ...... 54iii VI. ENVIRONMENTAL RISK ASSESSMENT ..........................................................................55

6.1 E NVIRONMENTAL F ATE

............................................................................................................................... ...... 55
6.1.1 Nanosilver .............................................................................................................. ................................... 55
6.1.2 Silver Ions ............................................................................................................. .................................... 56 6.1.3 Impacts to Wastewater Treatment/Septic Systems .......................................................................... ......... 56
6.2 E NVIRONMENTAL T OXICITY

.............................................................................................................................. 57
6.2.1 Silver Ions ............................................................................................................. .................................... 57 6.2.2 Aquatic Toxic ity of Nanosilver .......................................................................................... ....................... 57
6.3 A QUATIC R ISK A SSESSMENT

............................................................................................................................. 59 6.3.1 Aquatic Risk Quotient ................................................................................................... ............................ 60 6.3.2 Conclusions for Aquatic Risk ............................................................................................ ....................... 62
6.4 E COLOGICAL E FFECTS AND E NVIRONMENTAL F ATE D ATA R EQUIREMENTS

. .................................................... 63

VII. REGULATORY ACTION ......................................................................................................64

7.1 L EGAL F RAMEWORK

............................................................................................................................... ........... 64
7.2 F INDINGS AND R EGISTRATION D ECISION

........................................................................................................... 65
7.3 B ASIS FOR C ONDITIONAL R EGISTRATION

.......................................................................................................... 66
7.3.1 Data Genera tion ......................................................................................................... ............................... 66
7.3.2 Public Interest ......................................................................................................... .................................. 67
7.3.3 No Unreasonable Adverse Effects ......................................................................................... ................... 69

References

.............................................................................................................................. ...... 71

LIST OF TABLES

Table 1 – Product Testing for the Nanosilva Liquid Suspension ................................................... 5 Table 2 – Acute Toxicity Profile fo r the Nanosilva Liquid Suspension ......................................... 8 Table 3 – Toxicity Endpoints for Use in Nanosilver Risk Assessment ........................................ 23 Table 4 – Unit Exposures for Mixing and Loading of Liquids ..................................................... 28 Table 5 – Occupational Handlers Inhalation MOEs for the Nanosilver in the Nanosilva Liquid Suspension .................................................................................................................... ................ 30 Table 6 – Occupational Handlers Dermal MOEs for the Nanosilver in the Nanosilva Liquid Suspension .................................................................................................................... ................ 31
Table 7 – Concentration of Silver in Fluid Contacting the Nanosilva Containing Plastic Coupons at 40 degrees Celsius ......................................................................................................... ............ 35
Table 8 – Concentration of Silver Released from Shirts Incorporating Nanosilva during Modified
ISO Colour Fastness Test ...................................................................................................... ....... 38 Table 9 – Amount of Silver Released from Shirts Incorporating Nanosilva ................................ 39
Table 10 – Incidental Oral MOEs for Children Ex posed to Plastic Toys Incorporating Nanosilva

............................................................................................................................... ........................ 41 Table 11 – Dermal MOEs for Children Exposed to Flooring Incorporating Nanosilva ............... 43
Table 12 – Incidental Oral MOEs for Children Exposed to Flooring Incorporating Nanosilva ... 45 Table 13 – Inhalation MOE for Drying Te xtiles Incorporating Nanosilva .................................. 46iv Table 14 – Dermal Exposure to Te xtiles Incorporating Nanosilva .............................................. 47 Table 15 – Dermal MOEs for Children Exposed to Textiles Incorporating Nanosilva ................ 48 Table 16 – Incidental Oral Exposure to Textiles Incorporating Nanosilva .................................. 49
Table 17 – Incidental Oral MOEs for Children Ex posed to Textiles Incorporating Nanosilva ... 49 Table 18 – Aggregate MOEs for Children Exposed to Plastics and Textiles Incorporating
Nanosilva ..................................................................................................................... ................. 51 Table 19 – Nanosilver Toxicity to Aquatic Species ..................................................................... 58 Table 20 – Risk Quotients for Nanosilver in Surface Water ........................................................ 61

Appendix A: Evaluation of Nanosilv a LLC’s Data Waiver Requests Appendix B: Data Requirements, Enforceable Schedule, and Conditions of the Proposed Conditional Registration for Nanosilvav EXECUTIVE SUMMARY

EPA is proposing to grant a conditional regi stration for the Nanosilva LLC product named “Nanosilva” with a product code of NSPW-L30SS (hereafter referred to as “Nanosilva”) under section 3(c)(7)(C) of the Federa l Insecticide, Fungicide and Rodenticide Act (FIFRA). The Agency’s basis for the conditional registration is that:

 Nanosilva contains nanosilver as an active i ngredient and the nanos ilver in Nanosilva is not in any currently registered pesticide;

 Use of Nanosilva will not cau se unreasonable adverse eff ects on the environment during the period when newly required data are being developed;

 Insufficient time has elapsed for Nanosilva to generate and submit the newly required data; and

 Use of Nanosilva is in the public interest.

Nanosilva, which contains 1% nanosilver by weight , is the subject of an application submitted by Nanosilva LLC in August 2009 (MRID 47828900). Nanos ilva LLC requested th at the nanosilver in Nanosilva be registered as a new active ingred ient because it was not an active ingredient in any currently registered pesticide product. Nanos ilva is a silica-sulfur-n anosilver complex where the nanosilver active-ingredient is attached to cr ystalline silica via a thiolate bond. The diameter of the spherical silica core is 320 nm on average and the nanosilver particles that are attached to
the silica core have mean diameters between 6.9 and 10.6 nm. Nanosilva is proposed to be incorporated into plastics and textiles to suppress the growth of bacteria, algae, fungus, mold and mildew, which cause odors, discoloration, stains, a nd deterioration. The plastics and textiles will contain less than 0.003% silver by weight.

EPA is proposing to make a registration deci sion for non-food contact uses of Nanosilva incorporated into plastic films, sh eets, slabs, and molded parts, a nd textiles. Consistent with this, EPA is proposing to register Nanosilva for indoor use articles such as automotive parts, floor coverings, footwear, sportswear, uniforms and ou tdoor use articles such as furniture, decking, carpeting, and house siding.

EPA determined that workers, consumers, and the environment could be exposed to:
1) Silver ions released from Nanosilva; 2) the Nanosilva complex; and/or 3) Nanosilver that might break away from the Nanosilva complex.
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 text iles incorporating Nanosilv a are not of concern.

In evaluating the risk from short-term exposure to Nanosilva, Nanosilva LLC submitted results from acute animal-toxicity tests completed us ing high-level doses of Nanosilva showing thatvi there were no mortalities or abnormalities in test animals af ter administration of Nanosilva by oral, dermal, and inhalation exposur e routes. Nanosilva caused modera te irritation to the eyes of test animals, and was not a skin sensitizer. Base d on these results, shipping containers filled with Nanosilva are required to carry a label stating “CAUTION” where c ontact with eyes or clothing should be avoided. Nanosilva LLC submitted a reque st to waive the required intermediate-term toxicity studies because silver at concentrations greater than the analytical detection limit were not found leaching from plastic coupons incorpor ating Nanosilva and because of the lack of toxicity noted in the acute animal-toxicity test s completed using high-leve l doses of Nanosilva.

To evaluate occupational exposure to the nanosilv er that might break away from the Nanosilva complex, EPA calculated the daily-dose to worker s assuming that all the silver in Nanosilva was freely available as nanosilver. EPA believes that this assumption overestimate the daily-dose of nanosilver that a person could pot entially receive when working with Nanosilva. To evaluate consumer exposure to the nanosilver that might break away from plastics and textiles incorporating Nanosilva, Nanosilva LLC submitted stud ies showing that silver at concentrations greater than the analytical detection limit were not found leaching from plastic coupons and shirts incorporating Nanosilva. Thus, there is little exposure to nanosilver from plastics and textiles incorporating Nanosilva. EPA assumed th at silver leaching from plastic coupons and shirts incorporating Nanosilva was at one-half the analytical detection limit and was in the form
of nanosilver as found in Nanosilva.

There are no intermediate- or l ong-term human or environmental toxicity studies available for Nanosilva or for the nanosilver released from pr oducts incorporating Na nosilva. In the absence of intermediate-term toxicity studies, EPA eval uated the risk from occupational and consumer exposure using intermediate-term hazard data availa ble in the scientific l iterature for nanosilver. Because of the potential difference between the na nosilver in the available toxicity data and the nanosilver in Nanosilva along with the lack of an acceptable developmental and reproductive toxicity study for nanosilver, the Agency used a maximum 10-fold database uncertainty factor when evaluating the risk from exposure to Nanos ilva and the nanosilver released from products incorporating Nanosilva.

Impact to the environment was based on ecotoxicity studies available in the scientific literature for nanosilver and environmental exposure fr om indoor use was assessed assuming that 300 million people (U.S. population) each purchased one t-shirt containing Nanosilva and from outdoor use was assessed assuming that 1% of the projected yearly produc tion of plastic lumber
in the U.S. would contain Nanosilva.

Using robust toxicity data on nanosilver, cons ervative occupational e xposure assumptions, leaching data showing minimal consumer exposur e to nanosilver, and maximum values for risk uncertainty factors, EPA is able to determine that for the period of conditional registration, there is a low probability of adverse risk to huma n health and the environment from plastics and textiles incorporating Nanosilva. Thus, the Agen cy concludes that use of Nanosilva will notvii cause unreasonable adverse effects on the envi ronment during the period when newly required data are being developed.

As a condition of the proposed registration, EPA is proposing to requir e that Nanosilva LLC conduct studies during the period of conditional re gistration to better characterize the nanosilver in Nanosilva. In addition, EPA is proposing to require the following i nhalation route-specific
studies:

 90-Day Inhalation Toxicity (Rat) (OCSPP 870.3465) modified to include in vivo bone marrow assay and functional observationa l battery, motor activity and detailed neuropathology

 Reproduction/Developmental Toxicity Sc reening Test (Modified OCSPP 870.3550/ OECD TG 421)

Although there is no risk concern for workers w ho use close-system loading when mixing and loading the Nanosilva liquid suspen sion, EPA is proposing to require these tests to confirm the adequacy of the 10-fold database uncertainty factor, to reduce the uncertainties related to differences in the physical properties of the nanosilver, and because there are currently no acceptable studies on the reproductive and developmental toxicity for nanosilver.

These studies must be completed within four ye ars after issuing the registration. This time period was chosen to allow time for protocol reviews prio r to initiation of the studies, completion of the studies, and Agency review of th e study results. The Agency will evaluate these data as they are submitted to confirm the Agency’s determina tion that the use of Nanosilva will not cause
unreasonable adverse effects to human health an d the environment. 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 is proposi ng to issue a notice of intent to cancel Nanosilva’s registration under FIFRA section 6(e). In addition, the conditional registration for Nanosilva will automatically expire four years after being i ssued. 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 Nanos ilva in the United States.

EPA believes that the use of Nanosilva is in the public interest. Use of Nanosilva may lead to less environmental loading of silver as compared to currently registered products with the same use patterns. In addition, Nanosilva appears to o ffer prolonged ability to suppress the growth of odor and stain causing bacteria as compared to cu rrently registered produ cts containing silver salts with the same use patterns.1 

### I. REGULATORY DECISION SUMMARY

EPA is proposing to grant a conditional regi stration for the Nanosilva LLC product named “Nanosilva” under section 3(c)(7)(C) of the Federal Insecticide, Fungicide and Rodenticide Act (FIFRA). Nanosilva, which contai ns 1% nanosilver by weight, is the subject of an application submitted by Nanosilva LLC in August 2009 (M RID 47828900). Nanosilva LLC requested that the nanosilver in Nanosilva be registered as a new active ingredient beca use it was not an active ingredient in any cu rrently registered pesticide product.

As described in the risk assessment sections of th is document, EPA has determined that there is a low probability of adverse risk to human health and the enviro nment from plastics and textiles incorporating Nanosilva during the period required for developing and submitting protocols for review, conducting the studies, and submitting the re sulting data, as well as EPA’s review of the submitted data. Thus, the Agency concludes that use of Nanosilva will not cause unreasonable
adverse effects on the environm ent during the period when newly required data are being developed.

In August, 2009 Nanosilva LLC submitted acute toxicity studies required under 40 CFR part 161, a study that determined the amount of silver leaching from plastic coupons incorporating Nanosilva, and a request to waive required st udies. The waiver request was based on the low exposure to Nanosilva given that the product was proposed to be embedded in plastic and the submitted leaching study for a plastic incorpora ting Nanosilva indicated that silver was not released at concentrations above the analytical detection limit. In May 2013, Nanosilva LLC also submitted a study showing that silver was not rel eased at concentrations above the analytical detection limit from textiles incorporating Nanosilv a. EPA’s proposal is to grant the registration request with requirements for new data as te rms and conditions on the Nanosilva registration. Nanosilva LLC, however, has not had sufficient time to generate and submit these newly required data.

For a variety of reasons, EPA has determined that use of Nanosilva is in the public interest. EPA believes that Nanosilva LLC’s product may lead to less environmental lo ading of silver as compared to currently registered products contai ning silver salts with the same use patterns. In addition, Nanosilva appears to offer prolonged abili ty to suppress the growth of odor and stain causing bacteria as compared to currently regist ered products containing silver salts with the same use patterns. Moreover, because we can find that a section 3(c)(7)(C) registration is unlikely to cause unreasonable adverse effect s on the environment during the time while Nanosilva LLC is generating the newly required da ta, allowing such registration to be granted before the newly-required data is generated al lows the Nanosilva LLC product to compete with other like-situated products. Allowing Na nosilva LLC’s product on the market pending generation of data allows Nanosilva LLC to partic ipate in the plastics and textile economy along2 with the other registrants with like-situated products, and allows consumers of these products a new choice among the potential like-situated products.

EPA intends to require Nanosilva LLC to c onduct a number of studies as a condition of registration. These studies must be completed wi thin a time duration of four years which was chosen to allow time for protocol reviews prior to initiation of the studies, completion of the studies, and Agency review of th e study results. The Agency will evaluate these data as they are submitted during the period of the conditional registration to confirm the Agency’s risk assessment. If Nanosilva LLC fails to take appropriat e steps to initiate the required studies, or if Nanosilva LLC fails to submit the protocols or da ta, EPA has the authorit y to issue a notice of intent to cancel Nanosilva LLC’s registration under FIFRA section 6(e) . In addition, Nanosilva LLC’s conditional registration fo r Nanosilva will automatically e xpire four years after being
issued. Nanosilva LLC will have to submit an a pplication to obtain an unconditional registration if they wish to continue to sell and di stribute Nanosilva 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 Nanosilva, a nanosilver-based product that was proposed for use as a materials pres ervative additive with no food contact uses. Nanosilva LLC’s application claimed that Nanosilva was a new activ e ingredient not contai ned in any currently registered silver-based pesticide products and should be given a registration under FIFRA section 3(c)(5) (MRID 47828900).



### 2.2 FIFRA Scientific Advisory Panel (SAP) Meeting

In November, 2009 the Agency convened a meeti ng of the FIFRA Scientific Advisory Panel (SAP) to address a number of questions associat ed with assessing the hazard of and exposure to nanosilver and other nanoscale metal-based pestic ides (FIFRA SAP, 2009). In general, the SAP advised that the toxicity of na nosilver could differ from and might be higher than 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 betwee n silver ions released from nanosilver and the existing database for silver ions is feasible (FIFRA SAP, 2009). The SAP cautioned about extrapolating from one nanosil ver formulation to another when assessing hazards because differences in particle formulati on (e.g., coating and inert ingredients) are likely to affect biological activ ity, among other things.3 The SAP commented that not e nough literature is 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. On ly a few studies in rodents are known that investigate the toxicity of nanosilver from exposure by these routes . Nor is there much information on the level of human exposure to nanosilver by these routes, fo r either workers or consumers using products containing nanosilver. The same situation exists 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 are not well quantified. Furtherm ore, little or no information on the fate of
nanosilver in soils and sediments was found. As a result, the SAP recommended a case-by-case approach to hazard and exposure assessment (i .e., product-by-product). The SAP also advised that existing requirements may have to be adjusted to obtain data appropriate to assess the fate, degradation, metabolism, mobility, dissipa tion, and accumulation of nanomaterials.

The SAP report further suggested that existing information on conventional silver could be useful but would not necessarily be sufficient in assessing potential nanosilver risks. The SAP recommended that the Agency treat nanosilve r differently from its conventional silver counterpart in evaluating propos ed nanosilver product applications (in terms of both data requirements and the conduct of risk assessments ). Moreover, the Panel recommended that EPA require additional data on the physical chemistry, exposure potential, and the 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 determining 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 an ingredient that is a nanoscale version of a conventionally-size d active or inert 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 product, EPA necessarily will need to assess the nanoscale material and may require additional data to make the requisite stat utory findings.4 

### III. PRODUCT DESCRIPTIO N, TESTING, AND USE

3.1 Product Description

Nanosilva (NSPW-L30SS) is a liquid suspension c ontaining silica-sulfur-n anosilver particulates where the nanosilver active-ingredient is attached to crystalline silica via a thiolate bond. The particles are formed by reacting silver nitrat e and polyvinylpyrrolidone (PVP) with spherical silica particles that have been modified with th iol groups and are suspended in ethanol (Lee et al. 2007). The diameter of the spheri cal silica core particles are 3 20 nm on average (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 diam eter of 12 nm) after 10 minutes and 10.6 nm (minimum diameter of 5 nm and maximum diamet er of 18 nm) after 2 hours (Lee et al., 2007). The liquid suspension contains 1% nanosilver by weight with av erage diameters between 6.9 and 10.6 nm (minimum diameter of 3 nm and maximu m diameter of 18 nm), where the nanosilver
surface is coated with sulfur and PVP, and is attached to silica.

The proposed labels states that Nanosilva (N SPW-L30SS), which contains 1% nanosilver by weight, is to be formulated into a polymeri c intermediate known as a master batch. Nanosilva LLC proposed that the final product contain be tween 5 and 15% by weight of the polymeric
intermediate or master batch that contains Na nosilva. Although not stated on the proposed label, the final product that a consumer may be expos ed to will contain less than 0.003% nanosilver by weight. EPA is proposing that Nanosilva LLC state on the pesticide label that products incorporating Nanosilva contain a maxi mum of 0.003% nanosilver by weight.



### 3.2 Product Use

Nanosilva LLC is proposing that Nanosilva be in corporated into polymer and polymer based products to suppress the growth of bacteria, algae, fungus, mold and mildew, which cause odors, discoloration, stains, and de terioration. Nanosilva LLC propos ed a wide range of non-food contact use categories for Nanos ilva including house wares, bu ilding materials, bathroom fixtures and accessories, electronics and app liances, personal care products, automotive equipment, hospital and institutional facili ty equipment, sporting goods, and textiles.



### 3.3 Product Testing and Waivers

As an applicant for registration of a new activ e ingredient in 2009, Nanosilva LLC was required to submit all the applicable information and studies under 40 CFR part 161. Nanosilva LLC submitted results from testing of Nanosilva to determine the product identity and composition, physical and chemical properties, and acute toxicity as summariz ed in Table 1. Some of these tests were completed using st andard EPA test guidelines 1 prior to consulting with EPA. Although

1 Available at http://www.epa .gov/ocspp/pubs/frs/home/guidelin.htm5 these test results provided useful informati on for Nanosilva, the guidelines on which they are based have not been adapted generally for use wi th nanoscale particles. EPA anticipates that these guidelines will require revision going forwar d in terms of their application to nanoscale materials. As a result, it is recommended that fu ture applicants for products containing nanoscale
materials consult with the EPA prior to performing any tests.

Table 1 – Product Testing for the Nanosilva 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 production process 161.162 47828903 830.1650 Description of formulation 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 830.7200 Melting point/melting range 161.190 830.7220 Boiling Point/Boiling Range 47828915 830.7300 Density 161.190 47828916

830.7520 Particle size, fiber length, and
diameter distribution Not Required, information in Lee et al., 2006 and
2007 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 478289236 In addition to the test results listed in Table 1, Nanosilva LLC submitted the following study to support the registration of Nanosilva:

Plastic Leaching Study

 Leaching protocol for Nanosilva treated LLD PE 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:

1. Hydrolysis (OPPTS 835.2120) 2. Aquatic Invertebrate Acute Toxicity Te st, Freshwater Daphnids (OPPTS 850.1010) 3. Fish Acute Toxicity Test, Fres hwater and Marine (OPPTS 850.1075) 4. Avian Acute Oral Toxici ty Test (OPPTS 850.2100) 5. 90-Day Dermal Toxicity (OPPTS 870.3250) 6. Prenatal Developmental Toxicity Study ( OPPTS 870.3700) 7. Bacterial Reverse Muta tion Test (OPPTS 870.5100) 8. Detection of Gene Mutations in So matic Cells in Culture (OPPTS 870.5300) 9. In Vitro Mammalian Cyt ogenetics (OPPTS 870.5375) 10. Mammalian Bone Marrow Chromosome Aberration Test (OPPTS 870.5385) 11. Immunotoxicity (OPPTS 870.7800)

Waivers for the above studies were requested because of the lack of acute toxicity noted in the six acute toxicity studies (MRID 47828918 thro ugh 47828923), because Nanosilva has a specific gravity greater than water, because Nanosilva has low solubility in water as determined by the water column generator method (MRID 47828917), a nd due to the non-leaching characteristic of Nanosilva because silver at concentrations greate r than the analytical detection limit were not detected in leaching studies conducted with a plastic incorporating Nanosilva (MRID 47828925). As detailed in Appendix A, the data requireme nts listed above in items 1 through 11 are either waived or satisfied. In gene ral, the data requirement is met or satisfied because:

 certain guideline studies are not appropriate for nanosilver and where appropriate are being modified and required as terms and conditions on the registration (i.e., Reproduction/Developmental To xicity Screening Test)

 certain environmental data are satisfied because the environmental hazard labeling statement was determined using literatu re studies completed with nanosilver

 Dermal guideline studies are not appropr iate given there was no risk concern

 in vitro mutagenicity study requirements are satis fied and have shown that nanosilver is possibly mutagenic

 the immunotoxicity study is initially waived and only triggered after the potential immune toxicity of nanosilver is determin ed during the 90-day inha lation toxicity study required as a term and condition on the registration7 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 Nanosilva

 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 preparation st rategy using polyol process: Size-controlled immobiliz ation 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 Characterization of Silver Released from Textiles Treated with NanoSilva (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)8 

### IV. HAZARD CHARACTERIZAT ION 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 the Agency convened a meeting of the FIFRA Scientific Advisory Pa nel (SAP) to address a number of questions associated with assessing the hazard of and exposure to nanosilver and other nanoscale metalbased pesticides (FIFRA SAP, 2009). In general, the SAP advised that the toxicity of nanosilver 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 substant ial impact on particle properties, including rate and concentration of silver ion release, where the effects of size are generally most observable for particles with dimensions below 20 nm and largely below 10 nm (2009 SAP, p. 6). In addition to size, other properties such as shape, charge, and surface coating are also likely to impact the biological response to nanosilver.



### 4.1 Acute Human Toxicology of Nanosilva

Nanosilva LLC submitted results from guideline acute animal-toxicity tests completed using high-level doses of a liquid suspension containing Nanosilva with 1% nanosilver by weight. As outlined in Table 2, there were no mortalities or abnormalities noted in test animals after administration of Nanosilva by oral, dermal, and inhalation routes at dos e levels of up to 5,000 mg/kg, and 2.05 mg/L, respectively; Nanosilva caused m oderate to no irritation to skin or eyes at dose levels of up to 0.1 mL and 0.5 mL, respectively; and was not a skin sensitizer. According to the Agency’s Toxicity Category system, which is used for product la beling purposes, shipping
containers filled with Nanosilva are required to carry a label st ating “CAUTION” where contact with eyes or clothing should be avoided.

Table 2 – Acute Toxicity Profile fo r the Nanosilva Liquid Suspension Study Toxicity Category

Acute Oral Toxicity Category IV No mortality or abnormali ties after dose of 5,000 mg/kg

Acute Dermal Toxicity Category IV No mortality or abnormali ties after dose of 5,000 mg/kg

Acute Inhalation Toxicity Category IV No mortality or abnormali ties after dose of 2.05 mg/L

Acute Eye Irritation Category III
Moderate to not irritati ng after dose of 0.1 mL

Acute Dermal Irritation Category IV Mild or slight irritati on after dose of 0.5 mL

Skin Sensitization Not a sensitizer9 

### 4.2 Subchronic and Chronic Toxicologi cal Studies Available for Nanosilver

There are no repeated-dose subchronic or chronic toxicity studies available for Nanosilva or the nanosilver in Nanosilva. However, there are rep eated-dose toxicity studies available in the
scientific literature for nanosilver. Since na nosilver might be released from Nanosilva and articles incorporating Nanosilva, the Agency c onsiders the scientific literature studies on nanosilver toxicity relevant. The Agency believes the studies described in the following sections are sufficient for assessing the risks from the us e of Nanosilva 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 reported findings after 28 days of repeated administration of carboxymethyl cellulose-coated nanos ilver with aver age diameter of 60 nm (minimum diameter of 53 nm and maxi mum 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 hyperplasi a and increased foci), a coagulative effect on peripheral blood, and an increase in serum alka line phosphatase (ALP) an d 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 in the kidneys of female rats when compared with male rats was also re ported for all dose groups. A no observed adverse effect level (NOAEL) of 30 mg/ kg/day (lowest dose level) wa s reported based on the observed liver effects and increase in alkaline phosphatase (ALP) and cholesterol at 300 mg/kg/day (mid dose level). The second study was performed by th e same group using four-week old Fisher rats (n= 10 per dose) for 90 days (Kim et al., 2010). Th is study involved repeated administration of carboxymethyl cellulose-coated nanosilver with av erage diameter of 56 nm (minimum diameter of 25 nm and maximum diameter of 125 nm) to ra ts and reported similar findings as the 28-day study including gender-related distribution of si lver in the kidneys 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 day study.

The third study involved feeding nanosilver stab ilized with polyvinylpy rrolidone with average
diameter of 14 nm and silver acetate to four-wee k old male (n= 6 per dose) and female (n=10 per dose) Wistar Hannover Galas rats for 28 days (Had rup et al. 2012a). Consis tent to the studies above, investigators did not find in creases in ALP or cholesterol for nanosilver at doses of up to 9 mg/kg/day. They also reported no observable e ffects on the microbiological status of the rat’s gastrointestinal tract caused by ingesting nanosilver. However, Hadrup et al., (2012a) did report
effects for silver acetate at a dose of 14 mg/kg/day including an increase in ALP, decrease in plasma urea, and lower thymus weights. Hadrup et al. (2012b) compared the neurotoxic effects,
in vivo and in vitro , of polyvinylpyrrolidone stabilized na nosilver with average diameter of 14 nm and silver acetate. Following 28 days of oral administration, nanosilver (4.5 and 910 mg/kg/day) and silver acetate (9 mg/kg/day) significantly in creased the concentration of dopamine in the brains of Wistar female rats, while the brain concentration of 5- hydroxytryptamine (5-HT) was increased only by na nosilver at a dose of 9 mg/kg/day. However, in the 14-day range-finding study, the brain dopamine concentrati on decreased in rats treated with nanosilver at doses of 2.25 and 4.5 mg/kg/da y. Three solutions consisting of 1) nanosilver, 2) an ionic silver solution obtained by filteri ng a nanosilver suspension, and 3) silver acetate
were tested in neuronal-like PC12 cells in vitro. Nanosilver did not induce necrosis; however, cell viability was decreased and apoptosis (involving both the mitochondrial and the death receptor pathways) was found with all three soluti ons where silver acetate was relatively more potent.

There is one study for the repeated administration of nanosilver with aver age diameter of 42 nm (minimum diameter of 25 nm and maximum diam eter of 55 nm) to mice over 28 days (Park, et al., 2010). The study reported that, after oral admi nistration 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 levels of alkaline phosphatase (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 female s. Histopathological analysis wa s performed in the high-dose groups and revealed that tissue chan ge (i.e., slight cell in filtration) was observe d 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 examin ed. A NOAEL of 0.5 mg/kg/day was reported, based on the observed findings of elevated ALP, AST and ALT and the histopathological changes in the kidney s at the 1.0 mg/kg/day dose level.

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 DLS and 26.3 nm (size range from 10 to 70 nm) by TEM via intraper itoneal injection, 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 for the behavi oral studies. Investigators used the Morris water maze (MWM) test for spatial cognition along w ith bromodeoxyuridine to detect proliferating cells. The test results showed that both reference memory and working memory were not impaired in nanosilver exposed groups, compared with the c ontrol group, and no differences were revealed in hippocampal progenitor proliferation, new born cell survival, or differe ntiation in nanosilver treatment groups.

4.2.2 Inhalation
The subchronic inhalation toxicity of nanosilv er was determined in six-week old male and female Sprague-Dawley rats (n=10 per dose) ex posed to nanosilver with average diameters between 18 to 19 nm (minimum diameter of 2 nm and maximum diameter of 65 nm) in a whole11 body inhalation chamber for 13 weeks (90-day) using OECD test guid eline 413 (Sung et al., 2009). Male and femal rats exposed to the high-dose level of 515 μg/m 3 (3.03×10 6 particles/cm 3 )
showed toxic effects in the liver (bile-duct hyperpla sia) and lungs (chronic alveolar inflammation and macrophage accumulation in the lungs of males and females, and erythrocyte aggregation). A follow-on study was performed to determine lu ng-function recovery 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 (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 slig ht differences in particle si ze and concentration. During the 12-week exposure period, there was a decrease in lung function of male animals exposed to all doses of nanosilver including a de crease in tidal and minute volu me, and peak expiratory flow. After a 12-week recovery period, the ma le animals in the middle- (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 func tion. Remarkably, no adverse lung function effects were observed
for female animals. The decrease in l ung function was matched by histopathological
observations of mixed cell infiltr ate perivascular and chronic al veolar inflammation in male
animals but these effects were also observed in female animals even though they had no measurable decrease in lung function. Although female animals showed gradual clearance of silver from lung tissues and decreased inflam mation after 12 weeks of recovery, persistent inflammation in lung tissues was observed in the high-dose male animals throughout the 12- week recovery period. This lack of recovery i ndicates permanent or persistent organ damage. Significant increases in the amount of silver in tissues, such as lungs, liver, olfa ctory bulb, brain, kidneys, and blood, were also reported. Females ha d two to three times mo re silver accumulation 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 Dynamic Light Scattering (DLS) and with sizes from 50 to 100 nm by Transmission El ectron Microscopy (TEM) was administered to rats by nasal drops at doses of 3 mg/kg and 30 mg/kg once every two days for 14 consecutive days. Spatial cognition as determined by the Morris water maze (MWM) test showed that animals receiving nanosilver exhibited lower le arning 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) recording for these same animals. Further, the quantity of reactive oxygen species (ROS) in hippocampal homogenate was significantly increased in nanos ilver treated groups, and edem a, nuclear shrinking phenomenon, and necrobiosis were shown in hematoxylin and eo sin (HE) stains of pyramidal neurons in the PP (perforant path) and DG (dentate gyrus) regions of hippocampus.12 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 daily r ub exposure to aqueous solution using AgNO 3 as a positive control (Korani et al, 2011). The nanosil ver suspension was applied by daily rubbing to an area of 5 cm by 5 cm 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 we re restrained for 5 hours after a pplication of chemical, however,
the method of restraint was not discussed. During the 13 week study, dose dependent and nanosilver specific effects were seen in the liv er, 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 increased levels of macrophages in the endomysium. Liver effects included overproduc tion of Kupffer cells and degeneration of hepatocytes in a dose dependent fashion and necr osis at the 10,000 μg/ml concentration. In the liver, red capsules were thinner, inflammati on, accumulation of red blood cells, and white pulp atrophy along with hepatic cord destruction in a dose dependent fashion. It does not appear that gauze or other dressing was used to cover the do sed 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 e ffect of skin exposure to nanosilver (size = 20 and 50 nm, concentration = 0.34, 3.4 and 34 μg/ml aqueous solution) 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 a hair-clipped pig, allowed to air dry, then covere d with a Hilltop chambe r (occlusion pad) and
secured with non irrita ting 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 st ate, dependent effect was seen on the dermal layers under microscopic inves tigation. Low dose effects were s light intra and intercellular epidermal edema. Intermediate effects were a more focal intra and intercellular epidermal edema alongside focal dermal and epidermal inflammation. The highest dose caused severe edema, with severe focal dermal inflammation, epidermal hype rplasia and parakeratosis. Precautions were taken to prevent oral dosing, including restraint/ anesthesia and multiple covering 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 hist opathology panel performed, even in the cases where dose dependence effects of na no-silver particles was seen. None of these studies used the same size partic les, coating type or washing state, making it potentially difficult to generalize information from them onto ot her products. In Korani et al. (2011), the study13 lacked any clinical chemistry, he matology or urinalysis of the guinea pigs. This study also could not draw NOAEL/LOAEL conclusions given that eff ects were seen at all levels of dosing. In Samberg et al. (2010), no system ic histopathology would have b een possible, as multiple dose
concentrations were tested on the same anim al. This study would not suffice as a dermal
irritant/sensitizer as there is no induction or recovery period.

In addition, there is one publishe d report on nanosilver that is available for use by the Agency concerning nanosilver used in wound dressings for pa tients suffering from burns. In this report, a burn patient using nanosilver co ated wound dressing developed c linical signs of argyria and elevated serum liver enzymes i ndicative of liver toxicity along with elevated silver concentrations in blood and urine (Trop, 2006). This study indicates to the Agency that nanosilvers can be systemically absorbed when a large area of the skin barrier is severely compromised.

In the absence of any such dermal toxicity stud ies, the Agency normally uses extrapolation from another route of exposure (usually oral). However, use of the or al endpoint for evaluating dermal toxicity requires knowledge rega rding the amount absorbed through the skin. This information is typically provided by a dermal penetration st udy using whole animals (u sually rats), which
allows determination of the fract ion of topically app lied dose that is available for systemic absorption (i.e., the dermal absorp tion factor or DAF). There are in vitro techniques available that allow determination of dermal penetration of chemicals through isolated animal or human skin. However, the Agency does not rely on in vitro dermal absorption stu dy 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, the Agency has observed variation in test results among laboratories. As a result, the Agency
assumes a default DAF of 100% when no in vivo data are available. Therefore, if in vivo dermal penetration studies are available, either t hose studies alone or in combination with in vitro studies are used for deriving a DAF of less than 100%.

Here, there is no guideline or scientific lit erature study conducted in animals for the in vivo dermal absorption of the nanosilver in Nanosilv a or of nanosilver available to the Agency. However, there is a human clinical study, which is observational, that examined silver levels in serum and urine after ap plication 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 Bra ndt et al. (2012) demonstrated that nanosilver and silver sulfadiazine had similar skin absorption characte ristics in mice after normalizing for silver dose.
In addition an in vitro study with nanosilver in human skin is available in the scientific literature indicating that nanosilver penetr ation was very low for both int act and abraded skin at 0.00066% and 0.0033%, respectively (Larese et al., 2009).14 4.2.4 Reproductive and Developmental Toxicity of Nanosilver
There are studies showing significant, dose-dependent increases in the concentration of silver in
the testes of rats after oral ingestion, inhalation, 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 spermatagonia isolated from 6-day old mouse testes and immortalized with SV40 large T antigen (BraydichStolle et al., 2005). In this transf ormed (i.e. immortalized ) cell line, nanosilv er and silver ions caused altered cellular morphol ogy, decreased mitochondrial activity (as indicated by MTS assay), and increased apoptosis at doses up to 10 μg/ml; however, the affects from nanosilver were greater than observed for silver ions. The co nservative interpretation of this data is that nanosilver that reaches the testes may be able to cause decreased fertilit y due to toxicity to spermagonia and this effect would be more se vere for nanosilver than for silver ion.

Austin et al. (2011) investigated the distribution of citrate coated nanosilver with diameters between 30 and 60 nm and silver nitrate in preg nant mice (n= 6 to 12 per dose) and developing embryos. Nanosilver suspensions and silver nitrat e were administered by intravenous injection (i.v.) on gestation days (GD) 7, 8, and 9 at nanosilver concentrations of 0, 0.4, and 0.73 mg/kg/day. Austin et al. (2011) re ported a significant increase in nanosilver content as compared to silver nitrate treated animals in nearly a ll tissues; nanosilver accumulation was significantly higher in liver, spleen, lung, tail (i njection site), visceral yolk s ac, and endometrium. Nanosilver
was identified in vesicles in endodermal cells of the visceral yolk sac. This study demonstrated that nanosilver distributed to major maternal organs and extra-embryonic tissues, but the authors stated that very little silver reached developing embryos and no adverse morphological effects on the developing embryos were observed.

The teratogenicity potential of nanosilver in pregnant rats was investigated by Mahabady et al. (2012). Nanosilver of unknown size and surface coati ng was administered to pregnant rats via intraperitoneal injection (i.p.) on GD 8 and 9. Fetuses collected on GD 20 from animals that received nanosilver were reporte d to have reduced weight and le ngth but no effect on the skeletal system as compared to animals treated with a sa line control. Because Mahabady et al. (2012) did not report key information about the nanosilver particles size and surf ace coatings, it is not possible to compare these results to the nanos ilver in any other study or in any nanosilver containing product.

4.2.5 Mutagenicity of Nanosilver
Currently, there are no studies in the scientific literature that investigate the potential of nanosilver to cause cancer (i.e., ca rcinogenicity). However, the pot ential of nanosilver to induce changes in genetic material (i.e., mutageni city, genotoxicity) has been investigated in vitro using15 traditional mutagenicity tests including the bacterial reverse mutation assay ( i.e., Ames test), the mouse lymphoma forward mutation test, the ma mmalian cell chromosome aberration test in Chinese hamster ovary cells, and the mouse ly mphoma Comet assay fo r oxidative damage. Additionally, nanosilver wa s investigated in an in vitro human lymphoblastoid micronucleus assay as well as and in an in vivo rat bone marrow micronucleus assay. Results from these assays
are summarized below:

In vitro reverse gene mutation assay in bacterial cells: The mutagenicity of nanosilver (average diameter of 10 nm) suspended in a 1% citric aci d solution was determined at concentrations of up to 500 μg/plate in bacterial cells using the Am es test (Kim et al., 2012). Although cytotoxicity was observed at 31.25 μg/plate, nanosilver did no t induce a mutagenic effect in the histidinerequiring strains of Salmonella typhimurium TA98, TA100, TA1538 and TA1537 or in tryptophan-requiring Escherichia co li WP2uvrA with or without the metabolic 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 particles and for particles with a diameter of 1608.7 ± 175.4 nm, prepared in culture media. In this test, there was no
increase in revertant mutant colonies of the standard S. typhimurium tester strains, which
included the strain used to detect oxidative damage (TA102) up to cy totoxic concentration (31.25-62.5 μg/plate -S9; 125-250 μg/plate +S9). Ho wever, 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 muta genic 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 particles 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 of action was assessed using standard 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 na nosilver (average 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 statistica lly significant increase
in the number of cells with chromosome ab erration, polyploidy, or endoreduplication when compared with the control group at concentra tions causing approximately 50% cytotoxicity.16 In Vitro Mammalian Cell Micronucleus Test: An in vitro mammalian cell micronucleus test (OECD Test Guideline 487) was used to determin e the genotoxicity 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 medi a. Nanosilver at concentrations of up to 30 μg/mL induced a significant and dose-related increase in micronuc lei in human lymphoblastoid TK6 cells, indicating to the inves tigator that nanosilver has w eakly positive genotoxic potential (Li et al, 2012).

In Vivo Micronucleus Assay: In contrast to the above in vitro findings, oral administration of carboxymethyl cellulose-coated nanosilver with av erage diameter of 60 nm (minimum diameter of 53 nm and maximum diameter of 71 nm) caused ove rt toxicity but failed to induce an increase in micronucleated polychromatic erythrocytes (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, which was based on OECD Te st Guideline 474, reported that there was no statistically significant treatment-related increase in MN PCEs when compared to the negative control. This study indicates that nanosilver is neithe r clastogenic nor aneugenic in vivo

, although a limitation of this study is that no m easurements were performed to determine if
nanosilver reached the bone marrow.

Overall the results suggest that of the in vitro tests discussed above, the data from the Ames and chromosome aberration assays indica te that nanosilver is not expe cted to be mutagenic while the mammalian cell micronucleus and mouse lymphoma with 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 mutageni c properties of nanosilver (Bart ł omiejczyk et al., 2013). However, the lack of genotoxicity from the in vivo study suggests that the mutagenic potential
observed in some of the in vitro studies may be intrinsic to nanoparticles but may not be expressed in whole animals or th at nanosilver may be subjected to first-pass effects such as biliary elimination. At this time, there is inadequate informati on to assess mutagenic (and hence 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.

4.2.6 Silver Ions
Humans may also be exposed to silver ions that would be re leased by Nanosilva. Conventional silver, and the silver ions it releases, are defi ned as pesticides under FI FRA. The SAP concluded
that the hazards of silver ions would be the same, whether they ca me from conventional silver or from silver nanoparticles. With respect to silver i ons, the Agency evaluated the toxicity and exposure to silver ions and determined that un reasonable adverse effect s from use of silver containing products are unlik ely (U.S. EPA, 1993).17 

### 4.3 Absorption, Distribution, Metabo lism, 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 studies described in the following sections the Agency 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, heart, lungs and testes of animals dosed with nanosilver, silver ni trate, and silver acetate, where the organs of
animals dosed with silver nitrat e and silver acetate contained gr eater amounts of silver than in
animals dosed with nanosilver. Animals can cl ear silver from blood a nd most organs given enough time but retain silver in th e testes and brain. Animals treated with silver nitrate, silver acetate, and nanosilver all contain silver granules with dimensions on the nanoscale. However, it
is unclear if intact nanosilver is absorbed into tis sues or if nanosilver dissolves into ionic silver before being absorbed into tissu es 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 mi crosilver injected animals, which suggests that both nanosilver and microsilver were eliminated thr ough biliary excretion. Although there was no significant difference between th e amount of silver at the injection site or
in excrements after administrati on of nanosilver or microsilver, th e amount of silver in organs was significantly greater for nanosilver. Animals injected with nanosilver were found to contain significantly more silver in the liver, kidney, spleen, brain, lung and bl ood than in animals injected with microsilver. Hist opathological observations found that nanosilver injected animals contained elemental silver spheres that were ab sent from the microsilver treated animals. The elemental silver spheres were observed in different kinds of cells, such as renal tubular epithelial cells and hepatic cells. Moreover, these elemental silver spheres also induced blood-brain barrier (BBB) destruction and astrocyte swelli ng, 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 nanosilver 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 groups . Accumulation of silver was observed in all the tested orga ns including liver, kidney, spleen, lung, brain, testes, and thymus, where the liver and spleen contained the greatest amount of silver. As with the rat study above,
the amount of silver in feces be tween 1 and 28 days after injecti on was significantly greater than18 in urine, which suggests biliary excretion of silver is the ma jor route of elimination after injection of nanosilver.

Blood kinetics, tissue distribution, and organ accu mulation of silver after daily intravenous injections of between 23.8 and 27.6 mg/L over 5 consecutive days of nanosilver with average diameters of 20, 80, and 110 nm was determined in six week old male Wistar rats (n=21 in treatment groups; n= 2 in control) (Lankveld et al., 2010). The concentration of silver in blood rapidly decreased during the initial 10 minutes following both single and re peated injection of nanosilver and then remained stable for up to one hour after injection. Silv er was distributed to all organs evaluated including the liver, lungs, spleen, brain, heart, kidneys and testes, regardless of the size of nanosilver injecte d. After injecting 20 nm diameter nanosilver, silver was found to be distributed mainly in the liv er, followed by kidneys and splee n, whereas after injection of the 80 and 110 nm diameter nanosilver, silver was dist ributed mainly to spleen followed by liver and lung. Thus, there was a size dependent tissue dist ribution. Repeated administration of nanosilver resulted in accumulation of silver in liver, l ung and spleen, indicating th at these organs may be potential target organs for toxi city after repe ated exposure.

4.3.2 Oral Administration of Nanosilver
The organ distribution and cellu lar localization of silver was determined following 28 day repeated oral administration at 9.0 mg/kg/day of polyvinylpyrrolidone stabilized nanosilver with average diameter of 14 ± 4 nm a nd silver acetate to four week old 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 acetate was greater than it was for nanosilver treated animals. This was in agreemen t with the higher fecal excretion of nanosilver as compared to silver actetate. Besides the intestinal system, the largest silver concentrations
were detected in the liver a nd kidneys; however, silver was also found in the lungs and brain. Remarkably, silver containing granules in the same size range as that of the administered nanosilver were observed in the ileum and kidney ti ssues of rats exposed to nanosilver and silver acetate. Using transmission electr on microscopy (TEM), sulfur and selenium containing granules 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 lyso somes of macropha ges within the lamina propria. The results of the present study de monstrate that the organ distribution and form
of silver was similar when nanosilver or silver acetate were administ ered 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 with av erage diameter of 17.7±3.3 nm by TEM, polyvinylpyrrolidone coated nanos ilver 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 Da wley rats (n = 5 per group) (Van der Zande et al., 2012). Greater than 99% of the silver administered to the rats was19 excreted in their feces indicati ng that only a small fraction of silver from nanosilver and silver nitrate was absorbed. After normalizing for gavage dose, the concentration of silver in the blood of the silver nitrate treated animals was significantly higher than for the nanosilver treated animals at all time points during exposure. This clear ly illustrates a much hi gher uptake of silver when silver nitrate was administered as compared to nanosilver. One day after the oral gavage, a significant reduction in blood silver concentration was observed. One week after the oral gavage,
the concentration of silver in blood was reduced to nondetect able levels indicating rapid
clearance of silver from the blood for both nanosil ver and silver nitrate treated animals. After normalizing for gavage dose, silver was observed in all examined organs with the highest levels in the liver and spleen where animals treated with silver nitrate had accumulated significantly
more silver than in animals treated with nanosilv er. Silver was cleared fr om most organs eight weeks after the final gavage, but remarkably not from the brain and testes, where between 94 and 100% of the silver was still present in the brai n compared to the amount one day after the last gavage of nanosilver and silver nitrate, resp ectively. Using single-par ticle inductively coupled plasma mass spectrometry; nanosil ver was detected one day after the final gavage in the liver, spleen, lungs, and gastro intestinal contents of nanosilver gavaged rats. Remarkably, nanosilver was also detected in the liver, sp leen, lungs, and gastroin testinal contents one day after the final gavage from silver nitrate tr eated rats demonstrating the in vivo formation of nanosilver from silver nitrate. Blood enzyme levels were not significantly different from untreated animals indicating that there was no acute hepatotoxicity observed. Also, there was no indication that nanosilver caused nonspecific immune re sponses based on immunotoxic responses.



### 4.4 Safety Factor for Infants a nd Children (FQPA Safety Factor)

Under the Food Quality Protection Act (FQPA) , P.L. 104-170, which was enacted in 1996 to amend the Federal Insecticide, Fungicide, and Rodenticide Ac t (FIFRA) and the Federal Food, Drug and Cosmetic Act (FFDCA), the Agency was di rected to "ensure that there is a reasonable certainty that no harm will result to infants and children" from aggregate exposure to a pesticide
chemical residue. The law further states that in the case of threshold e ffects, for purposes of providing this reasonable certainty of no harm, "an additional tenfold margin of safety for the pesticide chemical residue and other sources of exposure shall be applied for infants and children to take into account potential pr e- and post-natal toxicity and completeness of the data with respect to exposure and toxicity to infants and children. 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 ba sis of reliable data, such margin will be safe for infants and
children."

There is no food use proposed for Nanosilva at this time, and therefore, no FFDCA aggregate exposure to Nanosilva residue is required.20 

### 4.5 Antimicrobial Resistance

Silver is currently used as a broad spectrum an tiobiotic in wound dressing s. There is a concern that increasing use of silver, such as nanosilver for preserving plastics, may result in more bacteria developing resistance to silver and limit its use as an antibiotic agen t for wound care (Gupta and Silver, 1998). In the wound care set ting, 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 th at release sublethal le vels of silver over a long period of time allowing bact eria to develop resistance.

In terms of environment, a recent study involved releasing 1 mg/L of nanosilver into microcosms containing estuary water overlying estuarine se diment cores (Bradford et al., 2009). The study found no impact to the microbial community over a 30 day monitoring period. Evidence for
antibacterial resistance was al so evaluated 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.

The Agency concludes that while development of antibacterial resistance due to the use of nanosilver in Nanosilva might be possible, the like lihood is low that the levels used in products incorporating Nanosilva will lead to the develo pment of silver resistant microbes who will spread throughout the environment and results in widespread bact erial 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 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 eff ects including chronic alveolar inflammation. There were potential neurotoxic effects identified with increases in neur otransmitter concentrations and loss of spatial cognition; however, these same effects were not observed in a follow-on study.
EPA believes that these neurotoxic ity studies are inadequate in th eir assessment of behavioral effects and do not use optimal methods to evaluate the potential toxicity to the nervous tissue
structure and function. The in vitro Ames and chromosome aberra tion assays indicate that nanosilver is not expected to be mutagenic while the mammalian cell micronucleus and mouse lymphoma with comet assay suggest that nanosilv er may have mutagenic potential. However, the lack of genotoxicity from the in vivo study indicates that there is inadequate information to assess mutagenic (and hence carcinogenic) poten tial of nanosilver. Finally, there is not enough information on the reproductive and developmen tal toxicity for nanosilver at this time.

Together, these studies indicate to the Agency th at, if sufficient quantiti es of nanosilver break away from Nanosilva, and if such nanosilver displays toxicity similar to the nanosilver used in21 the oral and inhalation route-spec ific studies, then route-specif ic exposure to Nanosilva derived nanosilver may result in a dverse health effects.

4.6.1 Point of Departures for Nanosilva
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 determine the risk associated with environmentally relevant hum an exposures. Commonly, this is a NOAEL from a laboratory animal toxicity study, which represen ts the dose at which no adverse affects were observed in laboratory animals. Oral or dermal subchronic and chronic t oxicology studies are not
available for Nanosilva or the nanosilver that might break away from articles incorporating Nanosilva. In place of these studies, the Agen cy is determining NOAELs and LOAELs from
subchronic inhalation and or al toxicity studies found in the scientific literature for nanosilvers to
evaluate the effects that could occur from e xposure to the nanosilver present in Nanosilva.

As stated in Section 2.2, th e FIFRA SAP was unsupportive of bridging among silver-based materials with different prope rties. However, the SAP indi cated that bridging would be appropriate for materials of similar size and es sentially identical physi cal properties and that bridging between silver ions released from nanosilv er and the existing databa se for silver ions is feasible (FIFRA SAP, 2009). The SAP cauti oned about extrapolating from one nanosilver formulation to another when assessing hazards be cause differences in particle formulation (e.g., coating and inert ingredients) are likely to affect biological activity, among other things.

The oral toxicity study by Hardrup et al. ( 2012a) used PVP coated na nosilver with average diameter of 14 nm, which is similar to the diam eter and surface coating of the nanosilver in Nanosilva. 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. Th e oral toxicity study by Park et al. (2010) used uncoated nanosilver with an average diamet er of 42 nm, which is different from the diameter and surface coating of the nanosilver in Nanosilva. In 2011, EP A used the NOAEL reported in the Park et al. (2010) study as the POD for evaluating the oral and dermal toxicity of the nanosilver in HeiQ AGS-20 (U.S. EPA, 2011a). EPA reviewed this st udy again as part of this assessment and now concludes that although the results of the Park et al. 2010 study showed evidence of changes in clinical chemistry, it lacks hist ological support for the effects us ed as the basis for the study NOAEL/LOAELs. In the absence of histopathological findings, the clinical chemistry changes
observed are insufficient eviden ce 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 Nanosilva. Th ese studies were stated as being completed22 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 signif icant increases in alkaline phosphatase and cholesterol, significant changes in hematology, and accompanied by histopathological evidences of liver toxicity (bile-duct hype rplasia around central vein, infiltra tion of inflammatory cells, and dilation of the central vein) at the LOA EL dose of 300 mg/kg/day. A NOAEL for the 90-day Kim et al. (2010) cannot be established because of adverse histological patterns evident at the lowest dose of 30 mg/kg/day. EPA has determined that the LOAEL from the 90-day Kim et al. (2010) study is 30 mg/kg/day based on histopathol ogical evidences of liver toxicity (bile-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 similar
to the diameter and but different than the surf ace coating of the nanosilver in Nanosilva. 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, perivascular mixed cell infilt rate and chronic alveolar inflammation and macrophage accumulation in the lungs in both male and female rats, and decreases in physiological measures of lung f unction (respiratory 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 reported in the Sung et al. (2009) study as the POD for evaluating the inhala tion toxicity of the nanosilver in HeiQ 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 newl y available Song et al. (2012) study is more appropriate for evaluating inhalation exposure to nanosilver.

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 Nanosilva (Table 3). EPA has al so determined that the LOAEL from the 90 day oral toxicity study by Kim et al., 2010 is the POD for intermed iate-term oral exposures (1 to 6 months) to the nanosilver in Nanosilva. EP A has determined that the NOAEL of 49 μg/m 3 derived from the Song et al. (2012) study is the POD for short- a nd intermediate-term inhalation exposures to the nanosilver in Nanosilva. These studies were conducted us ing OECD guidelines and the effects are consistent across studies, and therefore the NOAELs/LOAELs are protective
of effects seen in other studies.23 Table 3 – Toxicity Endpoints for Use in Nanosilver Risk Assessment
Dietary Risk Assessment – No Dietary Exposures
Nondietary Risk Assessments

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 Target MOE =
1000 Kim et al. (2008)

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

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

Dermal(Intermediateterm) LOAEL = 30 mg/kg/day
DAF = 6.7% UF A = 10
UF H = 10
UF D = 10
UF L = 3 Target MOE =
3000 Kim et al. (2010)

Inhalation (short- and intermediate- term) NOAEL = 49 μg/m 3 UF A = 10
UF H = 10
UF D = 10 Target MOE =
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.

There are no acceptable dermal toxicity studies on nanosilver available to the Agency. In the absence of any such dermal toxicity studies, the Agency normally uses extrapolation from another route of exposure (usually oral) and assumes a default derm al absorption factor (DAF) of
100% when no in vivo data are available (see Secti on 4.2.3). The available human in vivo study indicating absorption of na nosilver is 6.7% and the in vitro data indicating absorption of
nanosilver from intact and abraded human skin is substantially below 0.1% provide scientific support for setting a conservative DAF of 6.7% fo r the nanosilver that might break away from
the Nanosilva. EPA has determined that the derm al toxicity for the nanosilver in Nanosilva will be evaluated using the oral POD of 30 mg /kg/day and a DAFs of 6.7%. In 2011, EPA used a
DAF of 0.1% that was based on 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 a nd now concludes that the 0.1% DAF was an
underestimate because it was based on the Moie men et al. (2011) study which only reported silver concentrations in the blood of patients. The 6.7% DAF is based on the observational study by Wan et al. (1991) which reported silver concen tration in serum and e liminated through urine.24 There are currently no acceptabl e studies on the reproductive a nd developmental toxicity for 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 developi ng embryos were observed. Therefore, EPA
believes that the oral NOAEL and LOAEL of 30 mg/kg/day and inha lation NOAEL of 49 μg/m 3 are expected to be protective of developm ental 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, l acks histological support for determining NOAEL/LOAELs. The study by Liu et al. (2013) reported no effect s on the spatial cognition or hippocampal activity of mice after injecting nano silver into the body cavity (i.e., intraperitoneal injection) at concentrations of up to 50 mg/kg. Therefore, EP A believes that the oral NOAEL and LOAEL of 30 mg/kg/day are exp ected to be protective of neur otoxic effects of nanosilver.

The effects on spatial cognition and hippocampal act ivity observed by Liu et al. (2012) after administering nasal drops contai ning nanosilver at concentrations of 3 and 30 mg/kg to rats suggest possible neurotoxic eff ects from the inhalation of nano silver. However, EPA believes that the doses used in the Liu et al. (2012) study were 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.

The available in vitro studies suggest that na nosilver may have mutagenic potential. However,
the one available in vivo micronucleus assay performed on rats after oral administration of nanosilver at concentrations of up to 1,000 mg /kg/day (Kim, et al, 2008) indicated that
nanosilver is neither cl astogenic nor aneugenic in vivo ; however, there was no positive proof that
nanosilver reached the bone marrow in the micro nucleus assay. Therefore, EPA believes that the
oral short-term NOAEL and intermediate-term L OAEL of 30 mg/kg/day are expected to be protective of the potential muta genic effects of nanosilver.

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 sus ceptibility within the human population (intraspecies). In addition to the two standard uncertainty factors given ab ove, there is a third uncertainty factor (UF D )25 which accounts for the incomplete characterization of nanosilver toxicity (database). In this case, the Agency has determined that th e inhalation and oral t oxicity data for nanosilv er is sufficient to asses the potential health eff ects caused by nanosilver released from Nanosilva (Table 3). However, as the SAP concluded, there is uncertain ty on how particle parameters such as size, surface charge and coating affect the toxicity pot ential of nanosilver. In a ddition, the database is incomplete with respect to reproductive and deve lopmental effects. Therefore, the Agency is using the maximum 10 fold database uncertainty factor to cover the missing information on reproductive and developmental effects and the po tential for differences between the nanosilver in the 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 Nanosilva because the inhalation POD was based on a 90 day duration study by Song et al. (2012). The target MOE of 1,000 is for evaluating the short-term exposure (< 30 days) continuous daily oral exposures to Nanosilva because the oral POD was based on a 28-day oral toxicity 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 fold 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 Nanosilva 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 MOE is 3,000 (U.S. EPA, 2002), which has already been established for intermediate-term exposures. Therefore, a long-term target MOE cannot be determined from the existing toxicity database.

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, EPA may conclude that ex posure to the pesticide is unlikely to pose a risk concern and, therefor e, will not cause unreasonable adverse effects for26 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 the Agency uses when determining if there is a risk concern from expos ure to a pesticide. The Agency also considers
other scientific evidence in a wei ght of evidence evaluation such as the severity of toxic effects, the controls used to minimize exposures, and th e population exposed to th e pesticide. A risk concern, however, is not the equivalent of a dete rmination that the potential risk constitutes 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) fu rther refine its risk assessment analysis, particularly if conservative assumptions were used to produce the initial assessment; or (3) explicitly analyze any potential be nefits of the pesticide to determine whether, on balance, those benefits out weigh the identified risk.

The Agency’s MOE approach for nanosilver uses mass-based metrics, both for determining the POD and for calculating exposure. The Agency is aware of the ongoing debate within the scientific community that metrics other than ma ss (such as particle number or surface area) may be more suitable for assessing nanosilver risk s and therefore acknowle dges the potential for
limitations of mass-based risk estimates.27 

### V. OCCUPATIONAL AND CONS UMER RISK ASSESSMENT

Based on the proposed use pattern of Nanosilva in plastics and textiles, EPA anticipates humans could be exposed to th e following substances:

1) Silver ions released from Nanosilva; 2) Nanosilva complex; and/or 3) Nanosilver that might break away from the Nanosilva complex.

Because the levels of silver in plastics and textiles incorporating Nanos ilva are lower than for other currently registered products which use silver ions and because the Agency determined that
unreasonable adverse effects from use of silver containing products are unlikely (U.S. EPA, 1993), EPA concluded that the risk from exposure to silver ions derived from plastic and textile products incorporating Nanosilva is not of con cern. Nanosilva LLC has submitted a number of
studies and other information to support its application for re gistration, including information relevant to assessing the toxic ity of and exposure to Nanosilva. In addition, EPA has also
reviewed data and information on nanosilver from th e scientific literature. This section discusses EPA’s assessment of the potential risks to human health from the use of Nanosilva. The first section addresses potential levels of occupationa l exposure and risk to workers who handle the Nanosilva liquid suspension. The final sections a ddress potential levels of consumer exposure
and risk to children who come in contact with plastics and textiles incorporating Nanosilva.



### 5.1 Occupational Risk Assessment fo r Mixing and Loading the Nanosilva Liquid Suspension

EPA expects that occupational inhalation a nd dermal exposures to Nanosilva and the nanoparticles that might break away from Nanos ilva are likely to occur during the following use
scenarios :

1. Mixing and loading of Nanosilva duri ng preparation of a master batch

2. Mixing, loading, and applying the Nanosilva cont aining master batch during treatment of plastics and textiles

3. Handling plastics and textil es incorporating Nanosilva

EPA evaluates the risk of occupational expos ures from mixing, loading, and applying or handling pesticide products. A lthough exposure to Nanosilva duri ng subsequent work activities involving plastics and textiles in corporating Nanosilva can occur in occupationa l settings (items 2 and 3), the risk to workers for these scenarios is unlikely to exceed the risk during mixing and loading Nanosilva during preparation of the ma ster batch (item 1). Therefore, only the mixing and loading of Nanosilva during preparation of a master batch is evaluated in the subsequent
sections.28 5.1.1 Occupational Exposure when Preparing the Master Batch Containing Nanosilva
A master batch consists of polymer pellets (i.e ., plastic pellets) containing Nanosilva which are subsequently used in treating plastics (MRI D 48652901). A master batch is prepared by blending a polymer and the Nanosilva liquid suspension using a mixer and compounding this mixture using an extruder. Workers may be exposed to Nanosilva and nanosilver particles that break away from the Nanosilva complex while loading th e Nanosilva liquid suspension into the master
batch mixer.

Nanosilva LLC did not submit data or information quantifying the amount of Nanosilva that workers are exposed to during lo ading of the Nanosilva liquid suspension into a master batch mixer. To calculate worker exposure, the Agency 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 Pest icide Handlers Exposure Database (PHED) (U.S. EPA, 1998).

Table 4 – Unit Exposures for Mixing and Loading of Liquids Personal Protective Equipment (PPE)
Level Exposure
Route Unit Exposure Data Source

No respirator Inhalation 0.21 (μg/m 3 /lb) AEATF II

Engineering Control – Closed System Loading Inhalation 0.016 (μg/m 3 /lb) PHED

No gloves, use of long-sleeve shirt, long pants, shoes plus socks Dermal 10 (mg/lb) A AEATF II

Gloves, long-sleeve shirt, long pants, shoes plus socks Dermal 1.2 (mg/lb) B AEATF II

Engineering Control – Closed System Loading Dermal 0.0086 (mg/lb) PHED

A Arithmetic mean for conven tional liquid pour scenario

B 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 unit29 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 ai r concentration with units of μg/m 3 per lb of pesticide handled per day. This was done by dividing the air concentrations listed in the PHED 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 repr esent inhalation exposure during the mixing and loading of pesticide containi ng liquids while using closed-system loading.

5.1.2 Occupational Margins of Exposure
As detailed in the following sections, EPA evalua ted the risk to workers who load the Nanosilva liquid suspension into a master batch mixer.

Occupational Inhalation Daily-Exposure and MOEs
The inhalation exposure to the nanosilver in the Nanosilva liq uid suspension was calculated using the following:

Inhalation Daily Exposure = Amount of nanosilver in Nanosilva handled per day × Inhalation Unit Exposure

Where:

 Amount of nanosilver in Nanosilva handled per day is 1.2 lb/day. This value was calculated assuming that the Nanosilva 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. The 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).30 Table 5 – Occupational Handlers Inhalation MOEs for the Nanosilver in the Nanosilva Liquid Suspension

Mix/Load
Nanosilva

Liquid Suspension Amount of
Plastics Treated per day (lb) Application
Rate A (by wieght) Amount of
Nanosilver

Handled per day

(lb) Inhalation
Unit Exposure (μg/m 3 /lb) Daily Exposure C

(μg/m 3 ) MOE D

Without Respirator 40,000 0.003% 1.2 0.21 0.25 200

Closed System Loading (Without Respirator) 40,000 0.003% 1.2 0.016 B 0.02 2,500

A. Maximum amount of nanosilver in plastics incorporating Nanosilva B. PHED unit exposure value converted to air concentration units based on mean 8 hour TWA C. Daily Exposure = Amount nanosilver Handled × Unit Exposure D. MOE = POD / Daily Exposure (rounded to two significant digits)

The MOEs shown in Table 5 were calculated by dividing the inhalation exposure for nanosilver by the POD of 49 μg/m 3 , which is the NOAEL from a 90-da y inhalation toxicity study for nanosilver (Song et al., 2012). This assumes that all the nanosilver in Nanosilva is freely
available after inhalation and be haves like the nanosilver used in the Song et al. (2012) study even though the nanosilver in Nanos ilva is attached to a silica core partic le through a thiolate
bond and is covered with sulfur and polyvinylpyrrolidone. When no respirator is worn the MOE is 200, which is based on the AEATF II unit exposure data, and is less than the target MOE of 1,000 indicating the risk from shor t- and intermediate-term exposur es is of concern. The MOE is 2,500 for workers who use closed-system load ing when mixing and loading the Nanosilva suspension, which is based on PHED unit exposure da ta, and is greater than the target MOE of 1,000 indicating the risk from shor t- and intermediate-term exposur es is not of concern. These
MOEs indicate that the risk c oncern for inhalation exposures to the nanosilver in Nanosilva is mitigated when workers use closed-system load ing during mixing and loading of the Nanosilva liquid suspension.

Occupational Dermal Daily-Dose and MOEs
The dermal dose from exposure to the nanos ilver in the Nanosilva liquid suspension was calculated using the following:

Dermal Daily Dose = (Amount of nanosilver in Nanosilva handled per day × Dermal Unit Exposure × Dermal Absorption Factor) / Body Weight

Where:

 Amount of nanosilver in Nanosilva handled per day is 1.2 lb/day (Table 5).31  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 factor (DAF) for na nosilver is 6.7% (see Section 4.2.3).

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

Table 6 – Occupational Handlers Dermal MOEs for the Nanosilver in the Nanosilva Liquid Suspension

Mix/Load
Nanosilva

Liquid Suspension Amount of
Nanosilver
Added or
Handled A (lb) Dermal
Unit Exposure

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

Without

Gloves 1.2 10 12 0.010 3,000

With Gloves 1.2 1.2 1.4 0.0012 25,000

Closed System Loading 1.2 0.0086 0.01 0.0000086 >1,000,000

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 Factor)/ Body Weight D. Dermal MOE = POD / Daily Dose (rounded to two significant digits)

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 fr om a 28-day oral toxicity st udy (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 Nanosilva is freely available after dermal cont act and behaves like the nanosilver used in the Kim et al. (2008 and 2010) studies even though the na nosilver in Nanosilva is attached to a silica core particle through a thiolate bond and is covered with su lfur and polyvinylpyrrolidone. The MOEs for dermal exposure are 3,000 when no gloves are worn and 25,000 when gloves are worn, which are based on the AEATF II unit exposu re data, and are both gr eater than the target MOE of 3,000 indicating that the risk from shor t- and intermediate-term occupational dermal exposures are not of concern. The MOE is great er than 1,000,000 when closed-system loading is used, which is based on the PHED unit exposure data, and is greater than greater th an the target MOE of 3,000 indicating that the risk from shor t- and intermediate-term occupational dermal exposures are not of concern.32 5.1.3 Conclusions for Occupational Risk
The MOE for occupational inhalation exposur e to the nanosilver in the Nanosilva liquid suspension is 2,500 if workers use close-system loading when mixing and loading the Nanosilva liquid suspension, which is greater than the targetMOE of 1,000 indicating the risk from shortand intermediate-term inhalation exposures is no t of concern. The MOE for dermal exposure to
the nanosilver in the Na nosilva liquid suspension is greate r than 1,000,000 if workers use closedsystem loading when mixing and loading the Nanos ilva liquid suspension, which is greater than the target MOE of 3,000 indicati ng that the risk from short- and intermediate-term dermal exposures is not of concern. Ther efore, EPA proposes to require that closed-system loading as defined in 40 CFR part 170.240(d)(4) be used when mixing and loading the Nanosilva liquid suspension. The proposal to require closed-syste m loading is based on unit exposure data from PHED. The MOEs calculated using unit expos ure data from AEATF II were shown for comparison purposes only and were not relied on to determine that Nanosilva will not cause
unreasonable adverse effects to human health.

EPA does not typically consider long-term occ upational exposures to materials preservatives used to treat plastics or textil es because application of these chemicals does not typically occur on a daily basis for more than 6 months.

There are several uncertainties in the occupa tional risk assessment. The exposure analysis
assumes that all the nanosilver in Nanosilva is freely availabl e even though the nanosilver in
Nanosilva is attached to a sili ca core particle th rough a thiolate bond and the nanosilver in Nanosilva is covered with sulfur and polyvi nylpyrrolidone. The PODs used were based on inhalation and oral toxicity stud ies completed using nanosilver, not Nanosilva or the nanosilver that might break away from Nanosilva, and thus these PODs may not equal PODs determined from testing conducted with Nanosilva or th e nanosilver found in Nanosilva. Because the nanosilver in the toxicity database is different from the nanosilver in Nanosilva and because the database is incomplete with respect to reproduct ive and developmental effects, the Agency used a maximum 10-fold database uncertainty factor when evaluating the risk from occupational exposure to Nanosilva. There are al so uncertainties in extrapolati ng from effects observed after feeding test-animals nanosilver (i .e., oral route) to the effect s that might be observed after applying nanosilver to the skin of te st animals (i.e., dermal route).

5.1.4 Occupational Exposure and Health Data Requirements
EPA is not proposing to require that Nanosilva LLC conduct an indoor applicator study to quantify the unit exposure values during mixing and loading of the Nanosilva liquid suspension
into the master batch mixer. This is because closed-system loading, such as lock and load containers, where the Nanosilva liquid suspension is transferred to a pl astic extruder through piping minimizes occupational exposure to Na nosilva to the greatest extent possible.33 Although there is no risk concern for workers w ho use close-system loading when mixing and loading the Nanosilva liquid suspen sion, EPA is proposing to require an inhalation route-specific subchronic test (OCSPP 870.3465) to confirm the Ag ency’s determination that the risks to
workers who mix and load Nanos ilva are not unreasonable. Also, because workers of child bearing age could handle Nanosilva during mixing and loading, EPA is proposing to require a modified reproduction/developmental toxicity screening test since this data is currently missing from the toxicity database. In sum, EPA is proposing to require these tests to confirm the adequacy of the 10 fold database uncertainty factor, to reduce the uncertainties related to differences in the physical properties of the nanosilver, and because there are currently no acceptable studies on the reproductive and developmental toxicity for nanosilver.

In summary, the following guideline studies are proposed to be required for Nanosilva:

 90-Day Inhalation Toxicity (Rat) (OCSPP 870.3465) modified to include in vivo bone marrow assay and functional observationa l battery, motor activity and detailed neuropathology

 Reproduction/Developmental Toxicity Sc reening Test (Modified OCSPP 870.3550/ OECD TG 421)



### 5.2 Consumer Risk Assessment for Products Incorporating Nanosilva

EPA expects consumer exposures to Nanosilva, the silver ions derived from the Nanosilva complex, and the nanoparticles that break away from the Nanosilva complex could potentially occur during the following use scenarios :

1. Incidental oral exposure to plastics and textiles incorporating Nanosilva

2. Dermal exposure to plastics and te xtiles incorporating Nanosilva

Nanosilva is proposed to be mixed into polymer and polymer based products to suppress the growth of bacterial, algae, fungus, mold and mildew, which cause odors, discoloration, stains, and deterioration of plastics and textiles. Becau se plastics incorporating Nanosilva could be subsequently used to manufacture children’s toys and textiles worn by children, it is assumed that children will be exposed to products containing Nanosilva.

While children younger than 6 months may potentia lly be exposed to plastics and textiles incorporating Nanosilva, it is believed that exposure for children older than 6 months will be equivalent, if not greater, due to behavior al and anatomical/physiological development; therefore, EPA assessed exposure to children older than 6 months. EPA’s recently revised its standard operating procedures (S OPs) for residential pesticide exposure assessments (U.S. EPA, 2012b). Based on the combined quantitative and qualita tive analysis of the index lifestage, the Agency has determined that the 1 < 2 year old lifestage represents the most appropriate index34 lifestage for children for most exposure scen arios. In 2011, EPA evaluated the exposure to textiles containing the nanosilver based pesticide HeiQ AGS-20 usi ng children in the 2 to 3 year old lifestage based on the Agency’s determination, at that time, that the 2 to 3 year old child was likely the most vulnerable subpopulation from ch ewing on and wearing textiles treated with HeiQ AGS-20 (U.S. EPA, 2011a). After issuing the registration decision for HeiQ AGS-20, questions were raised about the use of the 2 to 3 year old lifestage. In light of those questions and in light of the 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 ye ar old age range from plastic toys and flooring and textiles incorporating Nanosilva for transparency.

5.2.1 Consumer Exposures to Plastics and Textiles Incorporating Nanosilva
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 textiles incorporating nanosilver. 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 m easuring 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 Nanos ilva and using laundry detergent and simulated
saliva for shirts inco rporating Nanosilva.

Plastics Leaching Study
Nanosilva LLC submitted a leaching study on the migration of Nanosilva from a linear lowdensity polyethylene (LLDPE) plastic (MRI D 478289-25). The study was based on Food and Drug Administration methodology to determine the migration of chemicals from plastics which contact food. The leaching study involved 44 LLDPE coupons with 14 being ashed to determine silver content and 28 used in the leaching tests. The 28 plastic coupons were prepared with four
Nanosilva master batch concentrations where seven coupons each contained 0, 2.5, 5, and 10%, respectively. 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 approximate mass of 15g. Each coupon was exposed to 50 mL of the fluids listed in Table 7 at temperatures of 40 and 100 degrees 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 was 0, 1500, 3000, and 6000 μg/L, respectively.

In general, the concentration of silver in th e fluids contacting plasti c incorporating Nanosilva was low with most values below the analytical detection limit. The analysis method, inductively coupled plasma atomic emission spectroscopy (ICP -AES), employed an acid digestion and thus
the silver concentrations are for total silver content. The Nanosilva LLC leaching study reported35 method detection limits that ranged from 0.8 to 2 μg/L, with the exception of the Oil trial where the detection limit was 240 μg/L fo r the times of less than 240 hours. However, because there was no method detection limit study provided to support these values , EPA is using the silver detection limit of 5 μg/L, which is the value repo rted as the estimated detection limit in U.S. EPA Method 6010C (U.S. EPA, 2007a) and in the Nanosilva LLC submitted leaching study (Table 1, Page 30).

Table 7 – Concentration of Silver in Fluid Co ntacting the Nanosilva Containing Plastic Coupons at 40 degrees Celsius

Fluid 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

Alcohol, 10% ethanol in water 14 of 15 samples < 5 μg/L (11 μg/L for the 5% coupon after 24 hr) 34 to 120

Salt, 10% sodium phosphate
monobasic in water 13 of 15 samples < 5 μg/L (9.9 μg/L for the 10% coupon after 24 hr) 80 to 174

Oil, 10% olive oil in water 12 of 12 samples < 240 μg/L 3 of 3 samples < 5 μg/L 5.7 to 7.8

Salt and Sugar, 10% sodium phosphate
monobasic and 10% sucrose in water 30 of 30 samples < 5 μg/L (14 μg/L for the 0% coupon after 168 hr at 40°C) 13.6 to 92

Salt and Sugar after scrubbing the plastic coupon 13 of 15 samples < 5 μg/L (27 μg/L for the 5% coupon after 48 hr) 3.6 to 6.8

Given that EPA is evaluating human exposure to plastics incorporating Nanosilva which occur at physiological temperatures (i.e., 98.6 degrees Fahr enheit 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 micr owaving plastics than of contact with humans. Silver was found above the detection limit during 4 out of the 105 coupon leaching trials conducted at 40 degrees Celsius where the maximu m silver concentration of 27 μg/L was found after 48 hours in a plastic c oupon containing 5% Nanosilva master batch (10 mg/kg nanosilver) (Table 7). A value of 27 μg/L represents 0.9% of the maximum concentration of silver that could
have leached from the 5% Nanosilva contai ning coupon. However, there was no clear36 correlation between Nanosilva content and the du ration or temperature of each experimental trial. In one analysis, silver was found in the sa lt and sugar fluid after 168 hours at 40 degrees Celsius in a coupon that did not contain Nanosilva (0% nanosilver) suggesting that the detections of silver may have been due to contamination fr om sources of silver ot her than from Nanosilva. Interpretation of these results is further compli cated by the wide range in silver matrix spike recoveries determined for the alcohol, salt, oil, and salt and sugar fluids. This range in silver matrix spike recoveries may indicate that nanosilver released from the coupons was not detectable by the analytical method.

The average concentration of silver was 3.3 μg/L , as calculated for the trials conducted at 40 degrees Celsius. This was based on the concentr ations reported and assuming that all results reported as being below the detection limit had a value of one-half the detection limit (i.e., 2.5 μg/L), which is consistent with how EPA assigns values to non-detect results when evaluating pesticide residues in food (US EPA, 2000). This average excludes the concentrations reported for
the oil trial because the detec tion limit for this trial was reported as 240 μg/L. The average concentration of 3.3 μg/L was determined for conditions where the plastic coupon incorporating Nanosilva was in contact with fluids having low and high pH, high salt and sugar content, and after mechanical abrasion of plastic incorpora ting Nanosilva. Thus, this average concentration represents a range of conditions that are expected to occur du ring residential use of plastics incorporating Nanosilva. However, EPA is only eval uating risk from human contact with plastics incorporating Nanosilva, and ther efore, only the results for low and high pH fluids, which are more representative of biological fluids, are included in this a ssessment. The concentration of silver in the low and high pH fluids were all below the analytical detection limit of 5 μg/L meaning that EPA used 2.5 μg/L as the concen tration of silver re leased from plastics incorporating Nanosilva. Since this value is belo w the detection limit, the form of silver is
unknown and was assumed to be in the form of nanosilver as found in Nanosilva. Using the assumed concentration of 2.5 μg /L equates to silver releas es of 0.17, 0.08, 0.042% from the coupons containing 5, 10, and 20 mg/kg of nanosilv er, respectively. Nanosilva LLC is proposing that plastic products incorporating Nanosilv a contain a maximum of 0.003% nanosilver by weight or 30 mg/kg of nanosilver. Assumi ng that a leaching study conducted using a coupon containing 30 mg/kg of nanosilver would also result in silver re leases below the detection limit
would result in a silver release rate of 0.028% for a coupon containing 30 mg/kg of nanosilver. However, Nanosilva LLC did not submit leaching results for coupons containing 30 mg/kg of
nanosilver.

The leaching study submitted by Nanosilva LLC for plastic coupons incorporating Nanosilva was initially determined to be unacceptable by EP A because less than 0.8% of the silver in the plastic coupons was recovered durin g the ashing process, the wide range in silver matrix-spike recoveries, and the lack of validation data for the silver analysis. Nanosilva LLC subsequently completed an additional study to determine the si lver content of identically prepared plastic37 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 Nanosilva liquid suspension, a master batch containing 2% Nanosilva, and plastic coupons containing 5% of the Nanosilva master batch. Using NAA, Nanosilv a LLC determined the amount of silver in coupons was within 99% of the amount added.

The available leaching study for plastic coupons containing Nanosilva was conducted using a protocol that was not reviewed by EPA prior to conducting the study. The leaching study did not employ physiological fluids such as artificial saliva, however, the study was completed at the physiological temperature of 40 degrees Celsius. The coupons tested contained nanosilver at concentrations of 5, 10, and 20 mg/kg, whic h are less than the maximum nanosilver concentration of 30 mg/kg that Nanosilva LLC is proposing for the Nanosilva pesticide label. Even though these issues preclude EPA from accepting the leaching study as final, the study does provide a reasonable first esti mate for the amount of silver transferred to the mouth while mouthing and transferred to skin while cont acting plastics incorporating Nanosilva.

Textile Leaching Study
Nanosilva LLC prepared and submitted a draft protocol titled “The Quantification and
Characterization of Silver Released from Te xtiles Treated with Nanosilva (NSPW-L30) as a Results of Washing” with the stated purpose to quantify and characterize silver released from textiles incorporating Nanosilv a during laundering. The prot ocol was based on methods developed by Geranio et al. ( 2009) and Lorenz et al. (2012) who modified the ISO Colour Fastness test (ISO, 1997) to determine the amount a nd form of silver released to the environment when washing textiles containing silver and nanosilver. Because EPA must also evaluate the
release of nanosilver when ch ildren chew and mout h textiles incorpor ating Nanosilva, EPA recommended including an additional test using simulated human saliva. EPA does not anticipate dermal exposure will re sult in a risk concern for tex tiles incorporating Nanosilva, and therefore a separate leachi ng study involving simulated huma n 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 nanosilv er from textiles in corporating Nanosilva.

Nanosilva LLC completed and submitted the m odified ISO Colour Fastness test (MRID 49045301) using shirts incorporati ng Nanosilva. The washing test s were conducted on shirts composed of polyethylene terepht halate (PET), which incorporated Nanosilva into the PET yarn
for a final nanosilver content of 0.00262% or 26.2 mg/kg per shirt. For the detergen t wash test, a
section was cut from each of three shirts and wash ed separately in 150 mL of distilled water with commercial laundry detergent and 10 hard rubber balls with diamet er of 10 mm for 30 minutes at 40 degrees Celsius. The detergent wash was follow ed by two rinse cycles with deionized water.
The concentration of silver in the wash and rins e water were determined after filtering samples through a 0.45 μm pore size filter using induc tively coupled plasma mass spectrometry (ICP-38 MS). The concentration of silver retained on the filter was also determined by ICP-MS. These samples were acid digested prior to analysis and t hus the silver concentrations are for total silver content and do not distinguish between silver ions, nanosilver , or the Nanosilva complex.

Although the Nanosilva wash test study reporte d an ICP-MS detection method of 0.0094 μg/L for aqueous samples and 0.94 μg/kg for filter samples, EPA determined that the detection limit for aqueous samples was 10 μg/L and 1,000 μg/kg for solids samples. EPA determined these detection limits based on a statistical analysis of seven ICP-MS calib ration curves and the analysis results from 100 μg/L quality control sa mples. The concentration of silver in the wash and rinse water from the detergent wash test fo r three shirt sections was below the analytical detection limit of 10 μg/L (Table 8). The concen tration of silver reta ined on 0.45 μm pore size filters was also less than the analyt ical detection limit of 1,000 μg/kg.

Table 8 – Concentration of Silver Released from Shirts Incorporating Nanosilva during
Modified ISO Colour Fastness Test

Number of
Shirt Sections Wash Medium Concentration of Silver

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

(μg/kg)

3 Distilled Water with Detergent < 10 < 1,000

9 Simulated Human
Saliva < 10 < 1,000

A separate study was conducted using three secti ons from each of three shirts and washed separately in 150 mL of simulated human saliva with 10 hard rubber balls with diameter of 10 mm for 45 minutes at 40 degrees Celsius. The concen tration of silver in the simulated saliva was determined after filtering through a 0.45 μm pore size filte r using ICP-MS where the
concentration of silver in the saliva for nine shirt s ections was below the analytical detection limit of 10 μg/L (Table 8). The concentration of si lver retained on filters was also less than the analytical detection limit 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 in corporating Nanosilva by replacing the non-detect values with half the detection limit, 5 μg/L fo r liquid samples and 500 μg/kg for solids samples (U.S. EPA, 2000). The amount of silver released from textile s 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).39 Table 9 – Amount of Silver Released from Shirts Incorporating Nanosilva

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 detection limit

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 simu lated human saliva was 94.9 μg,
therefore the amount of silver rel eased during the saliva wash was:

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

The value of 1.6% will be used in evaluating rele ases to the environment from wash water and the value of 0.9% will be used in calculating oral and dermal exposures to textiles incorporating
Nanosilva. Since these releases were determined using concentrations th at were below the ICPMS detection limit, the form of silver is unknown. EPA will assume the form of silver is identical to the nanosilver present in Nanos ilva in the absence of further information.

The results of these studies de monstrate that PET shirts wh ich incorporate Nanosilva at 26.2 μg/kg of nanosilver do not release silver at concentrations above the analytical detection limit. The ISO Colour Fastness test is thought to re present aggressive washing conditions with one wash cycle representing up to five domestic or commercial laundering cycles when the multiple test is employed. The amount of silver released during one ISO Colour Fa stness test is believed40 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 agit ation for 30 to 45 minutes. Thus, results from
studies which are based on the ISO Colour Fastness te st will be used to determine the daily dose of nanosilver for children who chew and mouth, adults who wear, and wo rkers 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 Nanosilva
EPA expects that consumers will be exposed to pl astics incorporating Nanosilva by the routes of incidental oral and dermal exposures. As detail ed 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 Nanosilva.

Consumer Incidental Oral Daily-Dose a nd MOE for Toys Incorporating Nanosilva
Incidental oral exposures to t oys incorporating Nanosilva were calculated using the following:

Incidental Oral Exposure = Surface Residue (mg/cm 2 ) × Saliva Extraction Efficiency (%) × Surface Area of Toy Mouthed per day (cm 2 ) Where:

 Surface area of a toy mouthed per day is 800 cm 2 for 6 to < 12 month old children, 560

cm 2 for 1 to < 2 year old children, and 396 cm 2 for 2 to < 3 year old children. 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 child ren contact objects 20 times per hour, 1 to < 2 year old children contact objects 14 times per hour, and 2 to < 3 year old children contact objects 9.9 times per hour (U.S. EPA, 2011a) for 4 hours per day.

The concentration of silver detected in low and high pH fluids, which are more representative of biological fluids, contacting Nanos ilva containing coupons represents both the surface residue and the saliva extraction efficiency. The concentra tion of silver in the low and high pH fluids were all below the analytical detection lim it of 5 μg/L for coupons containing 5, 10, and 20 mg/kg of Nanosilva. EPA is assuming that non-det ectable concentrations of silver would be found in a similar test conducted with coupons c ontaining 30 mg/kg of Nanosilva. EPA is using 2.5 μg/L, which is one-half the de tection limit of 5 μg/L, as the c oncentration of si lver released from plastic incorporating Nanos ilva. Using the concentration of silver, the volume of the leachate (50 mL), and the surface area of the test coupons, the amount of available nanosilver at the coupon surface is:

Surface residue and saliva extraction: ଴.଴଴ଶହ ௠௚

௅ ൈ ହ଴ ௠௅ ൈ ௅

ଵ଴଴଴ ௠௅ ൈ ଵଵଶ ௖௠ మ ൌ ଴.଴଴଴଴଴ଵଵଶ௠௚

௖௠ మ41 The incidental oral exposure for toys incorporating Nanosilva is:

Incidental oral exposure for 6 to < 12 month old children: ଵ.ଵଶൈଵ଴ షల ௠௚ ௡௔௡௢௦௜௟௩௘௥

௖௠ మ ൈ ଼଴଴ ௖௠ మ
ௗ௔௬ ൌ

଴.଴଴଴଼ଽଷ ௠௚ ௡௔௡௢௦௜௟௩௘௥ ௗ௔௬

Incidental oral exposure for 1 to < 2 year old children: ଵ.ଵଶൈଵ଴ షల ௠௚ ௡௔௡௢௦௜௟௩௘௥

௖௠ మ ൈ ହ଺଴ ௖௠ మ
ௗ௔௬ ൌ

଴.଴଴଴଺ଶହ ௠௚ ௡௔௡௢௦௜௟௩௘௥ ௗ௔௬

Incidental oral exposure for 2 to < 3 year old children: ଵ.ଵଶൈଵ଴ షల ௠௚ ௡௔௡௢௦௜௟௩௘௥

௖௠ మ ൈ ଷଽ଺ ௖௠ మ
ௗ௔௬ ൌ

଴.଴଴଴ସସଶ ௠௚ ௡௔௡௢௦௜௟௩௘௥ ௗ௔௬ The incidental oral daily-dose was calculated from the incidental or al exposure using the following:

Incidental Oral Daily Dose = Exposure / Body Weight

Where:

 Exposure is determined in the calculations 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 years is 13.8 (U.S. EPA, 2011b).

Table 10 – Incidental Oral MO Es for Children Exposed to Plastic Toys Incorporating
Nanosilva

Age Range of Child Exposure A
(mg/day) Daily Dose B
(mg/kg/day) MOE C

6 to < 12 months 8.93×10 -4 9.7×10 -5 310,000

1 to < 2 years 6.25×10 -4 5.5×10 -5 550,000

2 to < 3 years 4.42×10 -4 3.2×10 -5 940,000

A. Exposure = Surface Residue (mg/cm 2 ) × Saliva Extraction Efficiency × Surface Area of Toy Mouthed (cm 2 ) B. Dose = [Exposure (mg/day)] / Body Weight (kg) C. MOE = POD / Daily Dose (round ed to two significant figures)42 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 oral toxi city study (Kim et al., 2010). The MOEs for incidental oral exposures ar e 310,000 for 6 to < 12 month old children, 550,000 for 1 to < 2 year old children, and 940,000 for 2 to <3 year old children, which are greater than the target MOE of 3,000 indicating that the risk for short- and intermediate-term exposure to children who mouth plastic toys cont aining Nanosilva is not of concern.

Consumer Dermal Daily-Dose and MOEs to Flooring Incorporating Nanosilva
The dermal exposure to flooring incorporati ng Nanosilva was calculated using the following:

Dermal Exposure = Surface Residue (mg/cm 2 ) × Transfer factor from floor ing to skin (%) × Body surface area in contact with floor (cm 2 /day) Where the:

 Body surface area contacting flooring is 4,500 cm 2 /day for children between 6 and < 12 months of age, 5,300 cm 2 /day for children between 1 and < 2 years of age, and for a 2 to < 3 year old is 6,100 cm 2 /day (U.S. EPA, 2011b). These ar e the total surface area of children and don’t account for the porti on of child covered with clothing.

The concentration of silver detected in fluids contacting Nanosilva cont aining coupons (Table 7) represents both the surface residue and the frac tion of Nanosilva transfer red from flooring to
skin. The concentration of s ilver detected in low and high pH fluids, which are more representative of biological flui ds, were all below the analytical detection limit of 5 μg/L for coupons containing 5, 10, and 20 mg/kg of Nanos ilva. EPA is assuming that non-detectable
concentrations of silver woul d be found in a similar test co nducted with coupons containing 30 mg/kg of Nanosilva. EPA is using 2.5 μg/L, whic h is one-half the detection limit of 5 μg/L, as
the concentration of silver released from plastic incorporating Nanosilva. Using the concentration of silver, the volume of the leachat e (50 mL), and the surface area of the test coupons, the amount of available nano silver at the coupon surface is:

Surface Residue × Transfer factor from flooring to skin: ଴.଴଴ଶହ ௠௚

௅ ൈ ହ଴ ௠௅

ଵ଴଴଴ ௠௅ ൈ ଵଵଶ ௖௠ మ ൌ

଴.଴଴଴଴଴ଵଵଶ ௠௚

௖௠ మ The dermal exposure for floori ng incorporating Nanosilva is:
Dermal Exposure for 6 to < 12 month old children:

ଵ.ଵଶൈଵ଴ షల ௠௚

௖௠ మ ൈ ସହ଴଴ ௖௠ మ
ௗ௔௬ ൌ ଴.଴଴ହ଴ଶ ௠௚ ௡௔௡௢௦௜௟௩௘௥ ௗ௔௬ Dermal Exposure for 1 to < 2 year old children:

ଵ.ଵଶൈଵ଴ షల ௠௚

௖௠ మ ൈ ହଷ଴଴ ௖௠ మ
ௗ௔௬ ൌ ଴.଴଴ହଽଶ ௠௚ ௡௔௡௢௦௜௟௩௘௥ ௗ௔௬43 Dermal Exposure for 2 to < 3 year old children:

ଵ.ଵଶൈଵ଴ షల ௠௚

௖௠ మ ൈ ଺ଵ଴଴ ௖௠ మ
ௗ௔௬ ൌ ଴.଴଴଺଼ଵ ௠௚ ௡௔௡௢௦௜௟௩௘௥ ௗ௔௬ The dermal daily-dose was calculated from the dermal exposure using the following:

Dermal Daily Dose = Exposure × Dermal absorption factor (%) / Body Weight

Where:

 Exposure is determined in the calculations above.

 The DAF is 6.7% (see Section 4.2.3).

 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 years is 13.8 (U.S. EPA, 2011b).

Table 11 – Dermal MOEs for Children Exposed to Flooring Incorporating Nanosilva

Age Range of Child Dermal Exposure A

(mg/day) Daily Dose B
(mg/kg/day) MOE C

6 to < 12 months 5.02×10 -3 3.7×10 -5 820,000

1 to < 2 years 5.92×10 -3 3.5×10 -5 860,000

2 to < 3 years 6.81×10 -3 3.3×10 -5 910,000

A. Exposure = Surface Residue (mg/cm 2 ) × Floor to Skin Transfer Factor (%) × Skin Surface Area of Child (cm 2 ) B. Daily Dose = [Exposure (mg/day)] × Dermal absorption factor (%) / Body Weight (kg) C. MOE = POD / Daily Dose (round ed to two significant figures)

The MOEs in Table 11 are for dermal exposures to flooring incorporating Nanosilva 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) a nd the LOAEL from a 90 day oral toxicity study (Kim et al., 2010). The MOEs for dermal exposures listed in Table 11 are 820,000 for 6 to < 12 month old children, 860,000 for 1 to < 2 year old children, and 910,000 for 2 to < 3 year old children, which are greater than the target MOE of 3,000 indicating that the risk for short- and intermediate-term exposure to ch ildren who contact flooring inco rporating Nanosilva is not of

concern.

Consumer Incidental Oral Daily-Dose and MOEs to Flooring Incorporating Nanosilva
The incidental oral exposure to nanosilver in flooring incorporating Na nosilva was calculated using the following:44 Incidental Oral Exposure = Surface Residue (mg/cm 2 ) × Transfer factor from flooring to skin (%) ×
Surface area of hand that contacts th e floor and the child’s mouth (cm 2 /event) × Frequency of hand-tomouth contacts (events/hr) × Saliva extraction efficiency (%) × Exposure time (hr/day)

Where the:

 Surface area of the hands th at contact both the floor and the child’s mouth is 10

cm 2 /event for children from 1 to < 2 years of age (U.S. EPA, 2012b).

 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/hr for 2 to <3 year old children (U.S. EPA, 2011b).

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

 Exposure time is 101 minutes per day for 6 to < 12 month old children, 126 minutes per day for 1 to < 2 year old children, and 108 mi nutes/day for 2 to <3 year old children
which are the mean times for “Doers” in kitchens and bathrooms (Table 16-15, U.S. EPA, 2011b)

The concentration of silver detected in fluids contacting Nanosilva cont aining coupons (Table 7) represents both the surface residue and the frac tion of Nanosilva transfer red from flooring to
skin. The concentration of s ilver detected in low and high pH fluids, which are more representative of biological flui ds, were all below the analytical detection limit of 5 μg/L for coupons containing 5, 10, and 20 mg/kg of Nanos ilva. EPA is assuming that non-detectable
concentrations of silver woul d be found in a similar test co nducted with coupons containing 30 mg/kg of Nanosilva. EPA is using 2.5 μg/L, whic h is one-half the detection limit of 5 μg/L, as
the concentration of silver released from plastic incorporating Nanosilva. Using the concentration of silver, the volume of the leachat e (50 mL), and the surface area of the test coupons, the amount of available nanosilver at the coupon surface is:

Surface Residue × Transfer factor from flooring to skin: ଴.଴଴ଶହ ௠௚

௅ ൈ ହ଴ ௠௅ ൈ ௅

ଵ଴଴଴ ௠௅ ൈ

ଵଵଶ ௖௠ మ ൌ ଴.଴଴଴଴଴ଵଵଶ ௠௚

௖௠ మ

The incidental oral exposure for fl ooring incorporating Nanosilva is:

Incidental Oral Exposure for 6 to < 12 month old children: ଵ.ଵଶൈଵ଴ షల ௠௚

௖௠ మ ൈ ଵ଴ ௖௠ మ

௘௩௘௡௧ ൈ ଵଽ ௘௩௘௡௧௦

௛௥ ൈ

ହ଴%
ଵ଴଴% ൈ ଵ଴ଵ ௠௜௡ ௗ௔௬ ൈ ௛௥

଺଴ ௠௜௡ ൌ ଴.଴଴଴ଵ଻଼ ௠௚ ௡௔௡௢௦௜௟௩௘௥ ௗ௔௬

Incidental Oral Exposure for 1 to < 2 year old children: ଵ.ଵଶൈଵ଴ షల ௠௚

௖௠ మ ൈ ଵ଴ ௖௠ మ

௘௩௘௡௧ ൈ ଶ଴ ௘௩௘௡௧௦

௛௥ ൈ

ହ଴%
ଵ଴଴% ൈ ଵଶ଺ ௠௜௡ ௗ௔௬ ൈ ௛௥

଺଴ ௠௜௡ ൌ ଴.଴଴଴ଶଷସ ௠௚ ௡௔௡௢௦௜௟௩௘௥ ௗ௔௬45 Incidental Oral Exposure for 2 to < 3 year old children: ଵ.ଵଶൈଵ଴ షల ௠௚

௖௠ మ ൈ ଵ଴ ௖௠ మ

௘௩௘௡௧ ൈ ଵଷ ௘௩௘௡௧௦

௛௥ ൈ

ହ଴%
ଵ଴଴% ൈ ଵ଴଼ ௠௜௡ ௗ௔௬ ൈ ௛௥

଺଴ ௠௜௡ ൌ ଴.଴଴଴ଵଷଵ ௠௚ ௡௔௡௢௦௜௟௩௘௥ ௗ௔௬ The incidental oral daily-dose was calculated fr om the dermal exposure using the following:

Incidental Oral Daily-Dose = Exposure / Body Weight

Where:

 Exposure is determined in the calculations 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 years is 13.8 (U.S. EPA, 2011b).

Table 12 – Incidental Oral MOEs for Children Exposed to Flooring Incorporating
Nanosilva

Age Range of Child Oral Exposure A (mg/day) Daily Dose B
(mg/kg/day) MOE C

6 to < 12 months 1.8×10 -4 1.9×10 -5 >1,000,000

1 to < 2 years 2.3×10 -4 2.1×10 -5 >1,000,000

2 to < 3 years 1.3×10 -4 0.95×10 -5 >1,000,000

A. Exposure = Surface Residue (mg/cm 2 ) × Transfer factor from flooring to skin (%) × Surface area of hand that contacts the floor and the child’s mouth (cm 2 /event) × Frequency of hand-to-mouth contacts (events/hr) × Saliva extraction efficiency (%) × Exposure time (hr/day) B. Dose = [Exposure (mg/day)] / Body Weight (kg) C. MOE = POD / Daily Dose

The MOEs in Table 12 are for incidental oral exposures to flooring incorporating Nanosilva and
were calculated from the incidental oral dos e using the POD of 30 mg/kg/day, which is the NOAEL from a 28-day oral toxicity study (Kim et al., 2008) and th e LOAEL from a 90 day oral toxicity study (Kim et al., 2010). The MOEs for incidental oral exposures are greater than 1,000,000 for 6 to < 12 month old, 1 to < 2 year old children, and 2 to < 3 year old children, which are greater than the ta rget MOE of 3,000 indicating that the risk for short- and intermediate-term exposure to children who mouth their ha nds after contacting flooring incorporating Nanosilva is not of concern.

5.2.3 Consumer Margins of Exposure to Textiles Incorporating Nanosilva
EPA expects that consumers will be exposed to te xtiles incorporating Nanosilva by the routes of inhalation, dermal, and incidental oral exposures.46 Consumer Inhalation Daily-Dose and MOE
EPA recognizes the potential for inhalation expo sure to nanosilver during laundry drying of textiles incorporating Nanosilva. However, EPA lacks informa tion on the release rate of nanosilver from textiles incorporating Nanos ilva during laundry drying. While exposure may occur during laundry drying, EPA believes that wh en compared to exposure through dermal and
oral contact with textil es incorporating Nanosilva, exposure during laundry drying will likely be of lower significance. To support this, an estima te of the inhalation dose that might occur from exposure to textiles incorporat ing Nanosilva during laundry dryi ng was calculated using the following:

Inhalation Daily Dose = Amount of Nanosilva in clothing handled per day × Unit Exposure

Where:

 Amount of Nanosilva in clothing handled per day assumed that one t-shirt containin
Nanosilva 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).
Table 13 – Inhalation MOE for Drying Textiles Incorporating Nanosilva

Scenario Amount of
Nanosilver Laundered per day A Unit Exposure B (μg/m 3 /lb AI) Daily Dose C (μg/m 3 ) MOE D

Unload Clothes Dryer Without Respirator 4.5 mg 7.8 0.000077 640,000

A. Amount of AI laundered per day = Amount of Nanosilva 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 (round ed to two significant digits)

The MOE shown in Table 13 was calculated by dividing the inhalation dose by the POD of 49 μg/m 3 , which is the NOAEL from a 90-day inhalati on toxicity study for nanosilver (Song et al., 2012). This assumes that Nanosilva released from textiles during drying behaves like nanosilver after inhalation. This analysis further assumes that all of the nanosilv er from the textile incorporating Nanosilva becomes airborne dur ing 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 indicates that the risk for shor t- and intermediate-term exposure to laundering one textile incorporating Nanosilva per day is no t of concern. Up to 640 t-shirts incorporating Nanosilva could be laundered per day and the calculated MOE woul d be greater than the target47 MOE of 1,000, indicating that the risk for shor t- and intermediate-term exposure to drying textiles incorporating Nanos ilva is not of concern.

Consumer Dermal Daily-Dose and MOEs
The dermal exposure to textiles incorporating Nanosilva was calculated using the following:

Dermal Exposure = Amount of Nanosilva in Textile × Cloth Density × Surface Area Exposed × Transfer Efficiency

Where:

 The textile incorporating Nanosilv a 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 base d on the amount of silver released during the leaching study which wa s 0.9% (see Table 9).

Table 14 – Dermal Exposure to Textiles Incorporating Nanosilva

Age Range 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)

6 to <12 months 30 10 4,500 0.9% 0.0122

1 to < 2 years 30 10 5,300 0.9% 0.0143

2 to < 3 years 30 10 6,100 0.9% 0.0165

A. Exposure = Application Rate × Cloth Density × Surf ace Area Exposed × Cloth-to-Skin Transfer Efficiency × Dermal Absorption Factor

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

Dermal Daily Dose = Exposure × Dermal Absorption Factor / Body Weight

Where:

 Exposure is determined in the calculation above.48  The DAF is 6.7% (see Section 4.2.3).

 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 years is 13.8 (U.S. EPA, 2011b).

Table 15 – Dermal MOEs for Children Exposed to Textiles Incorporating Nanosilva

Age Range of Child Body Weight of Child (kg) Exposure (mg/day) Daily Dose A (mg/kg/day) MOE B

6 to <12 months 9.2 0.012 8.8×10 -5 340,000

1 to < 2 years 11.4 0.014 8.4×10 -5 360,000

2 to < 3 years 13.8 0.016 8.0×10 -5 370,000

A. Dose = [Exposure (mg/day)] × DAF / Body Weight (kg) B. MOE = POD / Daily Dose (round ed to two significant figures)

The MOEs for dermal exposures were calculate d from the dermal dose using the POD of 30 mg/kg/day, which is the NOAEL fr om a 28-day oral toxicity st udy (Kim et al., 2008) and the LOAEL from a 90 day oral toxic ity study (Kim et al., 2010). Th e MOEs for dermal exposures are 340,000 for 6 to < 12 month old, 360,000 for 1 to < 2 year old children, and 370,000 for 2 to < 3 year old children, which are gr eater than the target MOE of 3,000 indicating that the risk for short- and intermediate-term exposure to childr en from wearing textile s incorporating Nanosilva
is not of concern.

Consumer Incidental Oral Daily-Dose and MOEs
Incidental oral exposures were calculated using the following:

Incidental Oral Exposure = Amount of Nanosilva in Textile × Cloth Density × Surface Area Mouthed × Saliva Extraction Efficiency

Where:

 The textile incorporating Nanosilv a 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 th e area of blanket or shirt sleeve.49  The nanosilver saliva extraction efficiencies for mouthing fabric are based on the results of the leaching study which was 0.9% (see Table 9).

Table 16 – Incidental Oral Exposure to Textiles Incorporating Nanosilva

Application Rate (mg/kg) Cloth Density (mg/cm 2 ) Surface Area
Mouthed (cm 2 /day) Saliva
Extraction Efficiency Exposure A
(mg/day)

30 10 100 0.9% 0.00027

A. Exposure = Application Rate × Cloth Density × Surface Area Exposed × Saliva Extraction Efficiency

The incidental oral daily-dose was calculated from the incidental or al exposure using the following:

Incidental Oral Daily-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 years is 13.8, which is for children who mouth textiles (U.S. EPA, 2011b).

Table 17 – Incidental Oral MOEs for Children Exposed to Textiles Incorporating
Nanosilva

Age Range of Child Body Weight
of Child Exposure (mg/day) Daily Dose A (mg/kg/day) MOE B

6 to <12 months 9.2 0.00027 2.9×10 -5 >1,000,000

1 to < 2 years 11.4 0.00027 2.4×10 -5 >1,000,000

2 to < 3 years 13.8 0.00027 2.0×10 -5 >1,000,000

A. Dose = [Exposure (mg/day)] / Body Weight (kg) B. MOE = POD / Daily Dose (round ed to two significant digits)

The MOEs in Table 17 are for incidental oral ex posures were calculated from the incidental oral dose using the POD of 30 mg/kg/day, which is the NOAEL from a 28-da y oral toxicity study (Kim et al., 2008) and the LOAEL from a 90 da y oral toxicity study (Kim et al., 2010). The MOEs for incidental oral exposures are grea ter than 1,000,000 for 6 to < 12 month old, 1 to < 250 year old, and 2 to < 3 year old children, which are greater than the target MOE of 3,000 indicating that the risk for short- and intermediate-term expos ure to children w ho mouth textiles incorporating Nanosilva is not of concern.

5.2.4 Consumer Aggregate Margins of Exposure to Plastics and Textiles Incorporating
Nanosilver
In the Federal Food, Drug, and Cosmetic Act (FFDCA ), Congress specified th at, to establish an acceptable level of a given pesticide’s chemical residue that could be found in or on food products, EPA must determine that “there is a r easonable certainty that no harm will result from aggregate exposures to the pestic ide chemical residue, including a ll anticipated dietary exposures and all other exposures for which there is reliable information.” Furthermore, when enacting this provision of the FFDCA, Congress also amende d FIFRA’s definition of unreasonable adverse effects. Specifically, Congress redefined unreas onable adverse effects to include “a human dietary risk from residues that re sult from a use of a pe sticide in or on any food inconsistent with the standard under” the FFDCA. In other words, Congress explicitly requir ed the consideration of aggregate exposures for registration decisions under FIFRA for food-use pesticides, but chose not to similarly alter the st atutory requirements for non food-use pesticides.

For transparency, 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 in corporating Nanosilva. Inhalati on exposure to children during laundry drying of textiles incorporating Nanosilva is not anticipated and is not considered as part of the aggregate exposure. The aggregate daily dose was calculated by adding the daily oral and dermal doses using the following:

Aggregate Daily Dose = Incidental Oral Dose to Toys Incorporating Nanosilva + Dermal Dose to Flooring Incorporating Nanosilva + Inci dental Oral Dose to Flooring Incorporating Nanosilva + Dermal Dose to Textiles Incorpor ating Nanosilva + Incidental Oral Dose to Textiles Incorporating Nanoslva

Where:

 Incidental Oral Daily Dose to Toys Incorporating Nanosilva is from Table 10

 Dermal Daily Dose to Flooring Inco rporating Nanosilva is from Table 11

 Incidental Oral Daily Dose to Flooring In corporating Nanosilva is from Table 12

 Dermal Daily Dose to Textiles In corporating Nanosilva is from Table 15

 Incidental Oral Daily Dose to Textiles Incorporating Nanosilva is from Table 17

The oral and dermal daily doses can be summed because they are evaluated using the same POD of 30 mg/kg/day. The aggregate MOE in Table 18 for 6 and < 12 month old children is 110,000,51 is 140,000 for children who are 1 and < 2 years, and 170,000 for 2 to <3 year old children, which
indicates that the risk for shor t- and intermediate-term exposure to children is not of concern.

Table 18 – Aggregate MOEs for Children Expose d to Plastics and Textiles Incorporating
Nanosilva

Age Range of Child Incidental
Oral Dose to Toys Flooring Textiles Aggregate
Dose Aggregate
MOE A Dermal Dose Incidental
Oral Dose Dermal Dose Incidental
Oral Dose

6 to < 12 months 9.7×10 -5 3.7×10 -5 1.9×10 -5 8.8×10 -5 2.9×10 -5 2.7×10 -4 110,000

1 to < 2 years 5.5×10 -5 3.5×10 -5 2.1×10 -5 8.4×10 -5 2.4×10 -5 2.2×10 -4 140,000

2 to < 3 years 3.2×10 -5 3.3×10 -5 0.95×10 -5 8.0×10 -5 2.0×10 -5 1.7×10 -4 170,000

A. Aggregate MOE = POD / Daily Dose (rounded to two significant digits)

The assessment above is for the concurrent e xposure to the nanosilver in Nanosilva via the incidental oral and dermal routes of exposure while mouthing toys and textiles, and contacting flooring incorporating Nanosilva. Aggregate assessments can al so include other sources of exposure such as to Nanosilva in food and drinki ng water, and to other nanosilvers in the market place that are identical to the nanosilver in Nanosilver. There ar e no anticipated food exposures
to Nanosilva since Nanosilva LLC has not supplied the necessary information to support a food contact application and the pesticide label for Na nosilva states that it is only for non-food contact use. Neither the nanosilver that might break away from Nanosilva or Nanosilva is anticipated to enter drinking water because these particulates will be removed by gravitational sedimentation and adsorption (see Section 5.1.1).

EPA has determined that the only other pesticid e which was knowingly regi stered as containing nanosilver is HeiQ AGS-20 (U.S. EPA, 2011a); although, the nanosilver in HeiQ AGS-20 does not have the exact same size range and surface coatings as the nanosilver in Nanosilva. The incidental oral and dermal MOEs for HeiQ AGS-20 were all grea ter than 1,100 and the
incidental oral and dermal MOEs for Nanosilva are all greater than 310,000. None of the MOEs for HeiQ AGS-20 were less than the target MO Es for that assessment, which was based on conservative estimates expected to significantly overestimate exposures to the nanosilver in HeiQ AGS-20. Although EPA has not determined whether it’s appropriate to consider concurrent exposures to the nanosilver in Nanosilva and HeiQ AGS-20, the agency has concluded that even if exposures to these two active ingredients we re to occur conc urrently, they
would not result in MOEs that would be of c oncern. In addition, EPA is currently revising the HeiQ AGS-20 risk assessment based on newly subm itted leaching data and to be consistent with the risk assessment framework proposed in this decision document. EPA anticipates that the52 calculated MOEs for incidental oral and dermal exposure to textiles containing HeiQ AGS-20 will increase based on newly submitted data.

As to exposure to other nanosilvers in the mark et place, an aggregate risk assessment involves the analysis of exposure to a single chemical by multiple pathways and routes of exposure that
share a common toxic effect. For EPA to conduct such an assessment requires that the Agency 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 residentia l exposure scenarios ar e 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 triclosa n where population-based biological monitoring data were available to asse ss the co-occurrence of uses, EPA develops an aggregate exposure assessment for residential uses of a single chemical. However, in the case of nanosilver, with the excepti on of the HeiQ products, EPA does not yet have adequate information on the composition of the nanosilver or potential nanosilver in other silver based pesticides to determine if all nanosilvers should be trea ted as a single chemical.

5.2.5 Conclusions for Consumer Risk
The MOEs calculated for the incidental oral expos ure to nanosilver in plastic toys incorporating Nanosilva are 310,000 for 6 to < 12 month old child ren, 550,000 for 1 to < 2 year old children, and 940,000 for 2 to <3 year old children, whic h are greater than the target MOE of 3,000 indicating that there is no risk concern for short- or intermedia te-term exposure to children who mouth toys incorporating Nanosilva. The MOEs cal culated for the dermal exposure to nanosilver in flooring incorporating Nanosilva are 820,000 fo r 6 to < 12 month old children, 860,000 for 1 to < 2 year old children, and 910,000 for 2 to <3 year old children, which are greater than the target MOE of 3,000 indicating that there is no ri sk concern for short- or intermediate-term exposure to children who crawl on flooring incorporating Nanosilva. The MOEs calculated for the incidental oral exposure to nanosilver in fl ooring incorporating Nanos ilva are greater than 1,000,000 for 6 to < 12 month old children, 1 to < 2 year old children, and 2 to <3 year old children, which are greater than the target MOE of 3,000 indicating that there is no risk concern for short- and intermediate-term exposure to children who mouth fl ooring incorporating53 Nanosilva. The MOEs calculated for the dermal e xposure to nanosilver in textiles incorporating Nanosilva are 340,000 for 6 to < 12 month old child ren, 360,000 for 1 to < 2 year old children, and 370,000 for 2 to <3 year old children, whic h are greater than the target MOE of 3,000 indicating that there is no risk concern for short- and intermed iate-term exposure to children who wear textiles incorporating Na nosilva. The MOEs calculated for incidental oral exposures to nanosilver in textiles incorporating Nanosilva are greater than 1,000,000 for 6 to < 12 month old children, 1 to < 2 year old children, and 2 to < 3 year old children, whic h are greater than the target MOE of 3,000 indicating that there was no ri sk concern for short- and intermediate-term exposure to children who mouth and chew textiles incorporating Nanosilva.

The aggregate MOEs for children who are, within the same short time frame, mouthing a toy, flooring, and a textile while wearing a textile and crawling on floor ing all incorporating Nanosilva are greater than 100,000 for all lifestages, which indicates that the risk for short- and intermediate-term simultaneous exposure to plasti cs and textiles incorporating Nanosilva is not a

concern.

Although children’s short- and inte rmediate-term exposure durations to plastic toys and textiles treated with Nanosilva are expecte d, children are not anticipated to play with Nanosilva treated toys or contact Nanosilva treated textiles every day for a period of greater than six months, thus long-term exposures are unlikely. Nanosilva is pr oposed to be used in flooring meaning that long-term dermal and incidental oral exposures to children contacting flooring are possible. However, EPA does not believe that children will have continuous and daily exposure to flooring for a period of greater than 6 m onths. Even if these unlikely long-term exposures were to occur,
the calculated MOEs for children’s dermal and in cidental oral exposure to plastics and textiles incorporating Nanosilva are greater than 100,000 indi cating that daily exposure to plastics and textiles incorporating Nanosilva for greater than 6 months are not likel y to be 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 con centrations of silver would also be found if a leaching test was conducted with coupons cont aining nanosilver at 30 mg/kg. This assumption
was reasonable because the concen tration of silver in the text ile leaching studies, which were conducted on textiles incorporating nanosilver at 30 mg/kg, was also below the analytical detection limit, making it unlikely that Nanosilve r incorporated into plastic at 30 mg/kg will
release nanosilver at detectable concentrati ons. The PODs used were based on inhalation and oral toxicity studies completed using nanosilver, not Nanosilva or the nanosilver that might break away from Nanosilva, and thus these PODs may not equal PODs determined from testing
conducted with Nanosilva or the nanosilver f ound in Nanosilva. Because the nanosilver in the toxicity database is different from the nanos ilver in Nanosilva and because the database is incomplete with respect to reproductive and deve lopmental effects, the Agency used a maximum54 10-fold database uncertainty factor when evalua ting the risk from exposure to Nanosilva and the nanosilver that might be released from produc ts incorporating Nanosilva. There are also uncertainties in extrapolating from effects obser ved after feeding test-a nimals nanosilver (i.e., oral route) to the effects that might be observe d after applying nanosilver to the skin of test animals (i.e., dermal route).

EPA is able to determine that for the period of conditional registration, there is a low probability of adverse risk to children from plastics and textiles incorporating Nanosilva. Thus, the Agency
concludes that use of Nanosilva will not cause unreasonable adve rse effects on the environment during the period when newly requi red data are being developed.

5.2.6 Consumer Exposure and Health Data Requirements

EPA has determined that an additional leachi ng study is required for plastics incorporating Nanosilva using biological flui ds and physiological temperatur es, 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 Nanosilva are required.

EPA is proposing to require that Nanosilva LLC c onduct plastic leaching tests as part of Tier I studies because the available study was complete d using a protocol that was not reviewed by EPA prior to conducting the study, the coupons tested has less than the maximum nanosilver concentration of 30 mg/kg, and the study did not use physiological flui ds. The newly required leaching study should determine the nature and quantity of silver re leased from plastics incorporating Nanosilva under conditions of use. EP A will evaluate the results from the plastic leaching test along with results from Tier I t oxicity studies discussed in Section 5.1.4 using a weight of evidence approach based on physical a nd chemical properties, toxicity, and exposure. If the Agency identifies a concern, then the Agency may require additional to xicity data to assess potential adverse health outcomes in young children resulting from incidental oral exposure to Nanosilva-treated plastic toys ( i.e., children chewing on toys). If additional toxicity data are needed, the study should evaluate peri- and post-na tal exposure to Nanosilva. A more detailed description of these data requireme nts is provided in Appendix B.55 

### VI. ENVIRONMENTAL RISK ASSESSMENT

EPA anticipates the following substances co uld enter the environm ent through leaching of plastics and textiles in corporating Nanosilva:

1) Silver ions released from Nanosilva; 2) Nanosilva complex; and/or 3) Nanosilver that might break away from the Nanosilva complex.

There are no studies available to characterize the environmental fa te or ecotoxicity of Nanosilva. However, there are studies available in the scie ntific literature for nanosilver. Since nanosilver may be released from Nanosilva, the Agency has considered the scientific literature studies on nanosilver fate and ecotoxicity relevant to Nanosilva. The following sections cover the environmental fate of nanosilver, the environmen tal hazards posed by silver and nanosilver, and the potential risk to aquatic speci es from nanosilver. As part of these discussions, additional data that EPA is proposing to require in order to confirm its assessment of the environmental risks of
Nanosilva are also identified.



### 6.1 Environmental Fate

Nanosilva LLC has not conducted any studies to characterize the environmental fate of Nanosilva or the other particles that could be released during leachi ng or disposal of plastics and textiles incorporating Nanosilva. In lieu of this information, the Agency is relying on studies
available in the scientific li terature, discussed in the follo wing section, as the basis for determining the fate of nanos ilver in the environment.

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 one 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 natural organic matter such as humic acids, which will significantly dela y the rate at which nanosilver transforms into ionic silver. For example, Choi et al., (2009) provided spectroscopic evidence showing that nanosilver reacts with sulfide to produce stable silver-sulfide complexes, which were shown by Liu et al. (2010) and Levard et al . (2011) to have undetectable rate s of nanosilver to ionic silver transformation. These stabilized nanosilver complexe s are likely to partition to sediments, rather than remain suspended in water, due to gravita tional settling and coagulat ion processes (see Page 19 FIFRA SAP, 2009). Likewise, nanosilver is an ticipated to partition to biosolids during56 wastewater treatment but may also be released in the effluent. Thus, there is the potential for nanosilver to reside or persist in the envir onment for a significant period of time where these 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 chlo ride, sulfides, and natural orga nic matter (Andren and Armstrong, 1999). As with nanosilver, ionic si lver is found in sediments and associated with biosolids in wastewater treatment plants.

6.1.3 Impacts to Wastewater Treatment/Septic Systems
There is the potential for nanosilver that mi ght be released from plastics and textiles incorporating Nanosilva to reac h publicly owned wastewater tr eatment and privately owned septic systems where they will most likely complex with sulfide and partition to biosolids (Kaegi et al., 2011). Once entrained in th e biosolids, the nanosilver could serve as a long term source of ionic silver and could potentially adversely affect microorganisms th at are vital to the wastewater treatment process. There are contradictory reports in the scientific literature regarding the impact of nanosilver on wastewater treatment systems. For 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 wast ewater sludge. However, Burkhardt et al. (2010) found no impact to nitrification at nanosil ver dosages of 1 mg/L, the same dosage that Choi and Hu (2009a and 2009b) repo rted as inhibitory in munici pal wastewater sludge. These two research groups are reporting different findings with the Bu rkhardt group suggesting little impact of nanosilver to nitrifi cation and the Hu group suggesting that an impact to wastewater treatment systems from nanosilver is expected. A third group independently determined that nanosilver at concentrations from 0.5 to 1.5 mg/L had no detectable effect on the ability of the wastewater bacteria to biodegr ade organic material, as meas ured by chemical oxygen demand (COD) (Wang et al., 2012a). More recent work by the Hu group reported that nanosilver at concentrations of up to 40 mg/L had negligible impact on anaer obic digestion and methanogenic organisms (Yang et al., 2012).

While there are reports suggesti ng the potential for nanosilver to impact wastewater treatment operations, the Agency does not anticipate that registering Nanosilva will lead to negative impacts to wastewater treatment systems. This conclusion is based on the limited amount of silver released from plastics and textiles in corporating Nanosilva (see Section 5.2.1) and the small volume of nanosilver (i.e., < 1,123 kg/yr as estimated in Section 6.3.1) expected to be introduced into commerce from plastics a nd textiles incorporating Nanosilva.57 

### 6.2 Environmental Toxicity

Nanosilva LLC has not conducted any studies to ch aracterize the ecotoxicity of Nanosilva or the other particles that could be released during leaching or disposal of plastics incorporating Nanosilva. In lieu of this information, the Agency is relying on studies ava ilable 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 na turally present in surf ace waters in relatively low concentrations as compared to metals such as copper and zinc. However, 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 to 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 Nanosilva and aquatic organisms were submitted, there are studies in the scientific literature covering th e toxicity of nanosilver to aquatic organisms (Table 19). These studies indicate to the Agency that, if suff icient quantities of nanosilver break away from Nanosilva and reach surface water, and if such nanoparticles display toxicity similar to the
nanosilver used in these studies , then exposure of Nanosilva derived nanosilver may result in adverse effects to aquatic species. These results also indicate that, of the organisms tested, Daphnia magna is the most acutely sensitive to nanosilver . As with the toxicity for silver ions, the toxicity 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 the work by Laban et al. (2010).58 Table 19 – Nanosilver Toxicity to Aquatic Species

Aquatic Organisms

(Study Citation) Toxicity Silver Nitrate Nanosilver
Total Silver ( μ g/L) Total Silver ( 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 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 Not reported

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

Freshwater Flea 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

Freshwater Flea 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
Freshwater Flea D. magna (Asghari et al., 2012) 48-hr LC 50 0.23 2 to 187 E Not reported OECD 202 Media

Freshwater Flea D. magna (Hoheisel et al., 2012) 24-hr LC 50 0.81 4.3 to 10.5 3.5 to 8.1 Moderately
hard reconstituted water (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 Pimephales 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
Danio rerio (Wang et al., 2012b) 96-hr LC 50 77 to 82 72 to 223 G 0.7 to 22.3 H Not reported59 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 members of a tested population after the specified exposure. C. Range for eight different nanosilver formulations. D. Expressed as the fraction of suspended nanosilver.
E. For nanosilvers with diameters from 10 to 50 nm.
F. Three nanosilver formulations G. Rage of three different nanosilver formulations
H. Estimated

Based on the studies listed in Table 19, 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 fract ion of ionic silver present. This te st was conducted in moderately hard reconstituted water (MHRW), which is a test me dium 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 incorporat ing Nanosilva, the data represent short-term (e.g., 96 hr) acute exposures and do not characterize the effects from longerterm exposure to nanosilver. There is one l ong-term study on the eff ects of nanosilver on a freshwater fish ( Oryzias latipes ) that reported a no observed eff ect concentration (NOEC) of 100 μg/L based on the lethal effect observed at the dose of 250 μg/L during a 14-day exposure period (Wu and Zhou, 2013). Non-lethal effects were repor ted for all nanosilver concentration tested (50 to 500 μg/L) including histologi cal lesions in the fish tissue s along with silver accumulation
in the liver.



### 6.3 Aquatic Risk Assessment

EPA expects that Nanosilva, silver ions releas ed from Nanosilva, and nanosilver that might break away from Nanosilva coul d enter the environm ent through the following scenarios:

1. Leaching of plastics and te xtiles incorporating Nanosilva
2. Application of wastewater treatment biosolids to agricultural fields

Leaching of plastics and textiles incorporating Na nosilva is anticipated to be the primary route by which Nanosilva, silver ions, and nanosilver reach the environment. The silver released during leaching of plastics and textiles incorpor ating Nanosilva 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 scenario. Once Nanosilva, silver ions, or nanosilver reach wastewater treatment and septic systems they will most likely complex with sulfide and partition to biosolids. However, some frac tion of the silver compounds will60 reach surface water and may potentially impact a quatic organisms. Leaching of building material such as plastic siding and decks, and outdoor furniture and carpeting incorporating Nanosilva will be discharged directly into the environment.

As stated in Section 5.2.1, silver was not found above the analytical detection limit leaching from plastic coupons and shirts incorporating Nanos ilva. EPA does not expect unreasonable adverse effects to the environment from registered us es of silver ions, including the materials preservative use pattern (U.S. EPA, 1993). To evaluate the impacts on surface water from leaching of Nanosilva from plastic s and textiles, EPA is assumi ng that nanosilver as found in Nanosilva 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 the Agency's Level of Concern (LOC). An LOC is a policy tool that the Ag ency uses to interpret the RQ and to analyze potential risk to non-target organisms and the need to cons ider regulatory action (U.S. EPA, 2011c).

Indoor Use

 Mass of silver release per year: 1123 kg/year. The amount of silver released from textiles incorporating Nanosilva was derived assumi ng that 300 million people (U.S. population) purchase one t-shirt incorporating Nanosilva each year. Each t-shirt weighs 150 grams and contains 0.003% nanosilver by weight silver and releases 1.6% s ilver 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 nanos ilver removal of 88% with biomass present.

Outdoor use

 Mass of silver release per year: 570 kg/year. The amount of silver released from plastics incorporating Nanosilva was derived assuming that 1% of the yearly plastic lumber production contains Nanosilva 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 in corporating Nanosilva is releas ed during a year even though61 results from the plastic leaching study indicate that little silver is released from plastic incorporating Nanosilva.

 Release Days: 365 days per year. This assume s that each plastic incorporating Nanosilva
releases all its silver as nanosilver over the course of one year.

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

Down the Drain Model

The concentration of nanosilver in surface water re sulting from the use of Nanosilva in plastics was calculated using the Down the Drain M odule 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 dur ing 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 level of concern (LOC) is 0.05 for listed (i.e. endangered or threat ened) aquatic organisms and 0.5 for non-listed organisms.

Table 20 – 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?*

Indoor Use

(t-shirts) 85% 1.0 B 0.004 0.003 No

20.1 C 0.0002 0.0001 No

Outdoor Use (plastic lumber) 0% E 1.0 0.014 0.009 No

20.1 0.001 0.0007 No

*The LOC is 0.05 (listed species) and 0.5 (non-lis ted species). RQs that exceed the LOC are of concern A. Silver removed from wastewater during treatment be fore 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 Water Concentration / LC 50 for Daphnia magna (1.0 μg/liter) E. Assumes direct discharge to surface wate r with no rete ntion in soil or sediment.62 The down-the-drain modeling results are shown in Table 20 and these results were divided by
the 96-hr EC 50 of 1.5 μg/L for Daphnia magna to obtain acute risk quotients (RQs). The effect
level used to calculate the acute RQs (i.e., 1.5 ug/L ) was chosen to represent the most sensitive aquatic organism Daphnia magna and to represen t conditions that are re presentative of surface
water in the United States. The acu te RQ of 0.009 at the worst cas e stream dilution factor of 1.0 indicates that it is unlikely that the registration of Nanosilva as a plastics and textiles preservative
will lead to adverse effects for listed or non-list ed aquatic organisms during the four year period of conditional registration. However, this assessment does not consider long-term exposures or exposure to estuarine and marine species.

6.3.2 Conclusions for Aquatic Risk
The acute RQs for Daphnia magna exposed to nanosilver ranged from 0.0001 to 0.009, which are not of concern for listed or non-listed organi sms because they were less than the acute LOC of 0.5 and 0.05, respectively. The eff ect level used to calculate th e acute RQs (i.e., 1.5 μg/L) was chosen to represent the most sensitive aquatic organism Daphnia magna and to represent conditions of moderately hard reconstitute d water (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 Nanosilva at 0.003% resulting in a nanosilver production-volume of 5 70 kg/year. This may be an overestimate given
that the total mass of silver distributed as a mate rial preservative in th e U.S. during 2009 was less than 6,800 kg, based on EPA confid ential records. This analysis also assumes that all the nanosilver in plastic lumber incorporating Nanos ilva would be released into the environment during a one year period when in fact the leaching study for plastic coupons did not find silver at concentrations above the analytical detection li mit implying that signific antly less silver would be released from plastic lumber in corporating Nanosilva (see Section 5.2.1).

There are several uncertainties regarding the ex posure portion of this evaluation. The production
volume of Nanosilva is unknow n since 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% Nanosilva. The nanosilver that could be released from the lumber incorporating Nanosilva was assu med to directly enter the aq uatic environment without any
removal in soil or sediments. The rate at whic h nanosilver entered the sanitary sewer systems
was assumed to be constant in the down-the-dra in model, which does no t account for variability in the distribution of plastic products inco rporating Nanosilva among other factors. The ecotoxicity values shown in Table 19 are for acu te effects and mortalit y, and there is only one study on the long-term exposure to nanosilver. Finall y, this assessment only considers silver that could be released by Nanosilva and does not include other sources of silver which will contribute to the environmental loading of silver.63 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 Nanosilva.



### 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 Nanosilva during the period of conditional registration, 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
Nanosilva and confirm that the nanosilver in Nanos ilva is consistent with the risk from Table 19. The Tier I environmental fate testing categories include:

 Product Characterization and Testing

 Silver Release from Plastics: Form, Rate and Characteristics

Completion of these Tier I studi es will provide Nanosilva product characteristics and determine the substance or substances released during leaching of plastics incorporating Nanosilva.
Information from these studies will be used to substantiate Nanosilva’s claim that no nanosilver is released from plastics incorporating Nanosilva during use. However, if 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 then be used by EPA to re-run the environmental risk assessment for
Nanosilva. The Tier II environmental fate a nd ecological effects test ing 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.64 

### 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 ingredient are newly requir ed or identified. As discussed more thoroughly below, because EPA has not reached a final decision with regard to which types of data would be further required, the data re quirements for this regi stration are considered newly required.

In addition, we believe granting the proposed conditional registration with the terms and conditions identified in Appendix B is particularly appropriate given EPA suspects that some already-registered like-situated products on the market cont ain nanosilver as the active ingredient. While EPA approved th ese registrations without knowle dge that these products may
contain nanoscale silver and w ithout specifically assessing any potential risks that might be associated with any nanosilver contained in t hose products, they are none theless on the market. To avoid disparate treatment and consistent w ith the analytic decision framework in this document, EPA intends to seek similar data alon g with comparable terms on already registered products that are identified to contain nanosilv er. As part of this effort, EPA opened the Nanosilver Registration Review (docket EP A-HQ-OPP-2011-0370) on July 6, 2012, which is the first step towards issuing a Data Call -In for products containing nanosilver.



### 7.1 Legal Framework

The Federal Insecticide, Fungi cide, and Rodenticide Act (FIF RA) 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 issued under this Act, and on such other conditions as the Administrator may prescribe. A conditional registration under this subparagraph shall be granted only if the Administrator determines that use of the pesticide duri ng such period will not cause any unreasonable
adverse effect on the environmen t and that use of the pesticide 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:65 1) Nanosilva contains an active ingredient, silv er nanoparticles also known as nanosilver, that is not an active ingredie nt in any currently registered pesticide (i.e., a “new” active ingredient);
2) Use of Nanosilva will not cau se unreasonable adverse eff ects on the environment during the period when newly required data are being developed;
3) Insufficient time has elapsed for Nanosilva LLC to generate and submit the newly required data; and
4) Use of Nanosilva is in the public interest.



### 7.2 Findings and Registration Decision

The Agency proposing to issue a conditional, four year registration for Nanosilva in accordance with FIFRA section 3(c)(7)(C) and is proposing to require as a c ondition of registration specified use conditions as well as proposing that the compa ny provide data from a number of studies that will allow the Agency to confirm its current assessm ent of the risks. Specific data requirements are outlined in Appendix B. Although these data requirements are specific to Nanosilva, they form a starting point for identifying the type s of data the Agency 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 ppm (mg/kg) or 0.003% silver on a weight basis for plastics and textiles whic h incorporate Nanosilva.
2. Nanosilva may only be incorporated into linear low-density polyethy lene (LLDPE) plastic and polyethylene terephthalate (PET) based textiles consistent with the materials used in the leaching studies.
3. Nanosilva may only be used to preserve plas tics and textiles and ma y not be used for drinking water or food contact uses. 4. It is required that the following mitigati on measures be employed when mixing and loading Nanosilva during treatment of articles:

 Closed system loading of Na nosilva 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 al l of the components of the Nanosilva liquid suspension.

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

5. The label must have an environmental h azard statement consisting of the following:66 “This pesticide is toxic to fish and aquatic invertebrates, and birds, and can’t be used for irrigation purposes.”

“Do not discharge effluent containing this pr oduct into lakes, str eams, ponds, estuaries, oceans, or other waters unless in accordan ce with the requirements of a National Pollutant Discharge Elimination System (N PDES) permit and the permitting authority has been notified in writing prior to discharg e. Do not discharge e ffluent containing this product to sewer systems without previously notifying the local se wage treatment plant authority. For guidance contac t your State Water Board or Regional Office of the
EPA”.



### 7.3 Basis for Conditional Registration

The Agency’s basis for the proposed c onditional 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, they also submitted a request to waive some of the data requirements. In addition, Nanosilva LL C completed a leaching study using a protocol that was modified to be applicable for na nosilver. Although Nanosilva LLC provided EPA with some data in an effort to address Agency que stions and concerns, EPA had not reached a final decision with regard to which t ypes of data would be further required for Nanosilva. This was due in large part to the need to understand and apply 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 asse ss Nanosilva LLC’s application for Nanosilva LLC to have generated the data.

EPA is proposing to require the tests listed be low to confirm the adequacy of the 10 fold database uncertainty factor, to reduce the uncerta inties related to differences in the physical properties of the nanosilver, and because there are currently no acceptable studies on the reproductive and developmental toxicity for nanosilv er. Because this list of data requirements are only being proposed with today’s action, Nanosilva LLC has not yet been able to conduct these studies. Therefore, the Agency is proposing to re quire these studies as a condition of registration, allowing sufficient time for the study protocols to be agreed upon, the studies to be conducted, and for the Agency to review th em. The required studies include:67  90-Day Inhalation Toxicity (Rat) (OCSPP 870.3465) modified to include in vivo bone marrow assay and functional observationa l battery, motor activity and detailed neuropathology

 Reproduction/Developmental Toxicity Sc reening Test (Modified OCSPP 870.3550/ OECD TG 421)

 Product Characterization and Testing

 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 exposure. If th e Agency identifies a concern,
then one or a combination of the following tests will be triggered:

 Peri- and post-natal exposure to Nanosilva

 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

As discussed above, a listing of the studies that are proposed to be required as terms and conditions of this registration of Nanosilva are in Appendix B. If Nanosilva LLC does not meet
the conditions set forth in this Decision Document , EPA will issue a noti ce of intent to cancel Nanosilva LLC’s registration under section 6( e). In addition, Nanosilva LLC’s conditional registration for Nanosilva will automatically expire four years after 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 Nanosilva in the United States.

7.3.2 Public Interest
As required under FIFRA section 3(c)(7)(C), fo r the reasons summarized below, the Agency believes granting a conditional registration with the terms and conditions specified is in the public interest. EPA believes that use of Nanosilv a offers the potential for conservation of the
environment and consumer benefi ts which contribute to the pub lic interest. These points are
discussed in more detail below:68 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. Commonly, regardless of the silver additive type, upon contact with moisture, silver ions are released from the obj ect incorporating the additive. The antimicrobial potency of a silver additive is therefore directly related to the potential for releasing silver ions. The release potential differs between various si lver materials. As the size of silver particles decreases (e.g., from micro-size silver to nano -size silver), the potential for releasing silver ions increases, due to the increasi ng unit of surface 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 Nanosilva LLC’s product, most currently registered silver-based material s preservatives incorporate larger amounts of silver. Therefore, the overall potential environmental loading of silv er resulting from the lower-volume use of the Nanosilva product should be smaller than from a co mparable use of currently registered silverbased pesticides.

Consumer Benefits

A nanosilver materials preservative should mainta in its ability to reduce the number of odor and stain causing bacteria longer than other silver ac tive ingredients due to th e expected gradual and
controlled release of silv er ions from nanosilver as opposed to the rapid release of silver ions
from a zeolite structure or the im mediate dissolution of the silver salt. In the case of Nanosilva, the release of silver ions from plastics and tex tiles incorporating Nanosilva are at concentrations below the analytical detection limit while these sa me plastics and textiles are reported to retain the ability to reduce the number of odor and stain causing bacteria. While it may be that other silver active ingredients are presen t at greater silver concentrati ons, it is expected that their effects on odor and stain causing bacteria are only short-lived due to the rapid release of silver ions. Thus, plastics or textiles incorporating nanosilver may have longer-term ability to reduce the number of odor and stain causing bacteria as compared to other similar products on the
market that contain conventional silvers.

Innovation

EPA sees the emergence of nanotechnology as o ffering potential benefits for society in many different fields, including pest control products . The use of nanotechnolo gy in pesticide products may allow for more effective targeting of pests a nd use of smaller quantities of pesticide. These69 could contribute to improved human and environm ental safety and could lower pest control costs. Therefore, EPA seeks to encourage innovative work to realize these benefits.

In the case of the Nanosilva LLC’s applicati on, EPA’s proposed conditional registration of Nanosilva is in the public interest in that it will allow an innovativ e product to reach the market.

7.3.3 No Unreasonable Adverse Effects
In assessing the potential risks to human health and the environment associated with the
distribution and use of Nanosilva 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 Nanosilva during the period of the conditional registration are small. Moreover, EPA expects the overall quantity of silver used in plastics and text iles as a result of this conditional registration will be lower than the overall qua ntity of silver used in othe r materials preservative products containing conventional silver re sulting in expected reductions in environmental loadings of
silver and to humans. EPA conc ludes that the registration woul d not cause unreasonable adverse effects on the environment during the conditi onal 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 Nanosilva, and to nanosilver. With respect to silver ions, the Agency complete d a risk assessment (U.S. EPA, 1993) for silver salts and determined that the risks from register ed uses, including the materials preservative use pattern, were acceptable. The Agency concludes th at human exposure to silver ions from the use of Nanosilva and consumer exposures to plasti cs and textiles incorpor ating Nanosilva is not
unreasonable.

With respect to Nanosilva, the acute toxicity of Nanosilva is low by all routes of exposure.
Because there are no subchronic or chronic oral or dermal toxicity studies available for Nanosilva or on the nanosilver that might break away from products inco rporating Nanosilva, the
subchronic or chronic toxicities were estimated using studies on nanosilvers reported in the literature. The Agency relied on the results fr om studies submitted by Nanosilva LLC where the liquids in contact with plastic a nd shirts incorporating Nanosilva we re analyzed for silver content to estimate exposure the amount of Nanosilva and na nosilver that might be released from plastics and textiles. The Agency rec ognized the uncertainties and inco rporated multiple uncertainty factors and conservative assumptions when cal culating the Margin of Exposures (MOEs) for estimating the risk to children from plastics and textiles incorpor ating Nanosilva.70 Based on all of the foregoing, EPA concludes, wi th a level of confidence acceptable for the period of conditional registration, that there is a low probability of adverse risk to consumers from short- and intermediate-term exposure to toys, flooring, and textiles incorporating Nanosilva. Thus, the Agency concludes that use of Nanosilva will not cause unreasonable
adverse effects on the environm ent during the period when newly required data are being developed.

Risks to the Environment

The environment could be exposed to silver ions , to Nanosilva, and to nanosilver. With respect to silver ions, the Agency completed a risk a ssessment (U.S. EPA, 1993) for silver salts and determined that the risks from registered uses , including the materials pr eservative use pattern, were acceptable. The Agency concludes that envi ronmental exposure to silver ions from the use of Nanosilva and plastics and textiles incorporating Nanosilva is acceptable.

With respect to the fate and eco toxicity of Nanosilva, because th ere are no fate or ecotoxicity
studies available for Nanosilva or the nanosilver that might break away from plastics or textiles incorporating Nanosilva, the fate and ecotoxicity were estimated using studies on nanosilvers reported in the scientific literature. Direct re lease from leaching of plastics and laundering of textiles incorporating Nanosilva were anticipated to be the primary route by which Nanosilva, silver ions, and nanosilver reach the environm ent. The Agency assessed impacts to surface waters assuming that nanosilver was the only co mpound released. Environmental exposure from indoor use was assessed assuming that 300 million people (U.S. population) each purchased one t-shirt containing 0.003% nanosilver where 1.6% of the silver in the t-shirt incorporating Nanosilva was released during each weekly wa shing over a one year period. Environmental exposure from outdoor use was assessed assuming th at 1% of the projected yearly production of plastic lumber in the U.S. would contain Nanos ilva at 0.003% where all the silver would be
released into the environment as nanosilver during a one year period even though the leaching study for plastic coupons incorporating Nanosilva did not find silver at concentrations above the analytical detection limit. EPA estimates, derived from down-the-drain modeling, of the concentrations of nanosilver resulting from the use of Nanosilva do not exceed the Agency’s estimate of the highest concentration of nanos ilver in surface water to which an aquatic community can be exposed without resulting in an unacceptable effect. Thus, the Agency
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4186.

Wang, Y., Westerhoff, P., Hristovs ki, K.D. 2012a. Fate and biological effects of silver, titanium dioxide, and C60 (fullerene) nanomaterials durin g simulated wastewater treatment processes. Journal of Hazardous Materials 201-202:16-2277 Wang, Z, Chen, J., Li, J. et al. 2012b. Aquatic To xicity of Nanosilver Colliods 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. Poten tial nanosilver impact on anaerobic digestion
at moderate silver concentrations. Water Research 46: 1176-1187.Appendix A

- Page 1 

# Appendix A

– Evaluation of Nanosilva LLC’s Data Waiver Request s

August 2 7 , 201 3Appendix A

- Page 2 I. Introduction

On August 10, 2009, Nanosilva LLC submitted an application to register Nanosilva as a new active ingredient (PRIA A420)

. In 2009, a s an applicant for registration of a new active ingredient, Nanosilva LLC was required to submit all the applicable informati on and studies under 40 CFR part 161. EPA published a Notice of Propos ed Rulemaking on October 8, 2008

propos ing to revise and update the existing data requirements under 40 CFR part 161 for antimicrobial pesticides. These data requirements were made final as 40 CFR part 185W on May 8, 2013 ( 78 FR 26936). In 2009, t he Nanosilva LLC application included a request to w aive the requirement to generate data for Nanosilva using the following test guidelines :

1. Hydrolysis ( O PPTS 835.2120 ) 2. Aquatic Invertebrate Acute Toxicity Test, Freshwater Daphnids ( OPPTS 850.1010 ) 3. Fish Acute Toxic ity Test, Freshwater and Marine ( OPPTS 850.1075 ) 4. Avian Acute Oral Toxicity Test (OPPTS 850.2100 ) 5. 90

- Day Dermal Toxicity ( OPPTS 870.3250 ) 6. Prenatal Developmental Toxicity Study ( OPPTS 870.3700 ) 7. Bacterial Reverse Mutation Test ( OPPTS 870.5100 ) 8. Detection of Gene Mutations in Somatic Cells in Culture ( OPPTS 870.5300 ) 9. In Vitro Mammalian Cytogenetics ( OPPTS 870.5375 ) 10. Mammalian Bone Marrow Chromosome Aberr ation Test ( OPPTS 870.5385 ) 11. Immunotoxicity ( OPPTS 870.7800 )
The test guidelines in items 1 through 4 are cited in 40 CFR part 161 and were required at the time Nanosilva applied to registered Nanosilva. Guidelines in items 5 through 11 were proposed in 2008 to be included in 40 CFR part 158W, which was made final in 2013 and therefore, the test guidelines in items 5 through 11 w ere not required at the time Nanosilva LLC applied to register Nanosilva. 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 req uested because of the lack of acute toxicity noted in the six acute toxicity studies (MRID 47828918 through 47828923), because Nanosilva has a specific gravity greater than water, because Nanosilva has low solubility in water as determined by the
water col umn generator method (MRID 47828917), and due to the non

- leaching characteristic of Nanosilva because silver at concentrations greater than the analytical detection limit were not detected in leaching studies conducted with a plastic incorporating Nanosilv a (MRID 47828925).

The data requiremen t s listed above in items 1 through 11 are either waived or satisfied . In general, the data requirement is met or satisfied because :

• certain guideline studies are not appropriate for nanosilver and where appropriate ar e being modified and required as terms and conditions on the registration (i.e., Reproduction/Developmental Toxicity Screening Test )Appendix A

- Page 3 • certain environmental data are satisfied because the environmental hazard labeling statement was determined using literatur e stu dies completed with nanosilver

• Dermal guideline studies are not appropriate given there was no risk concern

• in vitro mutagenicity stud y requirements are satisfied and have shown that nanosilver is possibly mutagenic

• the i mmunotoxicity study is initially waived and only triggered after the potential immune toxicity of nanosilver is determined during the 90

- day inhalation toxicity study required as a term and condition on the registration

Each of the above studies is addressed 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 evaluation of each waiver request.

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, OPPTS 835.2120

This test is waived because nano silver undergoes dissolution with the generation of ionic silver rather than undergoing hydrolytic transformation.

B. Wildlife and Aquatic Organism Data Requirements

The following tests are required for solid

- formulation manufacturing use antimicrobials under 40
CFR 161.440:

2. Aquatic Invertebrate Acute Toxicity Test, Freshwater Daphnids ( OPPTS 850.1010 )
3. Fish Acute Toxic ity Test, Freshwater and Marine ( OPPTS 850.1075 )
4. Avian Acute Oral Toxicity Test (OPPTS 850.2100 )

The acute ecotoxicity testin g waiver request was based on the non

- leaching characteristic of
Nanosilva . However, the results of these tests are required for use in determining the appropriate precautionary labeling on packages containing the Nanosilva liquid suspension prior to
incorporating into a product. Therefore, the non

- leaching characteristic is not an appropriate basis for waiving these tests. As summarized in Table 19 of the Decision Document, EPA has information on the acute toxicity of nanosilver for Daphnids and Freshwater Fish. Because the
EC 50 values were less than 1 mg/L, as indicated in Chapter 8 of the Pesticide Label Review Manual, the following environmental hazard statement is required on the Nanosilva label:Appendix A

- Page 4 This pesticide is toxic to fish and aquatic invertebrate s.

Given that there are no test results for Avian species available for nanosilver, EPA requires that the environmental hazard statement for the Nanosilva label be expanded to include:

This pesticide is toxic to fish and aquatic invertebrates, and birds.

EPA is requiring that Nanosilva LLC place th e above environmental hazard statement on the pesticide label for Nanosilva. However, i f Nanosilva LLC wishes to remove birds from this statement then they can submit results from an Avian Acute Oral Toxi city Test (OPPTS 850.2100) showing that the nanosilver in Nanosilva is non

- toxic to birds with an LD 50 of greater than 100 mg/kg.

In addition, Nanosilva LLC is required to place the following statement on the label for Nanosilver liquid suspension:

“Do not discharge effluent containing this product into lakes, 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 ha s been notified in writing prior to discharge. Do not discharge effluent containing this product to sewer systems without previously notifying the local sewage treatment plant authority. For guidance contact your State Water Board or Regional Office of the EPA”.

Thus, the data requirements in items 2 through 4 are satisfied by data in the public literature for
nanosilver and required labeling.

C. Toxicology Data Requirements

The following test s are required for antimicrobials under 40 CFR part 158.2230 ( i.e., part 158W) :

5. 90

- Day Dermal Toxicity ( OPPTS 870.3250
6. Prenatal Developmental Toxicity Study ( OPPTS 870.3700 )

The requirement for the 90

- Day Dermal Toxicity study is waived because the calculated Margin of Exposures (MOEs) for dermal and incid ental exposure to products incorporating Nanosilva where 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 plastics incorporating
Nanosilva 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 riskAppendix A

- Page 5 concern, then the following route

- specific subchronic tests is proposed to be required to confirm
the risk assessment for the nanosilver in Nanosilva.

• Dermal penetration study in rats (OSCPP 870.7600)
The requirement for the Prenatal Developmental Toxicity Study is waived because the following study has been determined to be more appropriate for Nanosilva:

• Modified 870.3550/ OECD TG 421: Reproduction/Developmental Toxicity Screening
Test

The following tests are required for pesticides under 40 CFR part 158. 2230 (e.g., 158W) :

7. Bacterial Reverse Mutation Test ( OPPTS 870.5100 )
8. Detection of Gene Mutati ons in Somatic Cells in Culture ( OPPTS 870.5300 )
9. In Vitro Mammalian Cytogenetics ( OPPTS 870.5375 )
10. Mammalian Bone Marrow Chromosome Aberration Test ( OPPTS 870.5385 )
11. Immunotoxicity ( OPPTS 870.7800 )

The mutagenicity studies [ Bacte rial Reverse Mutation Test (OPPTS 870.5100)

, Detection of
Gene Mutations in Somatic Cells in Culture ( OPPTS 870.5300 ), In Vitro Mammalian Cytogenetics ( OPPTS 870.5375 ), Mammalian Bone Marrow Chromosome Aberration Test ( OPPTS 870.5385 )] are satisfied becaus e the re are in vitro studies available for nanosilver in the
scientific literature which addre ss/satisfy these data needs and show that nanosilver is possibly mutagenic. Th us, th e Agency is requir ing as a term and condition on the registration an in

- vivo bone marrow assay to clarify the mutagenic properties of the nanosilver in Nanosilva.

The i mmunotoxicity ( OPPTS 870.7800 ) is waived at this time because OPP has published a document (U.S. EPA, 2013) recommending that immune related endpoints be evaluated during the inhalation toxicity test , required as a term and condition on the registration, before determining if the immunotoxicity test is required. The currently available oral toxicity studies indicate that nanosilver causes liver and kidney toxicity i n laboratory animals where silver is distributed to all organs and tissues with accumulation of silver in the brain and male animal

testes. Inhalation toxicity studies also identified liver toxicity as well as lung effects including
chronic alveolar inflam mation. The toxicology database for nanosilver does not reveal any
evidence of treatment

- related effects on the immune system , which suggests that nanosilver does not directly target the immune system.

U.S. EPA, 2013. Part 158 Toxicology Data Requirements: Guidance for Neurotoxicity Battery, Subchronic Inhalation, Subchronic Dermal and Immunotoxicity Studies. May 1 st . Available at: http://www.epa.gov/pesticides/regulating/part158

- tox

- data

- requirement.pdfAppendix B

- Page 1 

# Appendix B

– Data Requirements , Enforceable Schedule, and Conditions of the Proposed Conditional Registration for Nanosilva

August 2 7 , 201 3Appendix B

- Page 2 I. Introduction

Nanosilva is a liquid suspension containing 1% nanosilver by weight where the nanosilver active

- ingredient is covalently bonded to crystalline silica via a thiolate bond. The diameter of the spherical silica core is 320 nm on average (Lee et al., 2006) and the nanosilver 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 sph erical silica particles modified with thiol groups with silver nitrate and polyvinylpyrrolidone (PVP) in ethanol (Lee et al. 2007). Thus, the resulting liquid suspension contains 1% nanosilver by weight where the nanosilver surface is coated with sulfur an d PVP, and is attached to silica. Reference to the nanosilver in Nanosilva (whether when part of the composite or when broken away from the complex) is to the “new active ingredient” and reference to “ Nanosilva ” or the Nanosilva “com plex ” is to the end

- use product.

II. Exposure Pathways

The human and environmental exposure s resulting from the use of Nanosilva , and use and disposal of plastics and textiles incorporating Nanosilva will largely be a function of what materials are available for inhalation or derma l exposure during treatment of plastics and textiles or what material s leach or break away from the plastics and textiles incorporating Nanosilva during use and disposal

. EPA anticipates that humans and the environmental will potentially be
expose d to the following materials :

1. Silver ions released from Nanosilva;
2. Nanosilva complex ; and /or
3. Nanosilver that might break away from the Nanosilva complex.

EPA expects that o ccupational inhalation and dermal exposures to Nanosilva and the nanoparticles that might break away from Nanosilva are likely to occur during the following use scenari os :

1. Mixing and loading of Nanosilver during preparation of a master batch

2. Mixing, loading, and applying the Nanosilva containing ma ster batch d uring treatment of plastics and textiles

3. Handling plastics and textiles incorporating Nanosilva

EPA expects c onsumer exposures to Nanosilva, the silver ions derived from the Nanosilva complex, and the nanoparticles that break away from the Na nosilva complex could potentially occur during the following use scenari os :

1. Incidental oral exposure to plastics and textiles incorporating Nanosilva

2. Dermal exposure to plastics and textiles incorporating Nanosilva

Leaching of plastics and textiles incorporating Nanosilva is anticipated to be the primary route by which Nanosilva, silver ions, and nanosilver reach the environment. The silver releasedAppendix B

- Page 3 during leaching of plastics and textiles incorporating Nanosilva could be discharged to the sanitary sewer system leading to publically owned wastewater treatment and privately owned septic systems, also known as the down

- the

- drain discharge scenario. Leaching of building material such as plastic siding and decks and ou tdoor furniture incorporating Nanosilva will be discharged directly into the environment. Once Nanosilva, silver ions, or nanosilver reach wastewater treatment and septic systems they will most likely complex with sulfide and partition to biosolids. Howeve r, some fraction of the silver compounds will reach surface water and may potentially impact aquatic organisms.

I II

. Data Needed to Confirm the Estimates for Risks of Exposure to Nanosilva

A s a condition of the registration , EPA is requiring the Nanosilva LLC to conduct a number of studies , based on a tiered approach, which will allow the Agency to confirm the findings of the risk assessment completed for the proposed conditional registration and discussed in the decision document. These tests inc lude Tier I studies to determine the nature and quantity of silver released from plastics incorporating Nanosilva under conditions of use, product characterization testing, and silver release characteristics from the Nanosilva complex. If nanosilver is det ermined to be released from plastics during the leaching tests as part of Tier I testing and the weight of evidence based on physical and chemical properties, toxicity, and exposure result in a risk

concern then route

- specific toxicity and environmental fa te 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 3 B to avoid cancellation of the conditional registration.

The durati on of four years was chosen to allow time for protocol reviews prior to initiation of the studies, completion of the studies, and Agency review of the studies following completion. The Agency will evaluate these data as they are submitted during the period of the conditional registration to confirm the Agency’s determination that 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 studies, or failed to submit the protocols or studies, as required pursuant to this Appendix , EPA will issue a notice of intent to cancel Nanosilva ’s registration under FIFRA section 6(e).

The following factors were considered in d eveloping the data requirements for Nanosilva :

• Submitted as a new active ingredient application, the Nanosilva nanosilver was subject to the data requirements for the registration of antimicrobial pesticides that are detailed in 40 CFR Part 161. These requ irements include studies on physical and chemical characteristics, residue chemistry, environmental fate, toxicology, reentry protection, spray drift, wildlife and aquatic organisms, plant protection, nontarget insects, and product performance.

• Although some studies, such as those dealing with 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 litera ture was used to tailor the data requirements to the proposed use pattern.

• Additional studies in the area of physical and chemical characterization that are notAppendix B

- Page 4 specifically included in 40 CFR Part 161 are needed because Nanosilva contains nanosilver. Thes e 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 the FIFRA SAP Report (FIFRA SAP, 2009) and by the MINChar In itiative (2008).
In addition, the following recommendations from the FIFRA SAP report were considered in the development of the data requirements as terms and conditions on the proposed conditional registration of Nanosilva :

• Both nano

- sized particles of s ilver (Ag) as well as ionic silver (Ag + ) can contribute to toxic effects of nanosilver. The rate of silver ion production, as well as the distribution of nanosilver in tissues and the environment, may differ substantially between nanosilver and other forms of silver , as nanosilver can potentially deliver silver ions directly to specific tissues, cell membranes or inside cells

– places where other forms of silver compounds cannot reach. Therefore, the hazard profile of nanosilver may 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 p hysicochemical properties, such as shape, surface area, surface charge or coating, are also likely to impact biological response and env ironmental fate.

• The Panel “disagreed that nanosilv er 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 sugges ted that the Agency 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 Nanosilva is incl uded in Tables 1 B and 2 B . The studies included in Table 1 B are considered to be Tier I, meaning that their need is not based on the results of other studies. The studies listed in Table 2 B are considered to be Tier II, because they may or may not be requir ed depending upon the results of the Tier I studies. The Nanosilva complex and plastics /textiles incorporating Nanosilva 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 leac hing and dissolution studies. Figure 1B contains a conceptual diagram outlining the Tier I and Tier II testing approach.Appendix B

- Page 5 Figure 1B

- Test m ateri al and tiered a pproach for Nanosilva and plastic incorporating Nanosilva

. S ee Tables 1 B and 2 B for the specific tests required for each material. Tier I I

- Testing

Nanosilva Complex

Environmental Fate Tier II

– Testing

Silver Nanoparticle

Product Chemistry
Health Effects Ecological Effects
Environmental Fate Tier I

- Testing

Nanosilva Comp lex

Product Chemistry

• U ltraviolent

- Vis ible Absorption

• Particle Size Distribution

• Particle Surface Area

• Particle Stability

Occupational Inhalation Exposure

• 90

- Day Inhalation Toxicity with Neuro and Muta

• Repro/Developmental Screening by Inhalation Route Tier I

- Testing

Nanosilva Treated Plastic

Leaching Study

W eight of evidence is
based on physical and chemical pro perties, toxicity, and exposure. Low risk potential, high confidence in available data, potential for no further testing.

Additional characterization of toxicity profile needed.Appendix B

- Page 6 I V. Non Data

- Related Terms and Conditions of Registration

Based on Nanosilva LLC’s original application, amendments to the proposed label language are required. The pesticide label must include the foll owing :

1. The application rate must be stated as being less than 3 0 ppm (mg/kg) or 0.00 3 % silver on a weight basis for plastics incorporat ing Nanosilva.

2. Nanosilva 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. Nanosilva may only be used to preserve plastics 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 Nanosilva during treatment of articles:

• Closed system loading of Nanosilva containing suspension.

• NIOSH c ertified full

- face respirators with P100 or equivalent filte r cartridges immediately available for use in an emergency

.

• Gloves which are chemically resistant to all of the components of the Nanosilva liquid suspension.

• A long

- sleeve shir t, long pants, shoes plus socks

.

5 . The label must have an e nvironmental h azard s tatement consisting of the following:

“This pesticide is toxic to fish and aquatic invertebrates, and birds, and can’t be used for irrigation purposes. ”

“Do not discharge effluent containing this product into lakes, streams, ponds, estuaries,

ocea ns, 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 discharge effluent containing this produ ct to sewer systems without previously notifying the local sewage treatment plant authority. For guidance contact your State Water Board or Regional Office of the EPA”.

V. Enforceable Schedule

Before EPA will issue a final determination on this application , Nanosilva LLC must provide information to satisfy the outstanding guideline items listed in Table 1 B under Product Chemistry. EPA will not issue a registration until all Product Chemistry information is submitted by Nanosilva LLC and accepted by the EPA.Appendix B

- Page 7 EPA has prepared an enforceable schedule that is presented in Table 3 B . This schedule is an estimation of the time required for developing and submitting protocols for review

, conducting the studies , and submitting the resulting data, as well as EPA’s review of the submitted data. However, unforeseen technical issues may arise due to the unique nature of Nanosilva (a difficult

- to

- test substance), which may cause a delay in testing. If such a case arises, Nanosilva LLC shall submit a written req uest 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 this case, the EPA shall 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 reviewing 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, N anosilva LLC shall:

1. S ubmit protocols modified for Nanosilva LLC to EPA’s satisfaction for each of the data requirements listed in Table 1 B

.

2. P erform each test and submit the results from each test as described in Table 1 B and 2 B

.

These items shall be submitted acc ording to the schedule provided in Table 3 B . If EPA determines that Nanosilva LLC has fail ed to initiate or to submit th e required studies by the dates indicated in Table 3 B , then EPA will issue notice of intent to cancel Nanosilva LL C ’s registration under FIFRA section 6(e). In addition, Nanosilva LLC’s conditional registration for Nanosilva will automatically expire four years after being issued. Nanosilva LLC will have to submit an application for an unconditional registration if th ey wish to continue to sell and distribute Nanosilva in the United States.

EPA will use this data to confirm EPA’s determin ation that the conditional registration of Nanosilva will not cause unreasonable adverse effects on the environment , taking into account the terms and conditions on the registration.

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 must meet 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 reportsAppendix B

- Page 8 Thus, the EPA expects that all studies performed to support, 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 stateme nt 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 registration or continued registration.

VI. Time

- Limited Conditional Registration

The conditional registration for Nanosilva will automatically expire four years after 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 dist ribute Nanosilva in the United States.

References:

FIFRA SAP, 2009. FIFRA Scientific Advisory Panel Meeting held November 3

- 5, 2009 on the Evaluation of Hazard and Exposure Associated with Nanosilver and Other Nanometal Pesticide Products Available at : http://www.epa.gov/scipoly/sap/meetings/2009/november/110309ameetingminutes.pdf

Lee, J.M., Kim, D.W., Jun, Y.D., Oh, S.G. 2006. Preparation of silica

- silver heterogeneous nanocomposite particles by one

- pot preparation strategy using polyol process: Size

- controlled 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 route for preparation of silica

- silver heterogeneous nanocomposite particles using alcohol reduction method. Materials Letters 61:558

- 1562. (MRID 48379902 and 48379904)

The Minimum Information for Nanomaterial Characterization (MINChar) Initiative, 2008. Available at : http://characterizationmatters.files.wordpress.com/2008/11/minchar

parameters

- list.pdfAppendix B

- Page 9 Table 1 B

- Summary of Ti er I Required Data for Nanosilva OSCPP Data Requirement (Note 1)

Guideline Number: Study Title Reason for Study Test Material Comments
Product Chemistry

830.1 620 : Production Process

Required for antimicrobial pesticides per 40 CFR part 158

. Nanosilva Completely describe quantities of reactants, reaction conditions, and reaction time.

830.1 6 50: Formulation Process Completely describe details regarding the formulation of the master batch.

830.1 670 : Formation of Impurities Describe all possible reaction by

- product impurities.

830.17 00: Preliminary Analysis Distinguish between the amount of silver and silver ions present.

830. 1800 : Analytical Method ICP

- AES only provides amount of silver present, also need method to quantify the amount of nanosilver present.

830.1900: Submittal of Samples Nanosilva LLC must provide samples of Nanosilva for EPA use in verifying analysis and identity of Nanosilva.

830.6314: Oxidation/Reduction; Chemical Incompatibility MSDS states that halogen salts and chlorides should be avoided; however, Nanosilva LLC states that the Nanosilva complex is stable. Need to fully describe stability of Nanosilva to other chemicals.

830.6317: Storage Stability Only the concentration of silver was determined. Need to describe changes in particle size and UV/Visible spectroscopy with storage time.

830.7050: UV

- Visible Light Adsorption UV/Visible spectroscopy is required to evaluate the assessment of the structural integrity of the active ingredient.

Nanosilva Characterization

Non

- Guideline: Particle Size and Diameter (size) Distribution Required to characterize product. Determine both the size of the silica core particle and the nanosilver particles.Appendix B

- Page 10 Table 1 B

- Summary of Ti er I Required Data for Nanosilva OSCPP Data Requirement (Note 1)

Guideline Number: Study Title Reason for Study Test Material Comments

Non

- Guideline: Surface Area Determination Required to characterize product. Determine both the surface area of the silica core particle and the nanosilver particles.

Non

- Guideline : Particulate Stability Required to assess stability of Nanosilva

. Nanosilva Nanosilva LLC claims that Nanosilva is a stable complex that does not release nanosilver. This test should be designed to demonstrate Nanosilva stability to mechanical stress (normal and shear) and common reactants and solvents. EPA will use this informati on to evaluate Nanosilva LLC claims of Nanosilva stability. Human Exposure

Non

- Guideline: Plastic Leaching Study (Confirm Nanosilva LLC study results) Required to confirm

assessment on human and environmental exposure.

This test will determine what test substance will be used for Tier II studies. Nanosilva Treated Plastics A leaching study is needed to d etermine the quantity and form of silver that is

r eleased from Nanosilva treated plastics under conditions of use. Nanosilva LLC submitted a leaching study using food simulants and determined the total silver concentration. EPA requires an additional, follow

- up study using physiological fluids and methods to determine the form of silver released from Nanosilva treated plastic. The exposure data will be used in to confirm the Agency’s evaluation of the risk of exposure to Nanosilva

.
Health Effects

870.3465: 90

- Day Inhalation Toxicity (Rat) (Replace S ong et al., 20 12 study results) Modified to include i n vivo bone marrow assay and functional observational battery, motor activity and detailed neuropathology Required to confirm the adequacy of the 10 fold database uncertainty factor, to reduce the uncertainties related to differences in the physical properties of the nanosilver

. Nanosilva The inhalation study is a route

- specific study required to evaluate the effects of inhaling nanosilver when mixing and loading Nanosilva.

Modified 870.3550/ OECD TG 421: Reproduction/Developmental Toxicity Screening Test 1 Required because there are currently no acceptable studies on the reproductive and developmental toxicity for nanosilver. Occupational exposure to workers of reproductive age. Nanosilva The combined repeated

- dose toxicity study with the reproduction/developmental toxicity screening test will provide initial information on possible effects on reproduction and/ or development . In addition, the study may also provide a toxicity endpoint applicable to a risk assessment for oral incidental expo sure.

Ecological Effects

– No Tier I Studies RequiredAppendix B

- Page 11 1 The use of a combined study that utilizes the 2

- generation reproduction study in rodents as a basic protocol for the addition of other endpoints or functional assessments in the immature animal is encouraged.

Table 2B

- Summary of Tier II Required Data f or Nanosilva OSCPP Data Requirement (Note 1)

Guideline Number: Study Title Reason for Study Test Material Comments Product Chemistry
830.7050: UV

- Visible Light Adsorption
These studies are required to characterize the nanosilver if released during Tier I stability, dissolution or leaching studies. Nanosilver Non

- Guideline: Particle Size and Diameter (size) Distribution
Non

- Guideline: Surface Area Determination 830.7840 : Solubility
Non

- Guideline: Zeta Potential and Surface Charge Determination Human Exposure

– No Tier II Studies are Required Health Effects

Non

- Guideline: peri

- and post

- natal exposure to Nanosilva Children’s exposure to nanosilver while contacting Nanosilva treated pl a stics and textiles

. Occupational exposure to nanosilver for workers of child bearing
age. See Note 2 Based on the results of the plastics leaching study , the Agency may require additional toxicity data to assess potential adverse health outcomes in young children resulting f rom incidental oral exposure to Nanosilva

- treated plastic toys (i.e., children chewing on toys).

Ecological Effects

850.1850: Modified Aquatic Food Chain Transfer Required to determine bioavailability and biomagnifications. See Note 2 Traditionally use Fish and Oyster BCF to estimate bioaccumulation for chemicals, however, mesocosm tests more likely to yield useful information.

850.4100: Terrestrial Plant Toxicity Required to determine effects to plants during early critical stages in their development. See Note 2 Nanosilver is likely to partition to biosolids during wastewater treatment. If those biosolids are then used in land farming, nanosilver may impact growth of plants in farm fields.

850.4400: Aquatic Plant Toxicity, Tier 2 Required to determine the toxicity to freshwater and aquatic plants

. See Note 2 Aquatic plants are the primary source of cellular carbon and chemical energy for aquatic environments. Impacts to these primary producers would have broad implications for the aquatic food chain.

850.5400: Algal Toxicity, Tier 2 Required to determine the phytotoxicity to freshwater and marine algae. See Note 2 Algae are the primary source of cellular carbon and chemical energy for aquatic environments. Impacts to these primary producers would have broad implications for the aquatic food chain.
Non

- Guideline: Measuring the Chronic Effects of Freshwater Sediment

- Associated Contaminants on Chironomus dilutes Required to determine chronic impact to freshwater sediment dwelling organisms. See Note 2 Silver nanoparticles if released into aquatic environments are likely to partition to sediment. Chronic tests on a freshwater benthic emergent insect ( Chironomus dilutes, formerly Chironomus tentans ) withAppendix B

- Page 12 Table 2B

- Summary of Tier II Required Data f or Nanosilva OSCPP Data Requirement (Note 1)

Guideline Number: Study Title Reason for Study Test Material Comments
epibenthic ecological niche will be used to estimate potential risks to freshwater benthic organisms.

Non

- Guideline: Measuring the Chronic Effects of Freshwater Sediment

- Associated Contaminants on Hyalella azteca Required to determine chronic impact to freshwater sediment dwelling organisms. See Note 2 Silver nanoparticles if released into aquatic environments are likely to partition to sediment. Chronic tests on a freshwater benthic amphipod ( Hyalella azteca ) with infaunal ecological niche will be used to estimate potential risks to freshwater b enthic organisms.
Non

- Guideline: Measuring the Chronic Effects of Marine and Estuarine Sediment

Associated Contaminants on Leptocheirus plumulosus Required to determine chronic impact to marine sediment dwelling organisms. See Note 2 Silver nanoparticles if released into aquatic environments are likely to partition to sediment. Chronic tests on an esuarine/marine benthic amphipod ( Leptocheirus plumulosus ) will be used to estimate potential risks to marine benthic organisms.

850.1075: Acute Toxicity Test w ith Freshwater and Marine Fish Required to confirm the chronic effects of nanosilver. See Note 2 A cute toxicity test with fish species is to help in the assessment of possible risk to similar species in natural environments, as an aid in determination of possible water quality criteria for regulatory purposes, and for use in correlation with acute testing of other species for comparative purposes

.

850.1300: Daphnid Chronic Toxicity Test Required to determine the chronic effects of nanosilver. See Note 2 T his guideline prescribes a chronic toxicity test in which daphnids are exposed to a chemical either in a static

- renewal or a flow through system.

850.1400 : Early

- L ife S tage Toxicity Test for Freshwater, Estuarine, and Marine Fish Required to determine the chronic effects of nanosilver. See Note 2 Tests with the early

- life stages of fish are intended to define the lethal and sublethal effects of chemicals on the stages and species tested. They yield information of value for the estimation of the chronic lethal and sublethal effects of the substance on other fish species. Environmental Fate

Non

- Guideline: Rate of Deposition The rates of aggregation and sedimentation are required for confirming estimates on the environmental fate and potential ecological impacts of these materials. See Note 2 Knowing the rate of aggregation and sedimentation of the products released from textiles would give primary information on the behavior of these compounds in the aquatic environment.

835.1100: Activated Sludge Sorption Isotherm Required to determine wast ewater treatment removal efficiency. See Note 2 The EPA uses Guideline 835.1110 test results to estimate the removal efficiency of a chemical as it passes through a wastewater treatment plant.Appendix B

- Page 13 Table 2B

- Summary of Tier II Required Data f or Nanosilva OSCPP Data Requirement (Note 1)

Guideline Number: Study Title Reason for Study Test Material Comments

835.1230 : Adsorption/Desorption (Batch Equilibrium) Required to determine partitioning to solids. See Note 2 If the material is removed during wastewater treatment, it may be deposited on land through the deposition of sludge (i.e. land farming). If the material is not removed during wastewater treatment it may be released into aquatic environments and may bind to sediment. Adsorption/desorption equilibrium studies required to determine the mobility of Nanosilva or nanosilver in the environment.

835.1240: Leaching Studies (Soil Column Tests) Required to determine the mobility in subsurface environment. See Note 2 The distance that nanoparticles move in soil and groundwater is thought to depend on interaction with soil grains. Attachment of nanoparticles to soil grains depends on the physical processes of sedimentation, interception, and diffusion rather than partitioning to natural organic matter. Nanoparticle to soil grain interaction depends on nanoparticle diameter, aqueous chemistry, and the arrangement of soil grains and must be measured using soil co lumn tests.

850.6800: Modified Activated Sludge, Respiration Inhibition Test for Sparingly Soluble Chemicals Required to determine impact to wastewater treatment systems. See Note 2 Silver from industrial processes (e.g., film processing) has been shown to reduce microbial activity in wastewater treatment systems. The purpose of the study is to assess the impact of Nanosilva or nanosilver on microbial activity during wastewater treatment.

Note 1: These guidelines only provide general guidance . Pro tocols shall be submitted prior to conducting these studies.

Note 2 : The test material shall include materials that are released during the stability and plastics leaching studies. These materials include nanosilver released from the Nanosilva comp lex or plastics treated with Nanosilva and or the Nanosilva complex released from plastics treated with Nanosilva

.Table 3 B

– Enforceable Schedule

The Nanosilva comp lex and plastics treated with Nanosilva will be the test material during Tier I studies. EPA anticipates that data will be developed in a phased approach. Thus, the schedule is separated into phases where Phase 1

– Product Characterization and Phase 2

– Product Testing will occur prior to devel oping protocols for Phase 3

– Release Characteristics/Exposure, and Phase 4

– Health Effects

.

Tier I : Nanosilva and Nanosilva Treated Plastics Testing

Prepare and Submit Protocols * Perform Study and Submit Results* Submittal of samples

Guideline Phase 1

– Product Characterization 2 6 Non

- Guideline Particle Size Distribution Non

- Guideline Surface Area

Phase 2

– Product Testing 5 9 Non

- Guideline Stability

Phase 3

– Release Characteristics/Exposure 21 27

Non

- Guideline Dissolution Kinetics Non

- Guideline Leaching Test of Plastic

Phase 4

– Health Effects 2 7 3 7 870.325 0 90

- Day Inhalation

870.3550 Modified Reproduction/Developmental Toxicity Screening Test

* Number of months after conditional registration is issued

.Appendix B

- Page 15 Table 3 B

– Enforceable Schedule

The test material for Tier II studies will depend on the results of the Tier I leaching and dissolution studies. EPA anticipates that data will be developed in a phased approach. Thus, the schedule is separated into phases where Phase 5

– Characterization will occur prior to developing protocols for Phase 7

– Health Effects, Phase 7

– Ecological Effects, and Phase 8

– Environmental Fate.

Tier I I : Testing for Nanosilver and /or Nanosilva Released during Tier I Tests
Prepare and Submit Protocols * Perform Study and Submit Results* Guideline Phase 5

– Characterization 19 † 24 830.7050 UV

- Vis Non

- Guideline Particle Size Distribution Non

- Guideline Surface Area 830.7840/7860 Solubility Non

- Guideline Zeta

- potential

Phase 6

- Health Effects 2 7 3 7 Non

- Guideline peri

- and post

- natal exposure to Nanosilva

Phase 7

– Ecological Effects 25 36
850.1850 Modified Aquatic Food Chain Transfer

850.4100 Terrestrial Plant Toxicity, Seedling Emergence
850.4400 Aquatic Plant Toxicity, Tier II
850.5400 Algal Toxicity, Tier II Non

- Guideline Measuring the Chronic Effects of Freshwater Sediment

- Associated Contaminants on Chironomus dilutes Non

- Guideline Measuring the Chronic Effects of Freshwater Sediment

- Associated Contaminants on Hyalella azteca
Non

- Guideline Measuring the Chronic Effects of Marine and Estuarine Sediment

- Associated Contaminants on Leptocheirus plumulosus
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 8

– Environmental Fate 25 36 Non

- Guideline Rate of Deposition
850.1100 Activated Sludge Sorption Isotherm
835.1230 Adsorption/Desorption
835.1240 Leaching Studies (Soil Column Tests)

850.6800 Modified Activated Sludge, Respiration Inhibition Test for Sparingly Soluble Chemicals

* Number of months after conditional registration is issued.