UNIT ED STATE S ENVIRONMENTAL PROTECTION AGENCY WASIDNGTON, D.C

. 20460



## FEB 27 2013 OFFICE OF CHEMICAL SAFETY AND POLLUTION PREVENTION

MEMORANDUM
SUBJECT:

FROM :

THROUGH :

TO: Environmental Risk Assessment for a FIFRA Section 5 Experimental Use Permit for MON 87410 and MON 87411 and other registered events expressed in single event and various combined even t corn lines (EPA File Symbol524-EUP-RNU); Decision No. 464546; DP Nos. 402221 and 40555 3; Contains Confidential Appendix


### Shannon Borges, Lead Biologist

# ~ ' Microbial Pesticides Branch

# \~~**~~** Biopesticides and Pollution Prevention Division, 7511P 

# i?r

Chris Wozniak, Ph.D., Biotechnology Special Assistant

# /J f

- (/ Microbial Pesticides Branch 

# L;tuJ.u Biopesticides and Pollution Prevention Division, 7511P
Shanaz Bacchus, Regulatory Action Leader Microbial Pesticides Branch Biopesticides and Pollution Prevention Division, 7511P

Monsanto Company (hereafter " Monsanto") has applied for a FIFRA Section 5 Experimental Use Permit (EUP) to allow evaluation ofnew transgenic corn (Zea mays L.) lines, containing events MON 87410 and MON 87411, that express a modified Bacillus thuringiensis (Bt) Cry3Bb1 protein and Dv49 double stranded RNA (dsRNA). MON 87410 and MON 87411 confer resistance to corn rootworm (CRW; Diabrotica spp.), and also express the 5- enolpyruvylshikima te-3-phosphate synthase (CP4 EPSPS) protein from Agrobacterium sp. strain CP4, which confers resistance to glyphosate.
The Dv49 dsRNA expressed by MON 87410 and MON 87411 is a new and novel PIP with a mode of action that differs from Bt-derived PIPs. MON 87410 and MON 87411 contain a suppression cassette with an inverted repeat sequence (Dv49), which is derived from Western corn rootworm (WCRW, Diabrotica virgifera virgifera). Expression ofthis cassette results in the formation of240 base pair dsRNA containing a fragment ofthe target WCRW Snj7 gene. When consumed by the target pest, the dsRNA is taken up within the insect gut and enters into native RNA interference (RNAi) machinery, wherein it is cleaved into 21-24 mer small interfering RNAs (siRNAs). These siRNAs bind to a RNA induced silencing complex (RISC), ultimately leading to suppression ofthe target 

### Snj7 gene. Snf7 is a protein that is part ofthe ESCRT(Endosomal Sorting Complex Required for Transport)-III complex, which is involved in sorting oftransmembrane proteins en route to lysosomal degradation (Teis et al. 2008, Rusten et al. 2008, Lee and Gao 2008, Vaccari et al. 2009, Kim et al. 2011, as cited in MRID 48919004). According to information submitted by Monsanto, it is conserved in many organisms, including yeast, humans, mouse, fruit fly (Drosophila sp.), nematode (Cdenorhabditis elegans), and Arabidopsis thaliana (see references within MRID 489 1900 4). Monsanto states that preliminary data show that Snj7 is an essential gene with significant roles in intracellular trafficking, and the function ofDvSnf7 in WCRW is consistent with those observed in other eukaryotes. Further details are provided in Bolognesi et al. (2012).
This EUP will evaluate MON 87410 and MON 87411 singly and in combination with other events that are currently registered for full commercial cultivation. The registered even ts involved in this EUP are listed below

. Some corn lines included in this EUP are single or combined event corn containing only these currently registered PIPs.
MON 89034 (EPA Reg. No. 524-576): produces two insecticidal proteins that protect against feeding damage caused by European corn borer (Ostrinia nubilalis) and other lepidopteran insect pests. MON 89034 produces two Bt proteins, Cry2Ab2 (subsp. kurstaki) protein and Cry1A.105, a modified CrylA Bt protein. The ecological effects associated with Event MON 89034 corn and the environmental fate ofthe two Cry proteins have been reviewed by EPA (USEPA 2008).
TC1507 (EPA Reg. No. 68467-2): produces the Bt subsp. aizawai Cry1F protein to control larvae ofthe European corn borer and other lepidopteran insect pests. The latest assessment of ecological effects and environmental fate is included in the updated Biopesticides Registration Action Document (BRAD) for Cry1F (USEPA 2010a).
MON 88017 (EPA Reg. No. 524-551 ): produces a modified Bt (subsp. kumamotoensis) Cry3Bb1 protein to protect against CRW larval feeding. The Cry3Bb1 expressed in MON 87410 and MON 87411 is stated to be identical to the Cry3Bbl expressed in MON 88017, and data submitted for Cry3Bb1 expressed in MON 88017 will be used to determine risk for the Cry3Bb1 expressed in MON 87410 and MON 87411 for purposes ofthis EUP. EPA's environmental risk assessment for Cry3Bb1 expressed in MON 88017 is found in the updated 2010 BRAD for Cry3Bb1 (USEPA 2010b).
DAS-59122-7 (EPA Reg. No. 68467-5): produces the Bt strain PS149Bl Cry34Ab1 and Cry35Ab1 proteins to protect against CRW. EPA's environmental ri sk assessment is found in the updated BRAD f or Cry34Abl and Cry35Ab1 (USEPA, 2010c).
MIR162 (EPA Reg. No. 67979-14): produces the Bt Vip3Aa20 protein to protect against feeding damage caused by lepidopteran insect pests such as com earworm (Helicoverpa zea). The Agency's environmental ri sk assessment is found in the BRAD for Vip3Aa20 (USEPA 2009).
5307 (EPA Reg. No. 67979-22): produces the eCry3.1Ab recombinant fusion protein, which contains domains from the Bt derived mCry3A and Cry 1 Ab proteins. The eCry3 .1 Ab protein has

2insecticidal activity against CRW. Event 5307 was registered in 2012, and the environmental risk assessment for eCry3.1Ab can be found in the eCry3.1Ab BRAD (USEPA 2012).
In addition to the data on environmental effects and fate contained in the BRADs for each registered event listed above, some additional data have been submitted to support the risk assessment for Dv49 dsRNA. All ofthese data are considered here in conjunction with potential exposure to estimate the environmental risks ofthe single and combined event PIPs included in this proposed EUP. This memorandum contains the environmental risk assessment for these events within this proposed EUP.
A. Nontarget Organism Exposure and Environmental Fate
This EUP is proposed to take place on a total of20,893 acres, across 22 states and Puerto Rico from its issuance (expected February 2013) to July 31, 2014. Ofthese, 17,740 acres will include PIPs (other acreages include corn planted in borders or non-PIP corn); 6,988 acres are planned for planting PIP corn in 2013 and 10,752 acres are proposed for 2014. The majority of these acreages will be planted with corn containing the single events listed above that are already registered or combinations ofthese events (24 combinations only utilizing currently registered PIP events are planned for planting in this EUP). For the entire EUP, approximately 7,600 total acres will be planted during 2013 and 2014 with event MON 87410 or MON 87411 com, either alone or in combination with one or more ofthe registered events listed above. Acreages planted with corn containing event MON 87410 or MON 87411 will vary across the 22 proposed states and Puerto Rico, with the greatest acreage proposed for Illinois and Iowa (> 1000 acres total). With the exception ofHawaii, South Dakota, Nebraska, and Kansas, the remaining states will be planted with< 300 total acres ofMON 87410 or MON 87411 over the two years planned for this EUP. An account ofthe number of acres proposed per year for each state was not provided.
In the description ofthe experimental program, Monsanto states that PIP test materials will be isolated to prevent persistence in the environment, and that isolation (pollen containment) methods will be implemented consistent with USDA standards of7 CFR § 340.3(c). This regulation describes performance standard requirements for introductions ofregulated articles under the notification procedure. Specific isolation methods or distances are not described in this cited regulation. Subsequent correspondence from Monsanto 1 states that 660ft isolation distances will be used to prevent outcrossing to other com. Approximately 95% ofthe experimental plots will have non-transgenic corn border rows that are intended as wind breaks and as part ofexperimental design to prevent "edge effects." While these rows are not planted to specifically prevent pollen movement outside ofthe experimental plots, it is expected to help contain pollen from MON 87410 and MON 87411 plants within the field, since a high percentage ofcom pollen is deposited within corn fields (Raynor et al. 1972). The majority of the remaining plots will be detasseled or destroyed before flowering or managed under controlled pollination per USDA field design protocol.

1 Letter from M.E. Groth (Monsanto Company) to K. Nesci (OPP) dated February 5, 2013; subject: Supplemental information in support ofthe application for an Experimental Use Permit to test MON 87410 and MON 87411 combined trait insect control corn, combinations for their production, and controls. 3Containment measures are described to ensure that com involved in this EUP does not grow outside ofthe area or time frame permitted by this EUP. Harvested ears, seed or grain will be placed in labeled containers of such construction to prevent loss ofplant material , and equipment will be thoroughly cleaned after harvesting. Leftover planting seed and harvested seed, grain, or plant material will be used for other research or future planting, placed in storage or returned to Monsanto or a third party facility, or destroyed. Plant material from non-harvested plots, ifnot destroyed prior to flowering, will be devitalized and volunteer plants will be routinely monitored and destroyed. In Hawaii and Puerto Rico, continuous cropping may occur, but must include at least a 30

-day irriga ted fallow period after each harvest, during which the plots will be monitored for germinating com plants.
Data are available on the expression ofthe 

### Bt proteins expressed in the single event com lines included in this EUP. Da ta that exist on currently registered events are contained in the BRADs listed above, and data on protein and dsRNA expression for MON 87410 and MON 87411 were reviewed for the product characterization assessment for this proposed EUP? Data contained in MRID 48919003 show that Dv49 dsRNA is expressed at quantifiable levels in leaf, root, forage, silk, and seed in MON 87410 and MON 87411 com, and is lowest in seed and pollen. Dv49 dsRNA is expressed at quantifiable levels in pollen in MON 87410 corn, but was below the limit ofdetection in MON 874 1 1 com. The routes by which nontarget organisms are exposed to 

### Bt proteins have been described in risk assessments performed for previous 

### Bt PIP registrations. Exposure to the 

### Bt proteins in the registered PIPs included in this EUP, and also the Cry3Bbl expressed in MON 87410 and MON 8741 1 corn, are expected to be similar; however, destruction ofleftover plant material and volunteers will reduce environmental exposure that may otherwise result from residual plant material. Dv49 dsRNA is expressed in similar locations in MON 87410 and MON 87411 plants (except it was below the limit ofdetection in pollen from MON 87411) , so exposure routes for Dv49 dsRNA expressed by MON 87410 and MON 87411 corn are also expected to be similar to that of

### Bt PIPs.
Short-term persistence of

### Bt proteins in soil is well-documented , and studies submitted to EPA have shown that the registered 

### Bt PIP proteins included in this EUP degrade rapidly in soil. The persistence ofthe dsRNA in the environment is not as well understood, but preliminary data submitted by Monsanto (Fisher et al. 20 12) show that degradation in soil is comparable to that of 

### Bt PIP proteins. DTsos ofdsRNA in various soil types have ranged from 19.7

- 24.1 hours. In soil containing com tissue spiked with dsRNA, RNA was not detected in soil and the soil had no toxicity to corn rootworm after 32 hours. Further data on environmental persistence will need to be collected to support a Section 3 registration ofMON 87410 and MON 87411, but these data provide sufficient preliminary evidence that the dsRNA degrades quickly in the environment.

2 See memorandum from J

. Facey to S. Bacchus dated January 10, 2013 , Re: Review ofproduct characterization and human health data for a combined trait plant incorporated protectant: a dsRNA transcript comprising a Dv49 inverted repeat sequence derived from Diabrotica virgifera and Bacillus thuringiensis (Bt) Cry3Bb 1 insect control protein and the genetic material necessary for its production in MON 87410 and MON 87411

...in support for an Experimental Use Permit. 4In summary, exposure ofnontarget organisms to the 

### Bt derived proteins and the Dv49 dsRNA is expected to be similar to that assessed previously for 

### Bt PIP proteins. Persistence is not expected to be greater for the Dv49 dsRNA as compared to the 

### Bt PIP proteins. This EUP involves a relatively limited acreage over which MON 87410 and MON 87411 will be planted, and crop destruct measures and volunteer monitoring will serve to reduce overall exposure to nontarget organisms. Corn pollen is relatively large among the grass pollens (90

- 100

**~m),** and density of corn pollen drops off very steeply within a few meters from the edge ofthe field , such that the majority sett les out within 10

- 15 meters (Pleasants et al. 2001, Raynor et al. 1972). The experimental design features described for this EUP are expected to reduce pollen movement. Additionally, since Bt-derived PIP proteins and dsRNA degrade rapidly, significant runoff is not expected. Based on the information available, the occurrence ofexposure to nontarget organisms is expected to be limited to the field and immediate margins, and is expected to be low for nontarget organisms. 

### B. Non

-Target Organism Effects Data
1. 

### Nontarget effects data for registered corn events Nontarget data are available for the com events listed above that are currently registered, and reviews ofthese data can be found in their respective BRADs. These data are sufficient for use in this EUP, and based on previous review ofthese data, EPA concluded that adverse effects are not expected for nontarget organisms exposed to the Cry2Ab2, Cry1A.105, Cry1F, Cry3Bb1, Cry34/35Ab1, Vip3Aa20, and eCry3.1Ab proteins expressed by these registered events in corn. Further, many ofthe stacked or pyramided combinations that will be tested in this EUP exist at least partially in other registered combined event com products. Protein expression equivalence and synergism has been assessed for those registered combinations , and indicate no potential increased exposure or effects related to their combination. Additional data on protein expression and interaction will be required to support a new Section 3 registration ofcombined event com containing any ofthese currently registered single events. 

### 2. Nontarget effects data for Cry3Bbl 

### expressed in MON 87410 and MON 87411 corn
No nontarget data with the Cry3B b1 protein expressed specifica lly in MON 87410 and MON 87411 have been submitted to support this EUP. However, nontarget data are available for the Cry3Bb 1 protein expressed in event MON 88017 corn (USEPA 201 Ob). Acceptab le data are available to show that adverse effects are not expected to occur for birds, w ild mammals, freshwater fish and invertebrates, honey bee 

### (Apis mellifera), nontarget insects and soil invertebrates. Additional supplemental studies on the insect activity spectrum, a preliminary Tier IV field evaluation, and summaries ofvarious higher tier laboratory and field studies were also submitted

. These da ta were sufficient to allow EPA to determine that the Cry3Bb1 expressed in MON 88017 corn presented minimal risk to nontarget organisms. Data to show the biochemical and functional equivalence ofthe Cry3Bbl as expressed 

### inplanta by events MON 88017, MON 87410 , and MON 87411 , has not been provided. However, according to information submitted with this application, the amino acid sequences for Cry3Bb1 expressed by MON 88017, MON 87410, and MON 87411 are identical. Additionally, that the expression and identity ofthe Cry3Bb1 protein from the expression cassette was confrrmed by SDS-PAGE and determination

5oftheN-terminus to be identical to MON 86 (see the memorandum cited in footnote 1)

. The available data on Cry3Bb1 as expressed in MON 88017 are sufficient to support this E UP.
3. Nontarget effects data for Dv49 dsRNA expressed in MON 87410 and MON 87411 corn Nontarget insect data have been submitted to support this EUP, and the data are meant to demonstrate the specificity ofDv49 dsRNA to the target pests. Monsanto submitted a preliminary study on the activity spectrum for Dv49 dsRNA (MRID 48919004). In this study, test material was fed to 14 species ofinsects from four insect orders, which were observed for mortality and various measures ofsubletha l effects (e.g., body weight, emergence). Two ofthe species tested were target pests for Dv49 dsRNA (WCRW and Southern corn rootworm, SCRW, [Diabrotica undecimpunctata howardi]) and six species were lepidopteran or other coleopteran pest species (Colorado potato beetle [Leptinotarsa decemlineata], Mexican bean beetle [Epilachna varivesti s], red flour beetle [Tribolium castaneum], fall armyworm [Spodoptera frugiperda], com earworm [Helicoverpa zea], and European corn borer [Ostrinia nubilalis]). The remaining six species included nontarget beneficial species (pink spotted lady beetle [Coleomegilla maculata], carabid beetle [Poecilus chalcites],jewel wasp [ Nasonia vitripennis] , eulophid wasp [Pediobiusfoveolatus], and insidious flower bug [Orius insidiosus]) and an economically important domesticated insect (silkworm [Bombyx mori]). Significant mortality was observed only in the two target species, and LCsos were determined to be 1.2 and 4.4 ng/mL diet for SCRW and WCRW, respectively. Significant mortality or sublethal effects were not observed in the remaining species, which were tested at 500- 5000 ng/mL diet. These concentrations were 300

- 3000 times the maximum expected environmental concentration. The study includes severa l deficiencies that prevent its classification as "acceptable" and introduces some uncertainty with the results (e.g., the test material is not fully characterized and its equivalence to the plant expressed dsRNA was not demonstrated), this study shows that the activity spectrum ofthe test material is limited among the species tested within the Order Coleoptera to the Chrysomelidae family under the test conditions. This preliminary study is supplemental, but does provide some informa tion to show that effects are expected to be limited to the target pests insects and closely related species. Further details ofthe study are contained in the study's Data Evaluation Record.
Monsanto subsequently provided additional unpublished data (no MRID at the time ofthis review) that included more details about the testing described above. This information also included data from reciprocal and indirect feeding bioassays, sequence alignments of orthologous Snj7 sequences from several insects, and phylogenetic trees based on these sequence alignments. These data provide stronger evidence for the specificity ofDv49 dsRNA for the target pests and show that the Snj7 orthologous sequences from several species closely related to the target species diverges rapidly across these species

. Since the data were provided in an unpublished manuscript and marked confidential, a summary ofthe information reviewed is included in the Confidential Appendix. Upon review, this informa tion provides stronger evidence ofthe specificity ofthe Dv49 dsRNA to the target gene and provides further support for a conclusion that effects on nontarget organisms are not expected.



### 6EPA has assessed the potential risk to humans (see footnote 2), and has determined that the potential for Dv49 dsRNA to produce toxic effects in humans is minimal for this EUP. EPA has also determined that the tolerance exemption for nucleic acids (40 CFR 174.507) applies to Dv49 dsRNA for this EUP. These are measures by which EPA determines the potential hazard to wild mammals, and the findings also support a conclusion that nontarget effects would not be expected as a result ofthis EUP.
In addition to the data considered above, there is also evidence ofbarriers to uptake that prevent effects in nontarget organisms. For example, one study with the tarnished plant bug (Lygus lineolaris) has shown that endonucleases present in saliva degrade dsRNA, which prevents RNAi effects in this species by orally delivered dsRNA (Allen and Walker 2010). The rationale also cites a review by Huvenne and Smagghe (20 1 0), in which sensitivity to dsRNA among different insects is discussed. Terenius et al. (20 11) also reviewed numerous studies with dsRNA and RNAi in lepidopterans, which documented the difficulties of achieving success with RNAi, particularly by feeding. As noted in the human health and product characterization review for this EUP, dsRNA is not stable within the digestive tract and blood because low pH depurinates nucleic acids, RNA is degraded quickly and cleared from the serum, and pancreatic and salivary nucleases metabolize dsRNA. Nucleic acids and sma ll dsRNA are also present in foods. In summary, while the database upon which to make a risk determination for MON 87410 and MON 87411 is limited relative to other PIP EUPs, the information presented here and available in the literature is sufficient to conclude that uses ofDv49 dsRNA as proposed for this EUP are not likely to produce adverse effects in nontarget species.
C. Outcrossing and Weediness
EPA has previously determined that there is no significant risk of gene capture and expression of any 

### Bt endotoxin by wild or weedy relatives ofcom in the U.S. , its possessions or territories (see BRADs cited herein and USEPA 2000). Since these conclusions are based on the nature of pollination, surviva l ofhybrid offspring, and weediness in com and its relatives, these onclusions can also be extended to Dv49 dsRNA.
D. Environmental Risk Characterization and Conclusions
EPA has previously concluded that the currently registered single event PIPs involved in this EUP present minimal risk of adverse effects to nontarget organisms, and these conclusions would also apply to their proposed use in this EUP. Protein expression and synergism data are not available for the combined event PIPs proposed for this EUP ; however, the current knowledgebase regarding the potential for several registered combinations ofthese events suggests the potential for unexpected nontarget effects among the combined trait com lines proposed for this EUP is unlikely. EPA has also concluded that the potential for outcrossing and development ofweediness are unlikely for the all com events included in this EUP.
Similar conclusions are made for the MON 87410 and MON 87411 single and combined event com lines proposed for this EUP. While the science supporting the use ofdsRNA for agricultural pest control is emerging and some uncertainties are recognized, the data presented to date and information available in the literatur e suggest strongly that effects on the targeted Snj7

7gene are likely to be limited to the target pests. As discussed above, any exposure to nontarget organisms is expected to be limited to consumption ofMON 87410 and MON 87411 plant material within the experimental field and surrounding margins (within 10-15 meters). Effects are concentration dependent and because MON 87410 and MON 87411 are expressed at very low levels, particularly in pollen and kernels, the likelihood ofexposure high enough to cause effects is expected to be low. The acreage ofcorn to be planted is also limited across the proposed states. Based on the lack ofexpected effects and the low potential for exposure, effects to nontarget organisms are expected to be minimal as a result ofMON 87410 and MON 87411 planted under this EUP.
E. Endangered Species Considerations
EPA has concluded that adverse effects are not expected to nontarget organisms as a result ofthe proposed EUP. Since EPA has determined that no effects are anticipated for nontarget species exposed to the Bt proteins and Dv49 dsRNA included in this EUP, effects to federally-listed threatened and endangered species and their designated critical habitats are also not expected. Therefore, a "No Effect" determination is made for direct and indirect effects to federally listed endangered and threatened ("listed") species and their designated critical habitats for the proposed experimental uses. Since Bt proteins and Dv49 dsRNA expressed by the events involved in this EUP target lepidopteran and/or coleopteran insect species, concerns are raised for listed lepidopteran and coleopteran species that are known to exist in the states included in this proposed EUP. Listed lepidopteran species in these states include Bay checkerspot butterfly (Euphydryas editha bayensis), Behren's silverspot butterfly (Speyeria zerene behrensii), Callippe silverspot butterfly (Speyeria cal/ippe cal/ippe), El Segundo blue butterfly (Euphilotes battoides allyni), Karner blue butterfly (Lycaeides melissa samuelis), Lange's metalmark butterfly (Apodemia mormo langei), Lotis blue butterfly ( Lycaeides argyrognomon !otis), Mission blue butterfly (Icaricia icarioides missionensis), Mitchell's satyr butterfly (Neonympha mitchellii mitchellii), Mertyle's silverspot butterfly (Speyeria zerene myrtleae), Oregon silverspot butterfly (Speyeria zerene hippolyta), Palos Verdes blue butterfly (Glaucopsyche lygdamuspalosverdesensis), Quino checkerspot butterfly (Euphydryas editha quino), Saint Francis ' satyr butterfly (Neonympha mitchellii francisci), San Bruno elfin butterfly (Callophrys mossii bayensis), Smith's blue butterfly (Euphilotes enoptes smithi), Uncompahgre fritillary butterfly (Boloria acrocnema), Blackburn's sphinx moth (Manduca blackburni), Kern primrose sphinx moth (Euproserpinus euterpe), Carson wandering skipper (Pseudocopaeodes eunus obscures), Laguna mountain skipper (Pyrgus ruralis lagunae), and Pawnee montane skipper (Hesperia leonardus montana). Listed coleopteran species in these states include American burying beetle (Nicrophorus americanus), Casey's June beetle (Dinacoma caseyi), Delta green ground beetle (Elaphrus viridis), Hungerford's crawling water beetle (Brychius hungerfordi), Mount Hermon June beetle (Polyphylla barbata), Ohlone tiger beetle (Cicindela ohlone), Salt Creek tiger beetle (Cicindela nevadica lincolniana), and Valley elderberry longhorn beetle (Desmocerus californicus dimorphus).

8Among the sta tes involved in this proposed EUP , one or more of these species occur in the following counties: 1) Arkansas: Franklin, Little River, Logan, Scott, Sebastian 2) California: Alameda, Amador, Butte, Calaveras , Colusa, Contra Costa, Del Norte, El Dorado , Fresno, Glenn, Kern, Kings , Lake, Lassen, Los Angeles, Madera, Marin, Mariposa, Mendocino, Merced, Monterey, Napa, Nevada, Orange, Placer, Riverside, Sacramento, San Bernardino , San Diego, San Francisco , San Joaquin, San Luis Obispo, San Mateo , Santa Clara, Santa Cruz, Shasta, Solano, Sonoma, Stanislaus, Sutter, Tehama, Tulare, Tuolomne, Yolo, Yuba 3) Colorado: Chaffee, Dolores, Douglas, Eagle, Gunnison, Hinsdale, Jefferson, La Plata, Lake, Mineral, Ouray, Park, Pitkin, Rio Grande, Saguache, San Juan, San Miguel, Summit, Teller 4) Hawaii: Hawaii, Kahoolawe, Kauai, Lana'! , Maui , Molokai, Oahu 5) Illinois: Lake 6) Indiana : La Porte, Lagrange, Lake, Porter 1) Kansas: Chautauqua, Elk, Montgomery, Wilson 8) Michigan: Allegan, Barry, Berrien, Branch, Cass, Emmet, Ionia, Jackson, Kalamazoo, Kent, Lake, Mason, Mecosta, Monroe, Montcalm, Montmorency, Muskegon, Newaygo, Oceana, Pr esque Isle, Saint Joseph, Van Buren, Washtenaw 9) Minnesota: Winona 10) Nebraska: Blaine, Boyd, Brown, Cherry, Custer, Dawson, Frontier, Garfield, Gosper, Holt, Keya Paha, Lancaster, Lincoln, Loup, Rock, Thomas, Wheeler 11) North Carolina: Craven, Hoke 12) Ohio: Athens, Hocking, Lucas, Portage, Vinton 13) South Dakota: Bennett, Brookings, Gregory, Haakon, Todd, Tripp, Union 14) Wisconsin: Adams, Barron, Burnett, Chippewa, Clark, Dunn, Eau Claire, Green Lake, Jackson, Juneau, Kenosha, Marquette, Menominee, Monroe, Oconto, Outagamie, Polk, Portage, Shawano, St. Croix, Waupaca, Waushara, Wood.
Not all cooperators tha t will be involved in this EUP are identified as ofthe writing ofthis memorandum , which is typical ofPIP EUPs, so experimental plot locations are not yet known and will need to be determined. Therefore, planting experimental plots in the states and counties listed above, as well as adjacent counties (where subcounty level species location information for the counties above is unavailable), must be limited to prevent exposure to listed lepidopteran and coleopteran species. Monsanto has proposed to perform a proximity assessment for listed species for planned trial locations prior to planting using data obtained from the FIFRA Endangered Species Task Force (FESTF) Information Management System (IMS). U pon completion ofthe assessment, Monsanto must submit a list ofcooperators to the EPA for proposed release sites. Monsanto must ensure that test plot locations are > 1320 feet (Y-1 mile) ofreported locations of listed lepidopteran and coleopteran species and their designated Critical Habitat, and provide sufficient detail on the proximity and the analysis performed to confirm the minimum Y4 mile distance from listed species and Critica l Habitat locations. This approach is consistent with the approach used in previous EUPs with Bt-derived PIPs to prevent exposure to listed species of these orders, and is acceptable for this EUP.

9F. References
Allen, M.L. and W.B. Walker. 2012. Saliva ofLygus lineolaris digests double stranded ribonucleic aci ds. Journal ofInsect Physiology 58: 391-396. Bolognesi R. , P. Ramaseshadri, J

. Anderson, P. Bachman, W. Clint on, et al. 2012. Characterizing the mechanism of action ofdouble-stranded RNA activity against Western com rootworrn (Diabrotica virgifera virgifera LeConte). PLoS ONE 7: e47534. doi: 10.1371/journal.pone.0047534 Huvenne, H. and G. Smagghe. 2010. Mechani sms ofdsRNA uptake in insects and potential of RNAi for pest control: a review. Journal oflnsect Physiology 56: 227

-235.
Pleasants, J.M. , R.L. Hellmich, G. Dively, M.K. Sears, D.E. Stanley-Home, H.R. Matilla, J.E. Foster, T.L. Clark, and G.D. Jones. 2001. Corn pollen depositionon milkweeds in and near corn fields. Proceedings ofthe National Academy of Science USA, 10.1073/pnas.211287498. Raynor, G.S., Ogden, E. C., and J .V.Hayes. 1972. Dispersion and deposition ofcorn pollen from experimental sources. Agron. 

### J. 64: 420-427.
Terenius, 0. , A. Papanicolaou, J.S. Garbutt, I. Eleftherianos, et al. 2011. RNA interference in Lepidoptera: an overview of successful and unsucces sful studies and implications for experimental design. Journal oflnsect Physiology 57: 231-245.
United States Environmental Protection Agency (USEPA). 2000. Biopesticide Registration Action Document: Bacillus thuringiensis Plant Pesticides. http:/ /www.epa.gov/scipoly /sap/meetings /2000/l 01800 mtg.htm
USEPA. 2008. Biopesticides Registration Action Document: Bacillus thuringiensis CrylA.105 and Cry2Ab2 insecticidal proteins and the genetic material necessary for their production in corn [PC Codes 006515 (Cry2Ab2), 006514 (Cry lA.10 5)]. http://www.epa.gov/pesticides/biopesticides/pips/mon-89034-brad.pdf.
USEPA. 2009. Biopesticides Registration Action Document

- Bacillus thuringiensis Vip3Aa20 Insecticida l Protein and the Genetic Material Necessary for Its Production (via Elements ofVector pNOV1300) in Event MIR162 Maize (OECD Unique Identifier: SYN-IR162

- 4). http: //www.epa.gov /oppOOOOl/chem search/reg actions/registration/decision G118 3-Apr-09.pdf
USEPA. 201 Oa. Biopesticides Registration Ac tion Document: Cry 1 Ab and Cry 1F Bacillus thuringiensis (Bt) Com Plant-Incorporated Protectants, http://www.epa.gov/oppbppdl/biopesticides/pips/ cry1 f-cryl ab-brad.pdf

.

10USEPA. 2010b. Biopesticides Registration Action Document: Bacillus thuringiensis Cry3Bb 1 protein and the genetic material necessary for its production (vector PV-ZMIR39) in MON 88017 com (OECD Unique Identifier: MON-88017-3). http://www.epa.gov/pesticides/biopesticides/pips/cry3 bb 1-brad.pdf
USEPA. 2010c. Biopesticides Registration Action Document

- Bacillus thuringiensis Cry34Ab1 and Cry35Ab 1 Proteins and the Genetic Material Necessary for Their Production (PHP17662 T-DNA) in Event DAS-59122-7 Corn (OECD Unique Identifier: DAS59122-7), http://www
.epa.gov/oppbppdl/biopesticides/pips/cry3435ab 1-brad.pdf.
USEPA. 2012. Biopesticides Registration Action Document: Bacillus thuringiensis eCry3.1Ab Protein and the Genetic Material Necessary for its Production (via elements ofvector PSYN12274) in Event 5307 Com (SYN-05307-1).

11Confidential Appendix
***This section contains information that has been claimed to be confidential***

12