UNITED STATES ENVIRONMENTAL PROTECTION AGENCY WASHINGTON, D.C. 20460

MEMORANDUM

DATE: August 27, 2013 OFFICE OF CHEMICAL SAFETY AND

POLLUTION PREVENTION

SUBJECT: Nanosilva: Summary of Hazard and Science Policy Council (HASPOC)

Meetin gs on June 20 and Augus t 15,2013: Recommenda tio ns on Data Requirements and Waivers for Nanosilva.

PC Code: 072599 DP Barcode: N/ A

Decision No.: N / A Registration No.: N I A

Petition No.: N / A Regulatory Action: N / A

Risk Assessment Type: N/A Case No.: NIA

TXR No.: 0056700 CAS No.: N/A



### MRID No.: N/A 40**C~** 

### NA •

FROM: Julie Van Alstine, MPTT

# ~**.~.1** ; 11

# 01/\...~+-a-.____ Executive

# Secretary,~~ Heal th Effects Division ( HED ; 09P)

THROUGH: Jes s Rowland, Co-Chair

Anna Lowit, Ph.D., Co-Chai

Hazard and Science Policy Counc · HED (7509P)

TO: Jed Costanza, Ph.D.

Antimicrobial Division (AD; 751 OP)

JUNE 20,2013 MEETING ATTENDEES:

HASPOC Members : Anna Lowit, Elizabeth Mendez, Jeff Evans, Jeff Dawson, Jess Rowland

, Jonathan Chen, Michael Metzger, P.V. Shah, Ray Kent

Presenter: J ed Costanza

Other Attendees: Joey Bever, Steve Weiss, Jonathan Leshin, Tim McMahon, Christopher Schlosser, Jaime D'Agostino, Minerva Mercado, Monique Perron, Yung Yang, Monica Hawkins

, Tim Leighton, Amber Aranda, Michelle Centra, Jenny Tao, Tim Dole, Uma Habiba, Anwar Dunbar, Kristin Rury, Julie

Van Alstine

Page 1 of9Page 2 of 9 AUGUST 15, 2013 MEETING ATTENDEES:

HASPOC Members: Anna Lowit, Elissa R eaves, Elizabeth Mendez, Jeff Dawson, John Kough, Michael Metzger, P.V. Shah, Ray Kent

Presenter: Timothy Leighton

Other Attendees: Jonathan Leshin, Timothy Leighton, Joey Bever, Seiichi Murasaki, Kristin Rury, Julie Van Alstine

I. PURPOSE OF MEETING

In August, 2009 Nanosilva LLC submitted an applic ation to register Nanosilva, a nanosilver containing pesticide, as a new active ingredient. Nanosilva LLC submitted acute toxicity studies, a study that determined the amount of silver leaching from plastic coupons incorporating Nanosilva, and a request to waive some of the required toxicity studies . The Antimicrobials Division (AD) requested that the Hazard and Sc ience Policy Council (HASPOC) evaluate two issues: 1) general considerations for toxicity data needs for nanoscale ma terials and 2) specific
data needs for Nanosilva. The HASPOC met on June 20 and August 15, 2013 to discuss all of
these issues.

II. GENERAL CONSIDERATIONS FOR NANOSCALE MATERIALS DATA NEEDS

In this memo the HASPOC delineates important considerations for toxicology data needs for
nanoscale materials to facilitate a transparent and consistent appr oach to evaluating pesticides containing nanoscale ingredients. The foundatio n for these considerations can be found in Appendix A of the HeiQ-AGS 20 Decision Document with further refinements based on insights gained from further review of the toxicol ogy database for nanosilver and on the HASPOC’s experience with conve ntional pesticides. The HASPOC generally uses a risk-based weight of the evidence (WOE) approach in considering toxicol ogy data needs. This WOE approach does not involve a “check-box approach,” but instea d evaluates and weighs the confidence and uncertainty associated with multiple scientific areas ( http://www.epa.gov/pesticides/regula ting/part158-tox-data-requirement.pdf ). The following four principles used by HASPOC for toxicity needs for conventional pesticides can be applied, with adjustments, to nanoscale materials.

1. Physical-Chemical Properties, Including Particle Information: Some important nanoscale properties for consid eration are: form (e.g., powder, liquid), composition (i.e., whether or not the material is a co mposite), size, shape, coating, and surface charge.

2. Use Pattern and Exposure Scenarios: Exposure patterns and pathways need to be
evaluated and described to ensure that toxic ity data match, to the extent possible, the appropriate duration and route of exposure in addition to the population of interest. This description needs to e xplicitly consider exposure to occupational handlers and consumers separately.Page 3 of 9 3. Toxicity Profile: First, the available experimental toxicology studies need to be summarized for the nanoscale ingredients in a specific pesticide product being evaluated. Then, available, relevant toxicology data on the same nanoscale ingredients from the open literature needs be considered. Uncertainty factors also
need to be described.

4. Margins of Exposure (MOEs) or Other Appropriate Risk Metrics (e.g., Percent Reference Dose): The WOE approach includes char acterization of risk metrics which incorporate toxicity and exposure toge ther. MOEs should be compared to the target MOE and associated confidence a nd uncertainties need to be described.

III. SUMMARY OF USE PROFILE AND RISK ASSESSMENT FOR NANOSILVA

Nanosilva is a liquid suspension containing 1% na nosilver particulates by weight where the nanosilver active-ingredient is attached to cr ystalline silica via a th iolate bond. Nanosilva LLC is proposing that Nanosilva be inco rporated into plastics and text iles at 30 mg per kg of article treated to suppress the growth of bacteria, algae, fungus , mold, and mildew, which cause odors, discoloration, stains, and deteri oration. Nanosilva LLC proposed that Nanosilva be used in a wide range of non-food contact categories including housewares, building materials, bathroom fixtures and accessories, electronics, appliances , personal care products, automotive equipment, hospital and institutional facility equipment, s porting goods, and textiles.

There are no product-specif ic intermediate- or long-term experi mental toxicity studies available
for Nanosilva. EPA evaluated the risk from occupational and consumer exposure using experimental toxicology data available in th e scientific literatu re for nanosilver.

Oral and dermal exposure is being asse ssed using oral toxi city studies by Kim et al. (2008) and

Kim et al. (2010). These studies used CMC coated na nosilver with average diameter of 56 and 60 nm, respectively; EPA acknowledges that the Ki m studies use nanosilver which is different from the diameter and surface coating of the na nosilver in Nanosilva. Unlike most toxicity
studies from the scientific li terature, however, thes e studies were completed according to Organization for Economic Cooperation and Devel opment (OECD) guidelines. Moreover, these studies identified histopathological patterns in the liver that were indicative of di stinct adverse
effects. EPA has determined that the no- observed adverse-effect level (NOAEL) of 30 mg/kg/day from the 28-day or al toxicity study by Kim et al. (2008) is the poi nt of departure (POD) for short-term oral exposures (<30 da ys) to the nanosilver in Nanosilva based on significant increases in alkaline phosphatase and cholesterol and si gnificant changes in hematology that were accompanied by histopathol ogical evidence of liver toxicity (bile-duct hyperplasia around central vein, infi ltration of inflammatory cells , and dilation of the central vein) seen at the lowest-observed adverse-e ffect level (LOAEL) of 300 mg/kg/day. EPA has also determined that the LOAEL of 30 mg/kg/ day based on histopathological evidences of liver toxicity (bile-duct hyperplasia with focal, multif ocal, or lobular necrosis) in both males and females from the 90-day or al toxicity study by Kim et al. (2010) is the POD for intermediateterm oral exposures (1 to 6 months) to the nano silver in Nanosilva. A dermal absorption factor (DAF) of 6.7% based on a human clinical study by Wan et al. (1991) is applied to extrapolate
from the oral to dermal routes.Page 4 of 9 EPA has determined that the NOAEL of 49 μg/m 3 is the POD for short- and intermediate-term inhalation exposures to the nanosilver in Na nosilva. The inhalation toxicity study by Song et al. (2012) used uncoated nanosilver with average diam eters of 14 to 15 nm, respectively, which is similar to the diameter, but different than the surface coating, of the nanosilver in Nanosilva. EPA has determined that the NOAEL is 49 μg/m 3 based on adverse histop athological patterns in lung tissue observed at the 117 μg/m 3 dose.

For short-term exposure, the target MOE of 1,000 is applied based on the standard 10-fold uncertainty factors for potential interspecies extrapolation and intraspecies variation, and a 10- fold database uncertainty factor. In addition, a 3-fold uncertainty factor (UF) was used to
account for the lack of NOAEL in the 90 day oral toxicity study by Kim et al. , 2010 making the
intermediate-term oral target MOE 3,000.

EPA does not typically consider long-term occupational exposures to antimicrobial 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. Howe ver, consumer exposure to Nanosilver may result in long-term exposure. The maximum reco mmended target MOE is 3,000 (U.S. EPA, 2002), which has already been established for intermed iate-term exposures. Therefore, an additional UF for extrapolation from a subchr onic study to chronic exposure durat ions has not been applied.

IV. STUDY WAIV ER REQUESTS

1. Physical-Chemical Properties:

Table 1. Physical-Chemical Properties of Nanosilva.

Parameter Nanosilva Form Liquid Suspension with 1% nanosilver by weight Composition Nanosilver-Silica Composite Transmission Electron Microscopy (TEM) Image

Overall Particle Size of Composite Average of 320 nm Shape of Nanosilver Spherical Size of Nanosilver (diameter) 3 to 18 nm Coating PVP and sulfur Surface Charge NegativePage 5 of 9 2. Exposure scenarios: EPA evaluates the risk of occupational exposures from mixing, loading, and applying or handling pesticide products. EPA expects that occupational inhalation and dermal expos ures are likely to occur during mixing and loading of Nanosilva during preparat ion of a master ba tch. 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 dete rioration of plastics and te xtiles. Because plastics incorporating Nanosilva could be subseque ntly used to manufacture children’s toys and textiles worn by children, it is assumed that children will be exposed to products containing Nanosilva. EPA expect s children’s exposures are likely to occur during incidental oral and derm al exposures to pl astics and textiles incorporating Nanosilva.

To evaluate the risk from exposure to na nosilver that might break away from the Nanosilva complex, Nanosilva LLC submitted studies showing that silver at concentrations greater than the analytical detection limit were not found leaching from plastic coupons and shirts incorpor ating Nanosilva (Table 2). Thus, based on the available data, there is little exposur e to nanosilver from plastic and textile incorporating Nanosilva. EPA calculated the daily-dose to workers assuming that all the silver in Nanosilva was freely avai lable as nanosilver. Likewise for the daily-dose to consumers, EPA assumed that silver leaching from plastic coupons and shirts incorporating Nanosilva, wh ich was at concentrations below the analytical detection limit, was in the fo rm of nanosilver as found in Nanosilva.

Table 2. Amount of Silver Released fr om Shirts and Plastics Incorporating
Nanosilva.

Parameter Nanosilva Source of Information Nanosilva Study Treatment Method Incorporated into
Shirts Incorporated into
Plastics Amount of Silver Released (% of silver in article) 1.6 to 0.9%* <0.1%*

Transmission Electron Microscopy (TEM) Image Not enough silver to image

Overall Particle Size of Composite Not enough silver Shape of Nanosilver Not enough silver Size of Nanosilver (diameter) Not enough silver Coating Not enough silver Surface Charge Not enough silver * Calculated assuming that silver at a c oncentration of one-half the detection limit was released during leaching studies.

3. Margins of Exposure (MOEs): The MOE for occupationa l inhalation exposure to
the nanosilver in the Nanosilv a liquid suspension is 2,500 if workers use close-system loading when mixing and loading th e Nanosilva liquid suspension (Target MOE=1,000). The MOE for dermal exposur e to the nanosilver in the NanosilvaPage 6 of 9 liquid suspension is greater than 1,000,000 if workers use closed-system loading when mixing and loading the Nanosilva liquid suspension, which is significantly greater than the target MOE of 3,000. The MOEs calculate d for the incidental oral exposure to nanosilver in plastic toys incorporating Nanosilva are 310,000 for children 6 to <12 months old, 550,000 for ch ildren 1 to <2 years old, and 940,000 for children 2 to <3 years old (Target MOE=3,000) . The MOEs calculated for the dermal exposure to nanosilver in flooring incor porating Nanosilva are 820,000 for children 6 to <12 months old, 860,000 for children 1 to <2 years old, and 910,000 for children 2 to <3 years old (Target MOE=3,000). The MOEs calculated for the dermal exposure to nanosilver in textiles incorporatin g Nanosilva are 340,000 for children 6 to <12 months old, 360,000 for children 1 to <2 y ears old, and 370,000 for children 2 to <3 years old (Target MOE=3,000). The other rout e-specific scenarios for incidental oral exposure to flooring and textiles incor porating Nanosilva provide MOEs of 1,000,000 or higher.

The aggregate MOEs for children who are, within the same short time frame, mouthing a toy, flooring, and a textile cont aining Nanosilva while wearing a textile containing Nanosilva and craw ling on flooring incorpora ting Nanosilva are greater than 100,000 for all lifestages, indicating that the risk for short- and intermediate-term simultaneous exposure to plastics and te xtiles incorporating Nanosilva is not a concern (Target MOE = 3,000; U.S. EPA, 2002). The calculated MOEs for dermal and incidental oral exposure to plastic s and textiles incorporating Nanosilva are greater than 100,000, indicating that daily exposure to plas tics and textiles incorporating Nanosilva for greater than 6 m onths are not likely to be of concern.

4. Toxicity: Nanosilva LLC submitted results from guideline acute animal-toxicity tests completed using high-level doses of a liq uid suspension containing Nanosilva with 1% nanosilver by weight. There were no mortalities or abnormalities noted in test animals after administration of Nanosilva by oral, dermal, and inhalation routes at dose levels of up to 5,000 mg/kg and 2.05 mg /L, respectively. Nanosilva caused
moderate 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 . The currently available subchronic oral toxicity studies indicate that nanosilver causes liver and ki dney toxicity in laboratory
animals where silver is distributed to a ll organs and tissues with accumulation of
silver in the brain and male animal testes. Subchronic inhalation t oxicity studies also identified liver toxicity as well as lung effects including chronic alveolar inflammation. There were pot ential neurotoxic effects identified with increases in
neurotransmitter concentrations and loss of spatial cognition; however, these same effects were not observed in a follow-on study. The in vitro Ames and chromosome aberration assays indicate th at nanosilver is not expected to be mutagenic while the mammalian cell micronucleus and mouse lymphoma with comet assay suggest that nanosilver may have mutagenic potential. However, the lack of genotoxicity from the

in vivo study indicates that ther e is inadequate information to assess mutagenic (and hence carcinogenic) potentia l of nanosilver. Finally, there is not enough information on the reproductive and developmental t oxicity for nanosilver at this time.Page 7 of 9 V. HASPOC WOE EVALUATIONS

1. Inhalation Study: Previously, the Office of Pesticide Programs (OPP) used a set of
criteria to determine whether an inhalati on study could be waived. These criteria
considered the scientific information avai lable for the chemical, including its: 1) degree of irritation and corrosiv ity; 2) volatility; 3) aerosol particle size; and 4) Acute Toxicity Category and extrapolated MO Es (e.g., MOEs 10 times higher than the target). In 2009, OPP developed an issue paper on risk assessment approaches for semi-volatile pesticides. As part of that issue paper, an analytical comparison was conducted of oral and inhalation experimental toxicology studies. In general, this analysis showed that the degree to which oral PODs were protective of potential inhalation toxicity va ried. In many cases the oral P OD was protective, but in some cases the inhalation PODs were significantl y more sensitive. Currently, OPP uses a WOE approach that builds upon OPP’s experien ce using the criteria listed above and conclusions from the 2009 SAP. As appr oaches for route-to-route extrapolation continue to evolve and impr ove, OPP may incorporate addi tional considerations into the WOE analysis.

The HASPOC has concluded that a subchr onic inhalation study with Nanosilva is
needed to reduce the uncertainties related to the differences in the physical properties of the nanosilver partic le used in the Song et al. (2012) study and the Nanosilva particles. This approach considered all of the available hazard and exposure information for nanosilver including: 1) the difference in the physical/chemical properties between the available nanosilv er inhalation toxici ty studies and the nanosilver in Nanosilva; 2) the sensitive effects noted in the inhalation study of decreases in physiological measures of lung function combined with chronic alveolar inflammation and macrophage accumulation in the lungs; and 3) the use of an
inhalation POD which results in an MOE that is near the Agency’s level of concern when exposure is assessed with a closed system.

2. Subchronic Dermal Toxicity Study: Based on a WOE approach, the HASPOC concludes that a dermal study is not require d because the use of an oral POD results
in MOEs that are ≥ 1,000,000 for occupational and great er than 300,000 for consumer exposure pathways. These MOEs are well above the Agency’s level of concern.

3. Prenatal Developmental Toxicity Study: HASPOC has concluded that a reproduction/developmental toxicity sc reening test (Modified OCSPP 870.3550/ OECD TG 421) conducted via th e inhalation route is neede d. This determination is based on a WOE analysis which provides: 1) female workers of child bearing age will handle Nanosilva during mixing and load ing; 2) as noted above, occupational MOEs are near the Agency’s level of con cern when exposure is assessed with closed system and additional information is need ed to reduce uncerta inty; 3) there are currently no acceptable studies on the reproductive and developmental toxicity for nanosilver. However, there were dose-depe ndent increases in th e concentration of
silver in the testes of rats after oral ingestion, inhalati on, and injection of nanosilver and, in another study, nanosilver was distribut ed to major maternal organs and extra-Page 8 of 9 embryonic tissues although no adverse mo rphological effects on the developing embryos were observed.

4. Bacterial Reverse Mutation Test, Detectio n of Gene Mutations in Somatic Cells in Culture, In Vitro Mammalian Cy togenetics, Mammalian Bone Marrow
Chromosome Aberration Test : The mutagenicity studies [Bacterial Reverse Mutation Test (OPPTS 870.5100), Detection of Gene Mutations in Somatic Cells in Culture (OPPTS 870.5300 ), In Vitro Mammalian Cy togenetics (OPPTS 870.5375),
Mammalian Bone Marrow Chromosome Aber ration T est (OPPTS 870.5385)] are
satisfied because the available in vitro studies suggest that nanosilver may have mutagenic potential. 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 clastogen ic nor aneugenic in

vivo ; however, there was no positive proof th at nanosilver reached the bone marrow in the micronucleus assay. Th e HASPOC has concluded that an in vivo bone marrow assay is needed and that this assay can be added as a component of the subchronic inhalation study.

5. Neurotoxicity Battery: Acute and subchronic neurot oxicity studies can provide important scientific information on potential nervous system effects from pesticide exposure. These studies offer data on a wi de range of functional tests for evaluating neurotoxicity including sensor y effects, neuromuscular effects, learning and memory and histopathology of the nervous system. The study completed by Hadrup et al. (2012b), which reported significant increases in neurotransmitter c oncentrations (e.g., dopamine) after oral administration of nanosilver to rats at concentrations of up to 9 mg/kg/day, lacks histological support for determining NOAEL/LOAELs. Liu et al. (2013) reported no effects on the spatial cognition or hippocam pal activity of mice after injecting nanosilver into the body cavity (i.e., intraperitoneal injection) at concentrations of up to 50 mg/kg. The e ffects on spatial cognition and hippocampal activity observed by Liu et al. (2012) after administeri ng nasal drops containing
nanosilver at concentrations of 3 and 30 mg/kg to rats suggest possible neurotoxic
effects from the inhalation of nanosilver. 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). Unless appropriate study designs are used, many guideline studies are consider ed inadequate in their assessment of
behavioral effects and do not us e optimal methods to evaluate the potential toxicity to
the nervous tissue structure and function. The HASPOC has concluded that there is potential for neurotoxicity based on the open li terature studies. However, in lieu of a standard guideline study, neurotoxicity evaluation (functional observational battery, motor activity and detailed neuropathology) can be added as a component of the subchronic inhalation study.

6. Immunotoxicity: The HASPOC recommends that a waiver can be granted for an immunotoxicity study for Nanosilva, ba sed on the following considerations:Page 9 of 9  The currently available oral toxicity st udies indicate that na nosilver causes liver and kidney toxicity in laboratory animals wh ere silver is distributed to all organs
and tissues with accumulation of silver in the brain and male animal testes. Inhalation toxicity st udies also identified liver t oxicity as well as lung effects including chronic alve olar inflammation.

 PODs from the most sensitive endpoints are currently used for assessing risks
from short and intermediate or al and inhalation exposures.

 The toxicology database for nanosilver does not reveal any evidence of treatmentrelated effects on the immune system. The overall WOE suggests that this chemical does not directly target the immune system.

IV. HASPOC CONCLUSIONS

In summary, the HASPOC concludes th at a subchronic inhalation study and a reproduction/developmental toxicity screening study are needed. In addition, data to evaluate the potential for neurotoxicity and in vivo bone marrow assay are needed. Immunotoxicity and
subchronic dermal studies are not needed and can be waived.

The HASPOC recommends that the registrant submit protocols fo r the needed studies for the Agency to review. If the registrant has additio nal exposure or toxicity information which could
be used to further refine the data needs descri bed here for Nanosilva, th e registrant may submit a
rebuttal for review.