UNITED STATES ENVIRONMENTAL PROTECTION AGENCY 

W ASIDNGTON, D.C. 20460 

FEB 2 7 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 event corn lines (EPA File 

Symbol524-EUP-RNU); Decision No. 464546; DP Nos. 402221 and 

405553; 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 of new 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-

enolpyruvylshikimate-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 of this cassette results in the 

formation of240 base pair dsRNA containing a fragment of the 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 of the 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 

of transmembrane 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 48919004). 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 events 

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 of the 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 of this 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 risk assessment is found in 

the updated BRAD for 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 risk 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 

2 
insecticidal 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 of the 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. Of these, 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 of these 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 of the 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 of Hawaii, 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 of the number of acres proposed per year for each state was not provided. 

In the description of the 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 of regulated articles 

under the notification procedure. Specific isolation methods or distances are not described in this 

cited regulation. Subsequent correspondence from Monsanto1 states that 660ft isolation 

distances will be used to prevent outcrossing to other com. Approximately 95% of the 

experimental plots will have non-transgenic corn border rows that are intended as wind breaks 

and as part of experimental design to prevent "edge effects." While these rows are not planted to 

specifically prevent pollen movement outside of the experimental plots, it is expected to help 

contain pollen from MON 87410 and MON 87411 plants within the field, since a high 

percentage of com 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. 

3 
Containment measures are described to ensure that com involved in this EUP does not grow 

outside of the 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 of plant 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, if not 

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 irrigated fallow period after each harvest, during which the plots will be monitored 

for germinating com plants. 

Data are available on the expression of the Bt proteins expressed in the single event com lines 

included in this EUP. Data 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 

of detection in MON 87411 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 of leftover 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 of detection 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 of the 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 of dsRNA 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 of product 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. 

4 
In summary, exposure of nontarget 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 of the field, such that the 

majority settles 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 of exposure 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 of these data can be found in their respective BRADs. These data are sufficient for use 

in this EUP, and based on previous review of these 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 of the 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 of combined event com 

containing any of these currently registered single events. 

2. Nontarget effects data for Cry3Bbl expressed in MON 87410 and MON 87411 corn 

No nontarget data with the Cry3Bb1 protein expressed specifically 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 (US EPA 201 Ob ). Acceptable data are 

available to show that adverse effects are not expected to occur for birds, wild 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 of various higher tier laboratory and field studies were also 

submitted. These data 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 

87 410, and MON 87 411 are identical. Additionally, that the expression and identity of the 

Cry3Bb1 protein from the expression cassette was confrrmed by SDS-PAGE and determination 

5 
of theN-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 EUP. 

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 of insects from four insect orders, which were observed for 

mortality and various measures of sublethal effects (e.g., body weight, emergence). Two of the 

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 varivestis], 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 [Pediobius foveolatus ], 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 several 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 of the 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 information to show that effects are expected to be limited 

to the target pests insects and closely related species. Further details of the study are contained in 

the study's Data Evaluation Record. 

Monsanto subsequently provided additional unpublished data (no MRID at the time of this 

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 of the information reviewed is 

included in the Confidential Appendix. Upon review, this information provides stronger 

evidence of the specificity of the Dv49 dsRNA to the target gene and provides further support for 

a conclusion that effects on nontarget organisms are not expected. 

6 
EPA 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 of this EUP. 

In addition to the data considered above, there is also evidence of barriers 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 dsRN A 

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 small 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 of com 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, survival of hybrid 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 of these 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 of weediness 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 of dsRNA for 

agricultural pest control is emerging and some uncertainties are recognized, the data presented to 

date and information available in the literature suggest strongly that effects on the targeted Snj7 

7 
gene 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 of exposure high enough to cause 

effects is expected to be low. The acreage of corn to be planted is also limited across the 

proposed states. Based on the lack of expected 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 of the 

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 lygdamus palosverdesensis), 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). 

8 
Among the states 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, Presque 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 that will be involved in this EUP are identified as of the writing of this 

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). Upon completion of the 

assessment, Monsanto must submit a list of cooperators to the EPA for proposed release sites. 

Monsanto must ensure that test plot locations are > 1320 feet (Y-1 mile) of reported 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 Critical 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. 

9 
F. References 

Allen, M.L. and W.B. Walker. 2012. Saliva of Lygus lineolaris digests double stranded 

ribonucleic acids. Journal of Insect Physiology 58: 391-396. 

Bolognesi R., P. Ramaseshadri, J. Anderson, P. Bachman, W. Clinton, et al. 2012. 

Characterizing the mechanism of action of double-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. Mechanisms of dsRNA 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 of the National Academy of Science USA, 

10.1073/pnas.211287498. 

Raynor, G.S., Ogden, E. C., and J.V.Hayes. 1972. Dispersion and deposition of corn 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 unsuccessful 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 (CrylA.105)]. 

http://www.epa.gov/pesticides/biopesticides/pips/mon-89034-brad.pdf. 

USEP A. 2009. Biopesticides Registration Action Document - Bacillus thuringiensis Vip3Aa20 

Insecticidal Protein and the Genetic Material Necessary for Its Production (via Elements 

of Vector 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 

USEP A. 201 Oa. Biopesticides Registration Action Document: Cry 1 Ab and Cry 1 F Bacillus 

thuringiensis (Bt) Com Plant-Incorporated Protectants, 

http://www.epa.gov/oppbppdl/biopesticides/pips/cry1 f-cryl ab-brad.pdf. 

10 
USEPA. 2010b. Biopesticides Registration Action Document: Bacillus thuringiensis Cry3Bb1 

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/oppbppd l/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 of vector 

PSYN12274) in Event 5307 Com (SYN-05307-1). 

11 
Confidential Appendix 

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

12